Cholix-derived delivery construct

Abstract

The present invention provides a delivery construct comprising: (a) a transport facilitator comprising an immunoglobulin CH2 domain; (b) a transporter comprising a cholix domain Ia; and (c) a therapeutic cargo; wherein the transporter is cleavably coupled to the therapeutic cargo and wherein the transport facilitator is N-terminal to the transporter. Also provided are pharmaceutical compositions comprising the delivery construct.

Description

[0001] Cholix-derived delivery construct

[0002] Field of the Invention

[0003] The present invention relates to delivery constructs for the systemic delivery of therapeutic cargo, and in particular, to delivery constructs comprising a transport facilitator comprising an immunoglobulin CH2 domain, a transporter comprising a cholix domain la and a therapeutic cargo.

[0004] Background

[0005] The exotoxin cholix (Chx) is a virulence factor secreted by Vibrio cholerae. The first domain of Chx, amino acids 1 -264, functions as a transporter to transport domains 2 (amino acids 265-386; translation function), 1 b (amino acids 387-423) and 3 (amino acids 424-634; ADP-ribosylation enzyme) of the toxin. In polarised intestinal epithelial cells, Chx domain 1 interacts with a series of host cell proteins to traffic within a series of endosomal compartments to efficiently undergo apical to basal (A^B) vesicular transcytosis without accessing the cytoplasm and avoiding routing to lysosomes (Liu et al., 2022). Steps of this A^B transcytosis pathway include: 1) receptor-mediated endocytosis at the apical plasma membrane, 2) entry into and exit from a vesicular compartment located in the apical region of the enterocyte, 3) vesicular trafficking through the endoplasmic reticulum Golgi intermediate complex (ERGIC), 4) entry into and exit from a vesicular compartment located in the basal region of the enterocyte, and 5) release from the basal surface of the enterocyte. The transcytosis of intact Chx across polarised intestinal epithelial cells allows the fully active toxin to selectively target non-polarised cells present in the lamina propria.

[0006] The transporter function of Chx domain 1 has previously been exploited for the transcytosis of therapeutic cargo. The first genetic chimera of this type composed the first 386 amino acids of the toxin (Chxsse) non- cleavably coupled to human interleukin 10 (IL-10). That construct, known as AMT-101 , was shown to efficiently transport across polarised intestinal epithelial cells to reach the lamina propria (Fay et al., 2020). However, systemic distribution was limited, due to the dual action of receptor mediated interactions with IL-10 receptors on macrophages and Chx receptor-positive cells present in the lamina propria.

[0007] Most biopharmaceuticals developed for therapeutic use provide subject benefit only after systemic exposure. There remains a need for biopharmaceuticals suitable for oral administration which achieve systemic delivery.

[0008] The present invention has been devised in light of the above considerations. 008866329 2

[0009] Summary of the Invention

[0010] The present invention is based on experimental work which sought to generate cholix-therapeutic cargo constructs which can be systemically delivered. The present inventors found that to enable systemic delivery, the Chx transporter needs to separate from the therapeutic cargo after completing apical to basal (A^B) transcytosis in polarised epithelial cells, and before uptake into Chx receptor-positive cells present in the lamina propria. This is achieved by coupling of the Chx transporter to the therapeutic cargo in a cleavable manner. Without this separation, the Chx receptor-positive cells present in the lamina propria consume the therapeutic cargo and prevent systemic delivery. However, a challenge with this cleavable approach is the pathway used by Chx for its A^B transcytosis. The coordinated processes of A^B vesicular transcytosis and cargo separation involve a series of specific steps, where the Chx intersects with vesicular compartments in both the apical and basal regions of polarised epithelial cells. These vesicular compartments process pre-pro forms of proteins destined for release from the apical or basal side of the cells, respectively. The inventors found that such cleavably coupled constructs were prematurely cleaved at the apical region. This resulted in the released cargo being subject to lysosomal degradation. Entirely surprisingly, the present inventors have found that this premature cleavage can be avoided by including an immunoglobulin CH2 domain in the construct. Even more surprisingly, the present inventors have found that the position of the CH2 domain, N terminal to the Chx domain, in the construct is necessary for its functionality. Without wishing to be bound by theory, inclusion of the CH2 domain enables the construct to undergo the A^B transcytosis pathway without intersecting the apical endosomal compartment. This avoids premature cleavage of the Chx domain from the cargo, ensuring that cleavage occurs primarily in the basal region. Advantageously, this results in improved systemic delivery of the therapeutic cargo compared to that achieved by constructs without an immunoglobulin CH2 domain.

[0011] Therefore, according to a first aspect, the present invention provides a delivery construct comprising:

[0012] (a) a transport facilitator comprising an immunoglobulin CH2 domain

[0013] (b) a transporter comprising a cholix domain la; and

[0014] (c) a therapeutic cargo; wherein the transporter is cleavably coupled to the therapeutic cargo and wherein the transport facilitator is N-terminal to the transporter.

[0015] In some embodiments, the transporter comprises or consists of SEQ ID NO: 5.

[0016] In some embodiments the transporter comprises or consists of SEQ ID NO: 3. In some embodiments the transporter comprises or consists of SEQ ID NO: 4. 008866329 3

[0017] In some embodiments, the delivery construct comprises, from N to C, the transport facilitator, the transporter and the therapeutic cargo.

[0018] In some embodiments, the transporter is cleavably coupled to the therapeutic cargo via a cleavable linker. The cleavable linker may comprise a furin cleavage sequence or a matrix metalloproteinase cleavage sequence. In some embodiments the cleavable linker comprises a furin cleavage sequence.

[0019] In some embodiments the immunoglobulin CH2 domain comprises an IgG CH2 domain. The IgG CH2 domain may comprise an lgG1 CH2 domain.

[0020] In some embodiments the immunoglobulin CH2 domain is a human or humanised immunoglobulin CH2 domain.

[0021] In some embodiments the immunoglobulin CH2 domain comprises a peptide having at least 80% identity to SEQ ID NO: 31 . The immunoglobulin CH2 domain may comprise SEQ ID NO: 31 .

[0022] In some embodiments the transport facilitator does not comprise any immunoglobulin variable domains.

[0023] In some embodiments the therapeutic cargo comprises a polypeptide or polynucleotide sequence. The polynucleotide sequence may comprise an RNA sequence, such as an siRNA.

[0024] In some embodiments the therapeutic cargo comprises a cytokine, a hormone, a single-chain Fv fragment (scFv), a Fab fragment, a diabody, a minibody or a nanobody. The therapeutic cargo may comprise a hormone. The hormone may comprise human growth factor hormone or glucagon-like- peptide-1 .

[0025] According to a second aspect, the present invention provides a pharmaceutical composition comprising a delivery construct according to the first aspect and a pharmaceutically acceptable carrier. In some embodiments the pharmaceutical composition is formulated for oral administration. In some embodiments the pharmaceutical composition is formulated in a capsule or tablet.

[0026] According to a third aspect, the present invention provides the delivery construct of the first aspect or the pharmaceutical composition of the second aspect for use in a method of treatment. 008866329 4

[0027] According to a fourth aspect the present invention provides one or more nucleic acid(s) encoding the delivery construct of the first aspect.

[0028] According to a fifth aspect, the present invention provides a vector comprising the nucleic acid(s) according to the fourth aspect.

[0029] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0030] Summary of the Figures

[0031] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0032] Figure 1. Construction and proteolytic testing of cholix (Chx) transporters composed of either the first 266 amino acids (Chx266) or 197 amino acids (Chxi97) of cholix and different linker sequences. A) Schematic to show the chimeric constructs tested. B) In vitro digestion of the chimeras noted in A).

[0033] Figure 2. Structural differences between Chx266 and Chxi97.

[0034] Figure 3. Comparison of in vivo and in vitro performance of Chx266 and Chxi97transporters to deliver human growth hormone (hGH) across intact intestinal barriers. A) Time-concentration profiles for systemic serum levels of hGH following the introduction of the constructs or hGH alone administered into the lumen of rat jejunum. B) Area under the curve (AUC) values calculated from data shown in A).

[0035] Figure 4. Process and outcomes to connect infliximab to Chx266 using a homo-bifunctional reagent. Diagram of steps involved for targeted coupling is shown where adjacent cysteine residues on infliximab and Chx266 with a modified C-terminal amino acid sequence of KCAACQ (SEQ ID NO: 13).

[0036] Figure 5. Structure of chemical coupling agent, and schematic denoting targeted site on an lgG1 antibody to be targeted to produce Chx266-R3-infliximab.

[0037] Figure 6. Systemic delivery of hGH is enhanced by incorporation of the CH2 element of the human Fc domain. A) Time-concentration profiles for systemic serum levels of hGH following the intraluminal injection (ILI) of hGH alone or Chx-based construct into the lumen of rat jejunum. B) Bioavailability of 008866329 5 hGH relative to the standard administration route (FREL%) of this protein by subcutaneous injection. C) In vitro digestion patterns of Cn2-Chxi97-L1 -hGH and CH3-Chxi97-L1 -hGH. D) Schematic of Ch2-F3-Chxi97 and L1 -hGH fragments.

[0038] Figure 7. An N-terminal position of the CH2 element is necessary for systemic delivery of hGH. A) Time-concentration profiles for systemic serum levels of hGH following the intraluminal injection (ILI) of Chxi97-L1 -hGH, Ch2-Chxi97-L1 -hGH, or Chxi96-Ch2-L1 -hGH into the lumen of rat jejunum. B) In vitro digestion patterns of Chxi96-Ch2-L1 -hGH and CH2-Chxi97-L1 -sfGFP.

[0039] Figure 8. MMP-cleavable linkers can effectively function as cleavable linkers to couple Chx to the therapeutic cargo. Rate of transport of hGH (PAPP) across human caco-2 monolayers following the application of MMP-cleavable linker containing constructs to the apical surface of the caco-2 monolayers for two hours.

[0040] Figure 9. hGH from constructs containing MMP-cleavable linkers reaches the basal compartment of cells. Contents of apical and basal compartments from caco-2 cells from Figure 8 were harvested and analysed for hGH levels by ELISA.

[0041] Figure 10. Chx transporters can deliver human growth hormone (hGH) across intact intestinal barriers when coupled using MMP-cleavable linkers. Bioavailability of hGH relative to the standard administration route (FREL%) of this protein by subcutaneous injection.

[0042] Figure 11. Pharmacokinetic profile of constructs comprising MMP-cleavable linkers. Timeconcentration profiles for systemic serum levels of hGH following the intraluminal injection (ILI) of Chx266- MMPL4-hGH, Chx266-MMPL5-hGH or Chx266-MMPL8-hGH into the lumen of rat jejunum.

[0043] Detailed Description of the Invention

[0044] In the context of the present invention, a transporter is an element which is capable of transporting a cargo intracellularly. A transporter may otherwise be referred to as a carrier.

[0045] The cholix A gene encodes a 666 amino acid length protein (SEQ ID NO: 1 ), which includes a 32 amino acid leader sequence. Mature cholix (SEQ ID NO: 2, SEQ ID NO: 1 without the leader sequence) has exotoxin functionality conferred by domain III (amino acids 424-634 of SEQ ID NO: 2), which is an ADP- ribosylation enzyme. It will be appreciated that in the present invention, the transporter is non-toxic. It will 008866329 6 therefore be appreciated that, typically, the transporter does not comprise domain III (amino acids 424- 634 of SEQ ID NO: 2). In some embodiments, the transporter does not comprise domain lb (amino acids 387-423 of SEQ ID NO: 2) of cholix. In some embodiments, the transporter does not comprise domain 2 (amino acids 265-386 of SEQ ID NO: 2).

[0046] Sequencing of cholix A gene subtypes has previously identified slight variations in sequence between subtypes and so has determined a consensus cholix sequence (Prasad Awasthi et al., 2012). Therefore, cholix domain la may comprise a consensus sequence, for example of amino acids 1 -264 of SEQ ID NO: 2 (SEQ ID NO: 3).

[0047] In some embodiments, Cholix domain la comprises SEQ ID NO: 4. SEQ ID NO: 4 comprises the consensus amino acids 1 -264 of the mature cholix sequence with the additional two first amino acids of domain II to form a 266 amino acid sequence. This may otherwise be referred to as “Chx266”.

[0048] In some embodiments Cholix domain la comprises a consensus sequence of amino acids 1 -193 of SEQ ID NO: 2 (SEQ ID NO: 5).

[0049] SEQ ID NOs 3, 4 and 5 each contain an X at position 180. X can be any amino acid. Preferably, X comprises the amino acid W or A. Exemplary cholix domain la sequences may comprise any of SEQ ID Nos 6 to 1 1 , which represent SEQ ID Nos 3-5 wherein X is A or W.

[0050] Cholix domain la may comprise a variant or fragment of any of SEQ ID Nos 3 to 1 1 which retains transporter function. A variant, as used herein, will be understood to have at least 80% sequence identity to any of SEQ ID Nos 3 to 1 1 (optionally at least 85%, at least 90%, at least 95% or at least 99% sequence identity to any of SEQ ID Nos 3 to 1 1 ).

[0051] Percent (%) amino acid sequence identity with respect to a reference sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. % identity values may be determined by WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span = 1 , overlap fraction = 0.125, word threshold (T) = 1 1 . A % amino acid sequence identity value is determined by the number of matching identical residues as determined by WU-BLAST-2, divided by the 008866329 7 total number of residues of the reference sequence (gaps introduced by WU-BLAST-2 into the reference sequence to maximise the alignment score being ignored), multiplied by 100.

[0052] The present inventors envisage that the claimed construct can be used for the efficient transcytosis of a cargo across polarised epithelial cells, thereby enabling systemic delivery of the cargo. Such transcytosis can occur across any anatomical location comprising polarised epithelial cells such as, but not necessarily limited to, sweat glands, ductal cells of the pancreas of breast, salivary glands, intestine or airway. In particular, the present inventors envisage that the claimed construct can be used for the efficient transcytosis of a cargo across polarised epithelial cells in the intestine or airway, thereby enabling systemic delivery of the cargo.

[0053] The term “therapeutic cargo” will therefore be understood to refer to any cargo which can have systemic therapeutic application.

[0054] Various suitable therapeutic cargos will be known to and available to the skilled person. For example, the therapeutic cargo may comprise a protein, a peptide, a polynucleotide sequence or a small molecule.

[0055] In some embodiments the therapeutic cargo comprises a polypeptide or polynucleotide sequence. The polynucleotide sequence may comprise RNA and / or DNA.

[0056] Exemplary polynucleotide sequences include, but are not limited to siRNA, PNA, miRNA, DNA, plasmids and antisense.

[0057] In some embodiments the therapeutic cargo comprises a cytokine, a hormone, a single-chain Fv fragment (scFv), a Fab fragment, a diabody, an antibody, a minibody or a nanobody.

[0058] In some embodiments the therapeutic cargo comprises a cytokine, a hormone, a single-chain Fv fragment (scFv), a Fab fragment, a diabody, a minibody or a nanobody. The therapeutic cargo may comprise an antibody. Alternatively, in other embodiments, the therapeutic cargo does not comprise an antibody. The therapeutic cargo may comprise a hormone. The hormone may comprise human growth factor hormone or glucagon-like-peptide-1 . In some embodiments, the hormone comprises human growth factor hormone. An exemplary peptide sequence for human growth factor hormone is SEQ ID NO: 12. 008866329 8

[0059] The construct may comprise a plurality of therapeutic cargos. Each therapeutic cargo may be the same or different.

[0060] It will be appreciated that the transporter is cleavably coupled to the therapeutic cargo. In the context of the present invention, “coupled” will be understood to mean that the transporter is bound or linked to the therapeutic cargo. “Cleavably” will be understood to mean that the bond or linker between the transporter and therapeutic cargo can be controllably broken, typically in a cell. In this way, the therapeutic cargo can be separated from the transporter, which ensures that the therapeutic cargo is not consumed by cells in the lamina propria, as typically occurs for the transporter. By “controllably broken”, this will mean that the break of the bond or linker occurs due to administration or exposure to a particular cleavage stimulus. Therefore, breakage of the bond or linker is controllable by way of timing and / or location (depending on when the bond or linker is exposed to the cleavage stimulus, and / or where the bond or linker is exposed to the cleavage stimulus.

[0061] Typically, the C terminus of the transporter is cleavably coupled to the N terminus of the therapeutic cargo.

[0062] In some embodiments, the transporter is covalently coupled to the therapeutic cargo. For example, the transporter may be covalently coupled to the therapeutic cargo via disulphide bridge(s). As the skilled person will be aware, a disulfide bridge is a covalent bond between the sulphur atoms of two cysteines. In some embodiments, the transporter is covalently coupled to the therapeutic cargo via a chemical crosslinker. Typically, the chemical cross-linker will form one or more disulfide bridges. The chemical crosslinker may comprise a bis-dibromomaleimide cross-linker.

[0063] In some embodiments, the cross-linker comprises the following formula:

[0064] In such embodiments, it will be appreciated that the transporter and therapeutic cargo are separate molecules bound by covalent interactions. Other cross-linkers are known and commercially available to the skilled person. 008866329 9

[0065] In some embodiments, the transporter comprises KCAACQ (SEQ ID NO: 13) at its C-terminus. SEQ ID NO: 13 is a short sequence which enables formation of disulphide bridges at its cysteine residues.

[0066] However, it will typically be more convenient for the transporter and therapeutic cargo to be cleavably coupled via a cleavable linker. In the context of the present invention, the cleavable linker will be understood to comprise a peptide sequence having a consensus cleavage sequence. In such embodiments, it will be appreciated that the transporter and therapeutic cargo form a fusion protein via the cleavable linker sequence.

[0067] The cleavable linker may comprise a furin cleavage sequence or a matrix metalloproteinase cleavage sequence. It will be appreciated that furin cleavage sequences and matrix metalloproteinase cleavage sequences are known consensus amino acid sequences at which furin or a matrix metalloproteinase, as appropriate, is capable of cleaving the sequence. They may otherwise be referred to as furin cleavage sites or matrix metalloproteinase cleavage sites. Furin is a calcium-dependent serine endoprotease which functions intracellularly to cleave precursor proteins at their paired basic amino acid processing sites. Matrix metalloproteinases are another group of calcium dependent endoproteases that also function intracellular to cleave proteins. In some embodiments, the matrix metalloproteinase (mmp) is selected from the group consisting of mmp 3, mmp 4, mmp 5, mmp 8 and mmp 12. An exemplary matrix metalloproteinase may comprise matrix metalloproteinase (mmp) 4, mmp 5 or mmp 8. In some embodiments, the mmp comprises mmp 5. In some embodiments, the mmp comprises mmp 4. In other embodiments, the mmp comprises mmp 8.

[0068] In some embodiments, the cleavable linker comprises a furin cleavage sequence. In such embodiments, it will be appreciated that the cleavable linker comprises a consensus furin cleavage sequence. The furin cleavage sequence may comprise the consensus sequence RXXR, where X can be any amino acid. Optionally, the furin cleavage sequence comprises the sequence RHKR (SEQ ID NO: 14). Alternatively, the furin cleavage sequence may comprise the sequence RQKR (SEQ ID NO: 15).

[0069] The cleavable linker may comprise a tryptophan (W) N-terminal or C-terminal to the furin or mmp cleavage sequence. For example, the cleavable linker may comprise a tryptophan C-terminal to the furin or mmp cleavage sequence.

[0070] In some embodiments the cleavable linker further comprises a spacer sequence. The spacer sequence may be C-terminal to the consensus cleavage sequence. In other embodiments, the spacer sequence is 008866329 10

[0071] N-terminal to the consensus cleavage sequence. In some embodiments, the cleavable linker comprises spacer sequences N and C terminal to the consensus cleavage sequence.

[0072] Various spacer sequences are known and suitable for use by the skilled person. Exemplary spacer sequences include, but are not necessarily limited to: SAAGGGGSGGGSGGGS (SEQ ID NO: 16), SAAGGGGSGEKSGGGS (SEQ ID NO: 17), WSAAGGGGSGGGSGGGS (SEQ ID NO: 18), GGGG (SEQ ID NO: 19), GGGGSGG (SEQ ID NO: 20), SVSGGG (SEQ ID NO: 21 ), SAAGGGGSGGGSGGGSGGGS (SEQ ID NO: 22) and SAAGGGGSGGGSGEKSGGGS (SEQ ID NO: 23).

[0073] In some embodiments, the cleavable linker comprises SEQ ID NO: 24 (RHKRSAAG), SEQ ID NO: 25 (PFETFTRHRQKRSAAG), SEQ ID NO: 26 (PFETFTRHRQPRGGG), SEQ ID NO: 27 (PFETFTRHRQPRSVSG), SEQ ID NO: 28 (RHKRSAAGGGGSGGGSGGGS), SEQ ID NO: 29 (RHKRSAAGGGGSGGGSGGGSGGGS) or SEQ ID NO: 30 (GGGGSPFETFTRHRQPRSVSGGG).

[0074] The cleavable linker may comprise SEQ ID NO: 24.

[0075] In some embodiments, the cleavable linker comprises SEQ ID NO: 28.

[0076] In some embodiments, the cleavable linker comprises SEQ ID NO: 29.

[0077] In some embodiments, the cleavable linker comprises SEQ ID NO: 30.

[0078] Transport facilitator

[0079] The transport facilitator comprises or consists of an immunoglobulin CH2 (constant heavy 2) domain. The present inventors have found that the immunoglobulin CH2 domain bypasses the apical compartments of the usual A-B transcytosis pathway, thereby “facilitating” transport to the basal regions, where cleavage and subsequent systemic delivery of the cargo can occur. This finding was entirely surprising, especially as antibodies containing a CH2 domain which were not coupled to cholix had minimal internalisation into cells and did not enter the A-B transcytosis pathway.

[0080] In some embodiments the immunoglobulin CH2 domain comprises an IgG CH2 domain. In some embodiments, the immunoglobulin CH2 domain comprises an IgG 1 , lgG2, lgG3 or lgG4 CH2 domain. The IgG CH2 domain may comprise an IgG 1 CH2 domain. 008866329 11

[0081] In some embodiments the immunoglobulin CH2 domain is a human or humanised immunoglobulin CH2 domain.

[0082] In some embodiments the immunoglobulin CH2 domain comprises a peptide having at least 80% e.g. at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 31 . The immunoglobulin CH2 domain may comprise SEQ ID NO: 31 .

[0083] In some embodiments, the immunoglobulin CH2 domain is non-glycosylated. Alternatively, the immunoglobulin CH2 domain may comprise an N-linked glycosylation, for example at Asn297.

[0084] In some embodiments the transport facilitator does not comprise any immunoglobulin variable domains.

[0085] In some embodiments, the transport facilitator comprises an immunoglobulin fragment crystallisable (Fc) region. As the skilled person will appreciate, an Fc region comprises CH2 and CH3 domains. An exemplary Fc region may comprise SEQ ID NO: 32.

[0086] In some embodiments the transport facilitator does not comprise any immunoglobulin CH3 domains.

[0087] Delivery construct

[0088] In the delivery construct, the transport facilitator is N-terminal to the transporter. The inventors have surprisingly found that the N-terminal position of the transport facilitator relative to the transporter is necessary for transport facilitation. Where the transport facilitator is C-terminal to the transporter, the construct did not bypass apical regions of the A-B transcytosis pathway and so did not avoid premature cleavage.

[0089] In some embodiments, the transport facilitator is at the N terminus of the construct.

[0090] In some embodiments, the delivery construct comprises, from N to C, the transport facilitator, the transporter and the therapeutic cargo.

[0091] The transport facilitator may be coupled to the transporter. In some embodiments, the transport facilitator is non-cleavably coupled to the transporter, for example, by a non-cleavable linker. Typically, the non- cleavable linker does not comprise a consensus cleavage site. 008866329 12

[0092] The non-cleavable linker may comprise a spacer sequence, such as any of SAAGGGGSGGGSGGGS (SEQ ID NO: 16), SAAGGGGSGEKSGGGS (SEQ ID NO: 17), WSAAGGGGSGGGSGGGS (SEQ ID NO: 18), GGGG (SEQ ID NO:19), GGGGSGG (SEQ ID NO: 20), SVSGGG (SEQ ID NO: 21 ), SAAGGGGSGGGSGGGSGGGS (SEQ ID NO: 22) and SAAGGGGSGGGSGEKSGGGS (SEQ ID NO: 23).

[0093] The delivery construct may comprise a hexa-histidine sequence (HHHHHH, SEQ ID NO: 33) at the N- terminus. Such sequences can assist with purification of constructs. In embodiments where the transport facilitator is at the N-terminus, SEQ ID NO: 33 may be at the N terminus of the transport facilitator.

[0094] In some embodiments, the delivery construct comprises, from N to C, the transport facilitator, a non- cleavable linker, the transporter, a cleavable linker and the therapeutic cargo.

[0095] In some embodiments, the delivery construct comprises, from N to C, a hexa-histidine sequence, the transport facilitator, a non-cleavable linker, the transporter, a cleavable linker and the therapeutic cargo.

[0096] In an exemplary construct, the transporter comprises SEQ ID NO: 5 and the transport facilitator comprises SEQ ID NO: 31 . For example, the transporter may comprise SEQ ID NO: 8 and the transport facilitator may comprise SEQ ID NO: 31 . Alternatively, the transporter may comprise SEQ ID NO: 11 and the transport facilitator may comprise SEQ ID NO: 31 .

[0097] In an exemplary construct, the transporter and cleavable linker comprise SEQ ID NO: 34. SEQ ID NO: 34 comprises SEQ ID NO:5 with an RHKR sequence (SEQ ID NO: 14) at the C terminus. The RHKR sequence will be understood to be an exemplary furin cleavage sequence. SEQ ID NO: 34 may additionally comprise a tryptophan (“W) at the C-terminus, which may otherwise be referred to as position 198.

[0098] In an exemplary construct, the transporter and cleavable linker comprise SEQ ID NO: 35.

[0099] In an exemplary construct, the transport facilitator, non-cleavable linker, transporter and cleavable linker comprise SEQ ID NO: 36.

[0100] In some embodiments, the delivery construct comprises or consists of SEQ ID NOs 37 or 38. 008866329 13 in some embodiments, the delivery construct does not comprise any immunoglobulin variable domains.

[0101] Preferably, the delivery construct is capable of delivering the therapeutic cargo to the basal vesicular compartments of a polarised epithelial cell. More preferably, the delivery construct is capable of avoiding the apical vesicular compartments of a polarised epithelial cell. It will be appreciated that the delivery construct may be capable of transporting the therapeutic cargo across a polarised epithelial cell. Once the therapeutic cargo has been transported across a polarised epithelial cell typically, it will enter a porous vascular bed and then into the circulation. This advantageously, enables systemic delivery of the therapeutic cargo.

[0102] The constructs described in this specification can be formulated in pharmaceutical compositions. These compositions may comprise, in addition to the delivery construct, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, pulmonary, intramuscular, intraperitoneal routes or topical application. Oral or pulmonary routes may be preferred.

[0103] In some embodiments the pharmaceutical composition is formulated for oral administration.

[0104] The term “oral administration” is intended to encompass any suitable delivery method by which the delivery construct is ingested in the mouth and processed in the digestive system. It is envisaged that oral delivery of the delivery construct will deliver the construct to the polarised epithelial cells of the intestine, at which the delivery construct can transport the therapeutic cargo across the cell, thereby enabling systemic delivery of the therapeutic construct.

[0105] Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Preservatives, stabilisers, buffers, antioxidants and / or other additives may be included, as required.

[0106] In some embodiments the pharmaceutical composition is formulated in a capsule or tablet.

[0107] Solid (e.g. powder) preparations may utilise carriers such as sugars, cyclodextrins, etc. They may be prepared by any suitable method including spray drying, spray freeze drying, solvent precipitation, jet milling, etc.. 008866329 14

[0108] Methods of treatment and medical uses thereof

[0109] The delivery constructs of the present invention are suitable for the treatment or prophylaxis of any disease, disorder or infection to which an appropriate therapeutic cargo can be used.

[0110] Therefore, the present invention provides a method of treatment or prophylaxis of a disease, disorder or infection in a subject, the method comprising administering a therapeutically effective amount of the delivery construct or pharmaceutical composition described above to the subject. Also provided is the delivery construct or pharmaceutical composition of the invention for use in a method of treatment or prophylaxis. Further provided is the use of the delivery construct of the invention for the manufacture of a medicament for the treatment of a disease, disorder or infection in a subject.

[0111] Administration is preferably in a “prophylactically effective amount” or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts, for example Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

[0112] Typically, the subject to be treated is a mammal. The subject is typically human, but may be any other primate (great ape, old world monkey or new world monkey), or a domestic, laboratory or livestock animal, such as a mouse, rat, guinea pig, lagomorph (e.g. rabbit), cat, dog, pig, cow, horse, sheep or goat.

[0113] Nucleic acids, vector(s) and host cells

[0114] Also provided are one or more nucleic acid(s) encoding the delivery construct of the first aspect.

[0115] The nucleic acid(s) encoding the delivery construct are typically provided as part of one or more nucleic acid expression constructs or vectors. Thus they may be provided on a single vector or on two or more separate vectors.

[0116] The skilled person will be capable of designing suitable nucleic acid expression constructs or vectors to obtain expression of the delivery construct as required. Typically such a vector comprises suitable transcriptional and translational regulatory sequences operably linked to a sequence encoding the 008866329 15 desired protein, to enable transcription and translation of the protein by the host cell. The vectors may contain other sequences such as selection marker genes as required, depending upon the particular host cell. The vectors may be intended to integrate into a host cell chromosome, or may exist and replicate independently of the host chromosomes as an episome, e.g. a plasmid.

[0117] Also provided is a host cell comprising the nucleic acid(s) or vector(s) described above. The host cell may be a bacterial host cell, e.g. an E. coli host cell.

[0118] In embodiments where the transporter and the therapeutic cargo are separate molecules in the final delivery construct which are, for example, covalently linked, they may be expressed as separate molecules within the host cell.

[0119] The invention further provides a method of producing a delivery construct comprising, providing a host cell as described and culturing said cell under conditions suitable for expression of the delivery construct. The method may further comprise a step of isolating the delivery construct, and optionally further steps of purification.

[0120] Sequences

[0121] SEQ ID NO: 1 Full Cholix Sequence with leader peptide

[0122] MYLTFYLEKVMKKMLLIAGATVISSMAHPTFAVEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEG VLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAI NWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSY KAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIE QKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSV FTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFC PDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTN HVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVY IPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAY EELAIDEEAVAKEQSISTKPPYKERKDELK

[0123] SEQ ID NO: 2 Mature Cholix Sequence without leader peptide

[0124] VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATV RATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKL YSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMD AIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLET LARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVT QVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPEN RAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYV 008866329 16

[0125] ATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPL

[0126] RNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERKDELK

[0127] SEQ ID NO: 3: 264 aa of Chx domain 1

[0128] LEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATV

[0129] RATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKL

[0130] YSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISXPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMD

[0131] AIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFS

[0132] SEQ ID NO: 4: 264 aa of Chx domain 1 + 2 aa from Chx domain 2

[0133] LEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATV

[0134] RATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKL

[0135] YSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISXPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMD

[0136] AIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKG

[0137] SEQ ID NO: 5: 193 aa of Chx domain 1

[0138] LEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATV

[0139] RATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKL

[0140] YSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISXPSVSYKAAQKEGS

[0141] SEQ ID NO: 6: 264 aa of Chx domain 1

[0142] LEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATV

[0143] RATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKL

[0144] YSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISAPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMD

[0145] AIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFS

[0146] SEQ ID NO: 7: 264 aa of Chx domain 1 + 2 aa from Chx domain 2

[0147] LEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATV

[0148] RATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKL

[0149] YSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISAPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMD

[0150] AIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKG

[0151] SEQ ID NO: 8: 193 aa of Chx domain 1

[0152] LEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATV

[0153] RATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKL

[0154] YSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISAPSVSYKAAQKEGS 008866329 17

[0155] SEQ ID NO: 9: 264 aa of Chx domain 1

[0156] LEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATV

[0157] RATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKL

[0158] YSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMD

[0159] AIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFS

[0160] SEQ ID NO: 10: 264 aa of Chx domain 1 + 2 aa from Chx domain 2

[0161] LEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATV

[0162] RATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKL

[0163] YSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMD

[0164] AIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKG

[0165] SEQ ID NO: 11: 193 aa of Chx domain 1

[0166] LEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATV

[0167] RATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKL

[0168] YSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGS

[0169] SEQ ID NO: 12: hGH sequence

[0170] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKS

[0171] NLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTY

[0172] SKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

[0173] SEQ ID NO: 13

[0174] KCAACQ

[0175] SEQ ID NO: 14

[0176] RHKR

[0177] SEQ ID NO: 15

[0178] RQKR

[0179] SEQ ID NO: 16

[0180] SAAGGGGSGGGSGGGS 008866329 18

[0181] SEQ ID NO: 17

[0182] SAAGGGGSGEKSGGGS

[0183] SEQ ID NO: 18

[0184] WSAAGGGGSGGGSGGGS

[0185] SEQ ID NO: 19

[0186] GGGG

[0187] SEQ ID NO: 20

[0188] GGGGSGG

[0189] SEQ ID NO: 21

[0190] SVSGGG

[0191] SEQ ID NO: 22

[0192] SAAGGGGSGGGSGGGSGGGS

[0193] SEQ ID NO: 23

[0194] SAAGGGGSGGGSGEKSGGGS

[0195] PFETFTRHRQPRGGG 008866329 19

[0196] SEQ ID NO: 27: Cleavable linker 4

[0197] PFETFTRHRQPRSVSG

[0198] SEQ ID NO: 28: FCS L 1 Linker

[0199] RHKRSAAGGGGSGGGSGGGS

[0200] SEQ ID NO: 29: FCS linker

[0201] RHKRSAAGGGGSGGGSGGGSGGGS

[0202] SEQ ID NO: 30: FCS linker

[0203] GGGGSPFETFTRHRQPRSVSGGG

[0204] SEQ ID NO: 31: CH domain

[0205] GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV

[0206] LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0207] SEQ ID NO: 32: Fc region

[0208] GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV

[0209] LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS

[0210] DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

[0211] SEQ ID NO: 33: Hexahistidine sequence

[0212] HHHHHH

[0213] SEQ ID NO: 34: Chx 193 + RHKR

[0214] LEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATV

[0215] RATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKL

[0216] YSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISAPSVSYKAAQKEGSRHKR 008866329 20

[0217] SEQ ID NO: 35: Chx 193 + L1 cleavable linker

[0218] LEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATV

[0219] RATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKL

[0220] YSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISAPSVSYKAAQKEGSRHKRSAAGGGGSGGGSGGGS

[0221] SEQ ID NO: 36: CH2 region + F3 non-cleavable linker + Chx 193 + L1 cleavable linker

[0222] GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV

[0223] LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGGGGSGGGSGGGSLEEALNIFDECRSPCSLTPEP

[0224] GKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDIT

[0225] TENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVS

[0226] FSVTRPEHNIAISAPSVSYKAAQKEGSRHKRSAAGGGGSGGGSGGGS

[0227] MHHHHHHGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

[0228] STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC

[0229] LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

[0230] YTQKSLSLSPGKGGGGSGGGSGGGSLEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS

[0231] MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLV

[0232] PIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISAPSVSYKAAQK

[0233] EGSRHKRSAAGGGGSGGGSGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQ

[0234] NPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLE

[0235] EGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGS CGF

[0236] SEQ ID NO: 38: CH2-Chx197-L1-hGH

[0237] MHHHHHHGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

[0238] STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGGGGSGGGSGGGSLEEALNIFDECRS

[0239] PCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAP

[0240] FGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQ

[0241] WKTQGNVSFSVTRPEHNIAISAPSVSYKAAQKEGSRHKRSAAGGGGSGGGSGGGSFPTIPLSRLFDNA

[0242] MLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQS

[0243] WLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDD

[0244] ALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF 008866329 21

[0245] SEQ ID NO:39.ChX!97 hGH cleavable linker 1 (LI)

[0246] MHHHHHHLEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESII

[0247] TIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQ

[0248] RNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISAPSVSYKAAQKEGSRHKRSAAGGGGSGG

[0249] GSGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNRE

[0250] ETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTG

[0251] QIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

[0252] SEQ ID NO: 40: Chx197-hGH- cleavable linker 2 (L2)

[0253] MHHHHHHLEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESII

[0254] TIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQ

[0255] RNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISAPSVSIPFETFTRHRQKRSAAGGGGSGEK

[0256] SGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREE

[0257] TQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQ

[0258] IFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

[0259] SEQ ID NO: 41: Chx266-hGH- cleavable linker 3 (L3)

[0260] MHHHHHHLEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESII

[0261] TIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQ

[0262] RNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLAL

[0263] CWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGGGGGSGGPFE

[0264] TFTRHRQPRGGGFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIP

[0265] TPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLED

[0266] GSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

[0267] SEQ ID NO: 42: Chx266-hGH- cleavable linker 4 (L4)

[0268] MHHHHHHLEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESII

[0269] TIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQ

[0270] RNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLAL

[0271] CWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGGGGGSGGPFE

[0272] TFTRHRQPRSVSGGGFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFS

[0273] ESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGR

[0274] LEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF 008866329 22

[0275] SEQ ID NO:43: CH3-Chx197-L1-hGH

[0276] MHHHHHHGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

[0277] GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGSLEEALNIFD

[0278] ECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVN

[0279] QDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQ

[0280] TLEQWKTQGNVSFSVTRPEHNIAISAPSVSYKAAQKEGSRHKRSAAGGGGSGGGSGGGSFPTIPLSRLF

[0281] DNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLL

[0282] LIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSH

[0283] NDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

[0284] SEQ ID NO: 44: Chx266-R3-KCAACQ

[0285] MHHHHHHHVEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGES

[0286] IITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQ

[0287] QRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGL

[0288] ALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGAEAAAKEAA AKEAAAKAKCAACQ

[0289] SEQ ID NO: 45: CH2-Chx197-L1-sfGFP

[0290] MHHHHHHGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

[0291] STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGGGGSGGGSGGGSLEEALNIFDECRS

[0292] PCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAP

[0293] FGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQ

[0294] WKTQGNVSFSVTRPEHNIAISAPSVSYKAAQKEGSRHKRSAAGGGGSGGGSGGGSVSKGEELFTGVVP

[0295] ILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDF

[0296] FKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITA

[0297] DKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFV TAAGITHGMDELYK

[0298] SEQ ID NO: 46

[0299] PFETFTRH

[0300] SEQ ID NO: 47

[0301] SGSPQALVAALD

[0302] SEQ ID NO: 48

[0303] GSGPAALVGAAD 008866329 23

[0304] SEQ ID NO: 49

[0305] SGGPAELIGPVD

[0306] SEQ ID NO: 50

[0307] TEVKTKSE

[0308] SEQ ID NO: 51

[0309] VHMPLGFLGP

[0310] SEQ ID NO: 52

[0311] NDAPGQ

[0312] SEQ ID NO: 53

[0313] EGDPANLVAPDP

[0314] SEQ ID NO: 54

[0315] ESTPLNLVGAYG

[0316] SEQ ID NO: 55

[0317] SEAPAALIGGAP

[0318] SEQ ID NO: 56

[0319] SVPASILDL

[0320] SEQ ID NO: 57

[0321] AAGFPGARGLPGQMPQNLRASG

[0322] SEQ ID NO: 58

[0323] ESTPANLVAPED

[0324] SEQ ID NO: 59

[0325] EDAPAGLVADAD 008866329 24

[0326] SEQ ID NO: 60

[0327] GEAPAGLVGPPN

[0328] SEQ ID NO: 61

[0329] EQRTYGLMDSH

[0330] Examples

[0331] Materials and Methods

[0332] Protein purification

[0333] All protein constructs were gene synthesised, with codon optimisation for E. coli expression (Genscript). Gene sequences were subcloned into pET26b (+) using the Nde1 and EcoR1 restriction sites. Plasmids were transformed into Shuffle cells (neb), a BL21 (DE3) strain of E. coli with enhanced disulphide bond formation. Cells were grown in TB medium (with the addition of kanamycin and glycerol) at 37°C with shaking at 220 RPM, to an OD 600 nm of 0.8-1 . The temperature was reduced to 18°C and protein expression induced by the addition of 0.5 mM IPTG. Expression was continued overnight, and the cells harvested by centrifugation. Cells were resuspended in His buffer A (20 mM Tris pH 8, 300 mM NaCI, 20 mM imidazole), supplemented with protease inhibitors, and lysed by sonication (3 x 1 min (5 sec on 5 sec off)). Soluble and insoluble fractions were separated by centrifugation (20,000 RPM, 45 min). The soluble fraction was filtered and loaded onto a 5 ml HisTrap™ column, equilibrated with His buffer A. The column was then washed, and protein eluted in His buffer B (20 mM Tris pH 8, 300 mM NaCI, 500 mM imidazole). Elution fractions were analysed by SDS-PAGE and protein containing fractions concentrated, filtered, and loaded onto a Superdex 200 16 / 600 size exclusion column, equilibrated with 20mM Tris pH 8, 150 mM NaCI. Peak fractions were analysed by SDS-PAGE and fractions containing pure protein were pooled, concentrated and frozen in aliquots at -80°C.

[0334] Furin cleavage studies

[0335] One unit of furin (NEB) was added to 25pg of protein. The reaction was carried out in 25 pl, at pH 5.5 (100 mM MES (pH 5.5), 0.5% Triton X-100, 1 mM CaCIz, 1 mM 2-mercaptoethanol) or at pH 7.5 (100 mM Hepes (pH 7the.5), 0.5% Triton X-100, 1 mM CaCIz, 1 mM 2-mercaptoethanol) at room temperature. Proteolytic cleavage was analysed over time, being stopped by addition of 2x SDS-PAGE buffer and heating at 95°C for 10 mins. Samples were analysed by SDS-PAGE. 008866329 25

[0336] Chemical coupling strategy

[0337] Antibody was partially reduced by incubation with 1 equivalence TCEP at 4°C for 2 hrs and reacted with an excess of linker (5 equivalence) for 3hr at room temperature. Excess linker was removed by size exclusion chromatography (Superdex ® 200 Increase 10 / 300 GL). In tandem, cholix transporter with a KCAACQ (SEQ ID NO: 13) sequence on the C-terminus, was reduced with 1 equivalence of TCEP. The reduced Chx266-KCAACQ was added to the linker-antibody complex and incubated overnight at room temperature. The conjugation product was isolated by SEC (Superdex ® 200 Increase 10 / 300 GL) and affinity chromatography (5 mL HisTrap™). All fractions were analysed by SDS-PAGE.

[0338] Measurement of serum hGH

[0339] A commercial ELISA kit (R&D Systems) was used to quantitate serum levels of hGH with samples being tested in triplicate. Absorbance values for the enzymatic reactions at 405 nm were registered in an ELISA microplate reader (Bio-Rad).

[0340] In vivo transcytosis

[0341] Male Wistar rats, housed 3-5 per cage with a 12 / 12 h light / dark cycle, were 225-275 g (approximately 6-8 weeks old) when placed on study. All experiments were conducted during the light phase using a nonrecovery protocol that used continuous isoflurane anaesthesia. A 4-5 cm midline abdominal incision exposed mid-jejunum regions. Stock solutions at 3.86 x 105M of test articles were prepared in PBS, with 50 pL (per 250 g rat) being administered by intraluminal injection (ILI) using a 29-gauge needle. The injection site mesentery was marked with a permanent marker. Systemic blood draws were collected up to 120 min after ILI and processed to serum that was store frozen prior to ELISA to quantitate the extent of systemic cargo delivery. At study termination, a 3-5 mm region that captured the marked intestine segment was isolated and processed for microscopic assessment. All experiments were performed in accordance with the U.K. Animals (Scientific Procedures) Act of 1986, the European Communities Council Directive of 1986 (86 / 609 / EEC), and the University of Bath’s ethical review procedures.

[0342] Immunofluorescence (IF) microscopy

[0343] IF microscopy imaging was performed as previously described (Taverner et al., 2020). Briefly, isolated intestinal tissues were fixed in 4% paraformaldehyde for 18 h at 4°C, processed using a Leica TP1020 tissue processor, dehydrated in increasing concentrations of ethanol, cleared with HistoClear (National Diagnostics), and infused with molten paraffin wax. Sections cut from tissue-embedded paraffin wax blocks (5-pm thickness; Jung Biocut2035 microtome) were mounted on glass microscope slides, rehydrated, and processed for antigen retrieval by boiling slides in 10-mM sodium citrate for 10 min. Processed tissue slices were permeabilised using 0.1 % Triton X-100 in PBS for 30 min, blocked using 2% donkey serum and 2% BSA in 0.1% Triton X-100 in PBS for 2 h, and incubated overnight at 4°C with primary antibodies diluted in 1% BSA and 0.05% Triton X-100 in PBS at 4°C. Tissue slices were washed 008866329 26 thrice with PBS, incubated for 2 h with secondary antibodies conjugated to AlexaFluor® fluorescent dyes, washed thrice with PBS, and incubated for 1 h with 200 nM DAPI, washed with PBS, dehydrated in ethanol, and covered by mounting a coverslip with Fluorshield (Abeam) mounting media; all steps performed at room temperature. After allowing the mounting media to dry at 4°C overnight, fluorescent images were obtained using a Zeiss 880 LSM confocal microscope using the following settings. For Alexafluor 488 the excitation wavelength was 488 nm and the emission wavelength was 562 nm. For

[0344] Alexafluor 564 the excitation wavelength was 561 nm and the emission wavelength was 602 nm. For

[0345] Alexafluor 633 the excitation wavelength was 633 nm and the emission wavelength was 693 nm. For

[0346] DAPI the excitation wavelength was 405 nm and the emission wavelength was 462 nm.

[0347] Antibodies and recombinant proteins

[0348] Primary antibodies: goat anti-human growth pAb (R&D Systems, AF1067); rabbit anti-Chx antibodies (Innovagen); Infliximab (MyBioSource MBS9232235); COPI (Abeam ab181224); COPII (Invitrogen PA1 - 069A); LMAN1 (Abeam ab32583); ATP5A1 (Proteintech 1467-1 -AP); furin (santa cruz sc-133142)

[0349] Secondary antibodies: Cy3- and Cy5-labeled donkey anti-mouse IgG (Jackson ImmunoResearch, 715- 165-151 , 715-175-150); donkey anti-rabbit lgG-aleafluore546 (Invitrogen, A10040); donkey anti-goat-IgG- alexafluore488 (Invitrogen, A1 1055).

[0350] Caco-2 transport assays

[0351] Caco-2 cells were seeded at 7 x 104cells / well and grown on Transwells until polarisation indicated by trans-epithelial electrical resistance (TEER) >~200Q.cm2> blank TEER. Constructs were then applied apically to Caco-2 Transwells for 2 hours, following which the contents of the apical and basal compartments of the cells were harvested and cells lysed for analysis by ELISA.

[0352] EXAMPLE 1: Assessment of furin cleavage site sequences to cleavably link a cholix transporter domain to a therapeutic cargo

[0353] Furin functions in the processing of prepro-forms of many proteins synthesised and trafficked through the ERGIC (from the ER to the Golgi network). Chx266-hGH+vesicles have also previously been observed as being sorted to the ERGIC (Taverner et al., 2020).

[0354] It was therefore decided to consider furin as a cleavable linker strategy. We examined the impact of transporter length and spacers located before and after a consensus furin cleavage sequence (FCS) to identify suitable construct organization for enzymatic cargo release from a Chx transporter. 008866329 27

[0355] Two lengths of the Chx transporter element were tested: amino acids 1 -266 (Chx266, SEQ ID NO: 10) where domain I naturally ends, or amino acids 1 -197 (Chxig?) that represents a 193 amino acid truncated form of domain I which naturally contains a furin cleavage sequence at positions 194-197 (SEQ ID NO: 34). All constructs were prepared with human growth hormone (hGH) as a cargo genetically conjoined through various linker sequences. Linker sequences for chx266 included a consensus FCS of RXXR, which is composed of two arginine (R) residues separated by non-specific (X) amino acids. Linker sequences for chx197 did not include this consensus FCS because of the FCS already present at positions 194-197 of chx197. Non-immunogenic spacers, composed primarily of serine (S) and glycine (G) amino acids, were introduced to increase the furin accessibility to the RXXR sequence. The impact of the wild-type tryptophan residue on FCS function was assessed by mutation to alanine in some constructs.

[0356] Chxi97-L1 -F3-hGH (C1 ) was composed of amino acids 1 -197, wherein Pisi to the FCS ending at R197 represented a cholix consensus sequence (SEQ ID NO: 34 or SEQ ID NO: 8 + SEQ ID NO: 14) and followed by trailing flexible linker (F3) SAAG(G3S)3 sequence of (SAAGGGGSGGGSGGGSGGGS, SEQ ID NO: 22). Chxi97-L2-N3-hGH (C2), tested the impact of the pre-furin region in Chxi97-L1 -F3-hGH by exchanging Chx amino acids with those from an exotoxin A binding domain-containing protein from Serratia fonticola (SF) prior the FCS (PFETFTRH, SEQ ID NO: 46), that was followed by a slightly more rigid linker sequence SAAGGGGSGGGSGEKSGGGS (SEQ ID NO: 23) (N3). Both Chxi97-L1 -F3-hGH and Chxi97-L2-N3-hGH contained an alanine residue (A) at position 180 to replace the tryptophan residue (W) at position 180 normally present in Chx.

[0357] The significance of retaining the tryptophan at position 180 (Wiso) was tested in Chxi97-(Wiso)-L1 -F3-hGH (C5) and in combination with another (W198) immediately following the FCS in construct Chxi97-(Wi8o,i9s)- L1 -F3-hGH (C6).

[0358] Optimally, the Chx-based approach would deliver a cleaved cargo that has minimal modifications from its native form. We tested if this could be achieved for hGH using a FCS with the Chxi97-L1 -hGH construct (C7), which lacked the F3 sequence present in the ChxwLI -F3-hGH (C5). Impact of short extensions of glycine (G) residues on FCS function were tested using Chxi97-(Wiso)-L1 -G-hGH (C8) and Chxi97-(Wiso)- L1 -G3-hGH (C9).

[0359] We also tested the value of amino acid extensions before and after the FCS in a format using the full- length domain 1 chx266 sequence, where such extensions were perceived to have less impact on carrier properties required for A^B transcytosis. Two constructs were prepared, both having Chx26eG4SG2-L2 formats with either a Gs-hGH or a SVSGs-hGH cargo: Chx26eG4SG2-L2-G3-hGH (C3) and Chx26eG4SG2- L2-SVSG3-hGH (C4). Schematics of these constructs are shown in Figure 1 A. 008866329 28

[0360] An in vitro time course study of FCS susceptibility was performed to compare accessibility of these different constructs at pH 5.5 and pH 7.5 (Fig. 1 B). These two pH values were selected to model conditions that might be found within intracellular endosomes and external to cells, respectively, for 10 min, 1 hr, 3 hr, and overnight (O / N) furin exposures (Table 1 ). While all sequences were cleaved following O / N incubation, some cleaved more efficiently than others, showing preference for cleavage between the two pH values tested (Table 2).

[0361] Table 1 : Summaries of digestive outcomes over time for Chx-hGH containing constructs. 008866329 29

[0362] Table 2. Relative rankings of cleaving efficiency at acidic and neutral pH of Chx-hGH containing constructs.

[0363] These studies demonstrated C xi97-L1 -F3-hGH (C1 ) to be efficiently cleaved at both pH 5.5 and 7.5, while Chxi97-L2-N3-hGH (C2) cut more efficiently at pH 7.5 than 5.5. Both Chx26eG4SG2-L2-G3-hGH (C3) and Chx266G4SG2-L2-SVSG3-hGH (C4) were cut by furin more efficiently at pH 5.5, but less efficiently at pH 7.5 than Chxi97-L2-N3-hGH (C2). Efficient cleavage was not achieved with Chxi97-(Wiso)-L1 -hGH (C7) or with a nominal extension on the hGH cargo: Chxi97-(Wiso)-L1 -G-hGH (C8) and Chxi97-(Wiso)-L1 -G3- hGH (C9). The presence of Wiso versus A did not have much of an effect on FCS function (Chxi97-(Wi8o)- L1 -F3-hGH (C5) vs. Chxi97-L1 -F3-hGH (C1 )), but Wi98did have an effect (Chxi97-(Wi8o,i98)-L1 -F3-hGH (C6). Overall, amino acid extensions before and after the FCS appeared to provide benefit for furin- mediated cleavage of these constructs, with some flexibility of their exact composition.

[0364] The L1 or L2 and L3 or L4 cleaving linker sequences were then tested using transporters of different lengths: Chx197 and Chx266, respectively (Fig. 2). This was to determine if the difference in transporter length could affect furin cleavage efficiency. All constructs generated comprised a hexa-histidine sequence (SEQ ID NO: 33) at the N-terminus to simplify purification. The construct Chx197-hGH- cleavable linker 1 (L1 ) (SEQ ID NO: 39), comprised the Chx197 transporter linked to hGH using cleavable 008866329 30 linker 1 (SEQ ID NO: 24). The construct Chx197-hGH- cleavable linker 2 (L2) (SEQ ID NO: 40) comprised the Chx197 transporter linked to hGH using cleavable linker 2 (SEQ ID NO: 25). The construct Chx266-hGH- cleavable linker 3 (L3) (SEQ ID NO: 41 ) comprised the Chx266 transporter linked to hGH using cleavable linker 3 (SEQ ID NO: 26). The construct Chx266-hGH- cleavable linker 4 (L4) (SEQ ID NO: 42) comprised the Chx266 transporter linked to hGH using cleavable linker 4 (SEQ ID NO: 27).

[0365] We investigated the in vivo performance of these constructs in a rat model of uptake from the small intestinal (jejunal) lumen and delivery of a therapeutic cargo, in this case hGH, to the systemic circulation. As the successful delivery of a cargo using the Chx carrier is a muti-stage process involving A^B transcytosis followed by proteolytic separation of the cargo from the carrier, we used two methods to examine these outcomes: pharmacokinetic (PK) profiles of serum cargo levels and distribution of the chimera, and its components, within the intestinal tissue where it was administered.

[0366] Comparison of PK profiles of L1 -L4 constructs demonstrated that Chx-based delivery results in hGH serum profiles observed after ~15 min with a peak serum level between 15-60 min after administration (Fig. 3A). Area under the curve (AUC) calculations from these time-serum concentration profiles demonstrated that the Chxi97-L2 and Chx266-L3 constructs did not result in any more hGH reaching the systemic circulation than that achieved by administration of the same molar amount of hGH (Fig. 3B). AUC calculations demonstrated that Chxi97-L1 -hGH achieved ~3-fold improvement in serum delivery of hGH compared to a similar intestinal administration of hGH alone; Chx266-L4-hGH administration resulted in ~2-fold improvement.

[0367] We next examined the potential for cleavage of these constructs at the designated furin site using an in vitro model of human intestinal epithelium: polarised Caco-2 cell monolayers, as described in Zhang et al., 2015. Based upon observations involving other proteins in the class of bacterial toxins, A^B vesicular transcytosis of a Chx-based construct without efficient cargo release will result in minimal systemic delivery of a therapeutic agent, as the intact construct will be consumed by locally by cells in the lamina propria (Hsieh et al, 2005). Basal media, collected 1 hr after apical application of a construct, was collected, concentrated, and submitted for SDS-PAGE separation followed by western blot analysis. Chxi97-L1 -hGH showed a greater amount of release of hGH cargo in the basal compartment compared to Chxi97-L2-hGH, and Chx266-L4-hGH showed a greater amount of hGH cargo in the basal compartment compared to Chx266-L3-hGH. While the size of hGH and Chx carriers detected in the basal compartment of these Caco-2 monolayers were consistent with values anticipated for these proteins, the sensitivity of this assay was insufficient to verify the exact site of separation for these chimeras. Some fraction of hGH applied to the apical surface of these cells also reached the basal compartment of Caco-2 monolayers in an intact form. 008866329 31

[0368] EXAMPLE 2: Investigating the intracellular lecaticn of furin

[0369] Example 1 demonstrated that furin cleavable linkers were capable of releasing hGH from the Chx transporter through a furin-dependent separation strategy. However, this did not necessarily result in systemic delivery of the therapeutic cargo.

[0370] It was next decided to investigate the location of furin in polarised enterocytes. Furin-mediated separation of the Chx transporter from the hGH cargo results in systemic delivery if it occurred only after the intact construct reached the ERGIC vesicular compartment. Cleavage at the apical surface would result in hGH not entering the A^B vesicular transcytosis pathway and cleavage in the apical vesicular compartment would lead to separated hGH being trafficked to lysosomes where it would be digested.

[0371] The human protein atlas (https: / / www.proteinatlas.org / ) is a repository of information that includes tissues stained using standard immunohistochemical methods to describe the specific location of a protein within a tissue. Tissues stains of human intestinal tissue for furin were therefore analysed. Furin was localised minimally at the apical plasma membrane but was present in both the apical and basal vesicular compartments of enterocytes. Cells within the lamina propria (Ip) demonstrated varying degrees of furin expression, consistent with the ultimate processing of the native Chx toxin in these non-polarised cells. The distribution of furin expression in rat jejunum tissue was also examined using immunofluorescence confocal microscopy. A distribution of furin in both apical and basal vesicular compartments was observed, along with limited expression in the microvilli at the apical surface of enterocytes.

[0372] EXAMPLE 3: Assessment cf a monoclonal antibody therapeutic cargo

[0373] A chemical, rather than genetic, coupling strategy was used to examine the potential for the Chx transporter to facilitate A^B transcytosis of a monoclonal antibody (mAb), in this case infliximab. The Chx266 transporter, prepared using bacterial expression, terminated with a linker sequence lacking a furin cleavage site followed by a rigid (R3) linker and then a short loop sequence (KCAACQ, SEQ ID NO: 13) that included two cysteine residues to form the sequence SEQ ID NO: 44. Using mild reducing conditions for cystine residues, free sulfhydryl moieties on both the Chx266 carrier and infliximab were conjoined using a bifunctional coupling agent to produce the construct Chx266-R3-infliximab. Figure 4 is a schematic detailing the method undertaken to generate this construct. Figure 5 is a schematic of the structure of the bifunctional coupling agent and an IgG 1 antibody. The arrow depicts the target site to be targeted to produce Chx266-R3-infliximab.

[0374] Chx266-R3-infliximab distribution in rat jejunal tissue following administration by intraluminal injection (ILI) was examined using immunofluorescence confocal microscopy. As a control, infliximab alone was introduced into rat jejunum. At 15 min post ILI, infrequent labelling of materials recognised by a monoclonal recognising infliximab alone was observed in enterocytes, with no labelling was detected in 008866329 32 the lamina propria. This suggested minimal internalisation from the intestinal lumen and no A^B transcytosis. These findings are consistent with the fact that monoclonal antibodies are not efficiently taken up across the intestinal epithelium (Abramson et al., 201 1 ). When coupled to the Chx266-R3 carrier, however, infliximab was extensively taken up into enterocytes, was detected in both apical and basal vesicular compartments of these cells and was observed in cells within the lamina propria at 15 min, consistent with efficient A^B transcytosis. These results demonstrated the ability of a Chx-based transporter to enable A^B transcytosis of a monoclonal antibody.

[0375] EXAMPLE 4: Chx-based A^B transcytosis of a mAb cargo is distinct from hGH cargo

[0376] We decided to investigate if the Chx constructs designed to deliver hGH or infliximab utilised the same or different A^B transcytosis pathways. To do this, the intracellular location of Chx266-R3-infliximab was compared to a Chx266-hGH construct lacking the furin cleavage site of RHKR (SEQ ID NO: 14).

[0377] In untreated rat jejunal enterocytes, COPI and COPII are known to be present in a subset of vesicles present in the supranuclear region (SNR), consistent with the location of the ERGIC, while LMAN1 distribution is limited to the apical compartment vesicles with a moderate level of co-localisation with COPP vesicles and no co-localisation with COPIP vesicles that are primarily present near the basal plasma membrane (Taverner et al., 2020). Previous work has shown that A^B transcytosis of Chx266- hGH involves extensive co-localisation with COPP in vesicles restricted to the SNR of enterocytes and COPIP vesicles observed in the apical vesicular compartment and at the basal membrane surface (Taverner et al. 2020). In addition, Chx266-hGH was found to co-localise with LMAN1 at both apical and basal vesicular compartments (Taverner et al. 2020). In contrast, in the present study, LMAN1 was found to remain in the apical vesicular compartment at 15 min post ILI of Chx266-R3-infliximab, where only limited co-localisation occurred (immunofluorescence image not shown).

[0378] In view of the differing distribution of LMAN1 following Chx266-R3-infliximab administration, it was decided to examine the distribution of other potential elements of the A^B transcytosis pathway. Infliximab distribution during its A^B transcytosis mediated by Chx266-R3-infliximab showed little to no colocalisation with a subunit of mitochondrial ATP synthase (ATP5A1 ), suggesting limited distribution at sites where mitochondria are present in the apical and basal intracellular compartments of enterocytes (immunofluorescence image not shown). By comparison, A^B transcytosis of Chx266-hGH showed extensive co-localisation of hGH with ATP5A1 in both apical and basal intracellular compartments of rat jejunal enterocytes at 15 min post ILI (immunofluorescence image not shown).

[0379] We next examined furin distribution during A^B transcytosis of Chx266-hGH with regard to the apical and basal intracellular compartments defined by ATP5A1 . Prior to Chx266-hGH exposure, there was very limited co-localisation of ATP5A1 and furin, ATP5A1 being distributed in a compartment closer to the 008866329 33 apical plasma membrane and furin being distributed in an area closer to the basal plasma membrane. At 15 min post ILI of Chx266-hGH, co-localisation of ATP5A1 and furin was observed, occurring to a greater extent in the apical region of the cell compared to the basal region. In contrast, at 15 min post ILI, Chx266- R3-infliximab was not observed to extensively co-localise with furin at in the apical region of rat enterocytes but did intersect furin in the basal region of the cell (immunofluorescence images not shown).

[0380] The results in Example 4 indicated that soon after apical entry, Chx266-R3-infliximab moves from its internalisation receptor to a cellular element not involved in the A^B transcytosis observed for Chx266- hGH. We tested the idea that a cellular element recognising the mAb, i.e., the Fc domain of the IgG 1 framework, could participate in this event.

[0381] We focused on the Fc receptor-like A (FCRLA) protein, which is an ER resident protein that can bind Fc domain of multiple Ig isotypes (Santiago et al., 201 1 ). Untreated rat jejunal enterocytes were found to express FCRLA in both apical and basal vesicular compartments (immunofluorescence image not shown). At 15 min post ILI application of infliximab alone, a small fraction of this mAb entered enterocytes but failed to co-localise with or affect the cellular distribution of FCRLA and was not observed in the lamina propria (immunofluorescence image not shown). At 15 min post-ILI of Chx266-R3-infliximab, infliximab was observed to co-localise with FCRLA in both apical and basal vesicular compartments and be present in cells within the lamina propria, demonstrating the completion of A^B transcytosis (immunofluorescence image not shown). This was not observed for infliximab alone (immunofluorescence image not shown).

[0382] We next investigated if the Fc domain of a mAb could be involved in the intersections between Chx266-R3- infliximab and FCRLA. Three chimeras were prepared that contained a generic human Fc sequence or one of the two Fc domain elements (CH2 or CH3): Fc-Chxi97-L1 -hGH (SEQ ID NO: 37), Cn2-Chxi97-L1 - hGH (SEQ ID NO: 38), and Cn3-Chxi97-L1 -hGH (SEQ ID NO: 43) . A flexible linker composed of glycine and serine residues was used to conjoin the Fc-domain element at the N-terminus of Chxi97 and the FCS L1 linker (SEQ ID NO: 22, with the RHKR (SEQ ID NO: 14) provided by amino acids 194-197 of Chxi97) was included in all three chimeras. The Chxi97 and the L1 linker sequence were selected based upon studies described in Example 1 .

[0383] At 15 min post ILI of Chxi97-L1 -hGH, Chx and hGH were distributed in both the apical and basal vesicular compartments with occasional co-localisations (immunofluorescence image not shown). By comparison, Chx and hGH present in the apical vesicular compartment of enterocytes at 15 min post ILI of Fc-Chxw L1 -hGH showed more extensive separation of Chx and hGH elements (immunofluorescence image not 008866329 34 shown). A similar ILI study with Cn3-Chxi97-L1 -hGH showed Chx and hGH being co-localised in both apical and basal vesicular compartments, as well as in cells within the lamina propria (immunofluorescence image not shown). Cn2-Chxi97-L1 -hGH showed extensive amounts of Chx-hGH colocalisation, with hGH separated from Chx also readily observed within the lamina propria (immunofluorescence image not shown).

[0384] The hGH element of the Cn3-Chxi97-L1 -hGH chimera was observed to co-localise with FCRLA in the apical, but not the basal, region of enterocytes (immunofluorescence image not shown). By comparison, the hGH element of Cn2-Chxi97-L1 -hGH co-localised with FCRLA in both the apical and basal regions of enterocytes (immunofluorescence image not shown), like that observed for infliximab and FCRLA following ILI of Chx266-R3-infliximab.

[0385] EXAMPLE 7: The CH2 element of IgG 1 Fc enhances furin-mediated cargo release

[0386] We hypothesised that cleavage of the furin cleavage site linker present in Chxi97-L1 -hGH in the apical compartment would result in release of the hGH cargo and its trafficking to the apical cell surface or lysosomal degradation pathway. We further hypothesised that only cleavage in the basal vesicular compartment would result in the efficient release of the hGH cargo from the basal surface of the enterocyte to enable systemic delivery of the cargo.

[0387] A study involving ILI administration of hGH alone, Chx266-L1 -hGH, Fc-Chxi97-L1 -hGH, Cn2-Chxi97-L1 - hGH, and Cn3-Chxi97-L1 -hGH was used to test the hypothesis that deviation from the normal apical routing of Chx could improve systemic cargo delivery of an FCS-containing construct. We observed that serum concentration-time profiles for hGH peaked between 15-30 min after administration for all the Chx- based chimeras, as would be expected for a A^B transcytosis process (Fig. 6A). Comparison of these results to serum concentration-time profiles obtained following subcutaneous injection of hGH, were used to determine bioavailability relative to the standard administration route (FREL%) (Fig. 6B). ILI administration of hGH alone resulted in a FREL% of -0.2%, consistent with a small fraction of non-specific intestinal uptake. Chxi97-L1 -hGH dosing resulted in a FREL% of ~1 .4%, consistent with the AUC improvement noted in Example 1 . ILI administration of Fc-Chxi97-L1 -hGH demonstrated a doubling of the FREL% achieved with Chxi97-L1 -hGH. While Ch3-Chxi97-L1 -hGH resulted in a FREL% of ~1 .5%, CH2- Chxi97-L1 -hGH demonstrated a FREL% of 4.5%.

[0388] To ensure that the benefit observed for Cn2-Chxi97-L1 -hGH versus Cn3-Chxi97-L1 -hGH was not due to a reduced potential for hGH separation by furin, we performed an in vitro cleaving study at pH 7.5. A time course assessment of these chimeras exposed to furin showed comparable rates of proteolytic separation, with both quickly and comparably being cut into ~36 kDa and ~23 kDa fragments (Fig. 6C), that were consistent with the Cn2-F3-Chxi97 and L1 -hGH, respectively (Fig. 6D). 008866329 35

[0389] These results show that the Fc domain of human IgG 1 can increase the efficiency of Chx-mediated A^B transcytosis and subsequent systemic cargo delivery from a construct that contains a consensus site for furin-based separation. Further, the CH2 element within the Fc domain of human IgG 1 was sufficient to achieve this increase in FREL%.

[0390] EXAMPLE 8: Assessment of the position of the immunoglobulin CH2 domain in the construct

[0391] To assess the importance of CH2 position within the construct, a construct was prepared where this element was positioned C terminal to, instead of N terminal to, the Chxi97 component. Due to the construction design, this repositioning of the CH2 domain resulted in the first 196 amino acids of Chx being used in the construct to produce Chxi96-Ch2-L1 -hGH. ILI administration of Chxi96-Cn2-L1 -hGH resulted in only negligible amounts of serum hGH levels over the same time course where ILI of Chxi97- L1 -hGH or Cn2-Chxi97-L1 -hGH resulted in consistent serum level profiles (Fig. 7A).

[0392] We explored whether the reduced hGH delivery to the systemic circulation following ILI dosing of Chxi96- CH2-L1 -hGH construct was because it could not be cleaved by furin. In vitro incubation of Chxi96-Cn2-L1 - hGH showed that the cut site in this construct remained accessible, with furin effectively cutting the material within 10 min of incubation and comparable to a construct where the CH2 preceded the Chx domain, in this case using a construct with super folding green fluorescent protein (sfGFP) as a cargo, CH2-Chxi97-L1 -sfGFP (SEQ ID NO: 45) (Fig. 7B).

[0393] We next examined the distribution of Chx and hGH within rat jejunal tissue over a 30 min time course following ILI of Chxi96-Ch2-L1 -hGH. At 5 min, both the Chx and hGH elements of Chxi96-Cn2-L1 -hGH were detectable and co-localised throughout enterocytes demonstrating that the construct was readily endocytosed at the apical plasma membrane and trafficked throughout the enterocyte (immunofluorescence image not shown). By 15 min, the extent of Chxi96-Cn2-L1 -hGH within enterocytes appeared to have increased and remained almost exclusively co-localised (immunofluorescence image not shown). At 30 min post-ILI, Chx and hGH detection continued to demonstrate extensive colocalisation with very little material detectable in cells within the lamina propria (immunofluorescence image not shown). Overall, there did not appear to be much separation of the Chx and hGH components of Chxi96-Cn2-L1 -hGH within enterocytes following apical endocytosis and trafficking.

[0394] These results demonstrate that placement of the CH2 moiety C-terminal to the Chx transporter element does not support release of the cargo from the basal surface of enterocytes. 008866329 36

[0395] EXAMPLE 9: Assessment of MMP cleavage sequences as cleavable linkers

[0396] It was next decided to assess if MMP cleavage sequences would be suitable as cleavable linkers.

[0397] Table 3 shows the MMP cleavage sequences tested. These sequences were selected as MMP consensus cleavage sequences based on Eckhard et al., 2016. Each “L” name corresponds to the specific MMP which can cleave the sequence. Therefore, the sequence “L1” is cleavable by MMP1 , “L2” by MMP2, “L3” by MMP3, “L4” by MMP4, and so on.

[0398] Table 3: MMP cleavage sequences tested 008866329 37

[0399] Constructs tested were composed of Chx266 linked at the C-terminus linked to hGH using an MMP cleavable linker as defined above in Table 3. These constructs were applied to the apical surface of human Caco-2 monolayers for two hours, following which the basal sample was collected and analysed by ELISA for hGH. hGH alone and the MMPLC construct were used as controls. As Figure 8 shows, all constructs enhanced hGH transport.

[0400] The ratio of hGH in basal versus intracellular compartments of the Caco-2 cells was investigated. Results are shown in Figure 9. In all constructs, the majority of hGH taken up into the cell reaches the basal compartment. We observed that the ratio of hGH in the basal versus the intracellular compartment remained similar across different MMP linkers.

[0401] We investigated the in vivo performance of these constructs in a rat model of uptake from the small intestinal (jejunal) lumen and delivery of a therapeutic cargo, in this case hGH, to the systemic circulation.

[0402] MMP constructs 1 , 2, 5, 6, 7, 8 and 1 1 (containing hGH cargo), were injected into rat jejunum and compared to serum concentration-time profiles obtained following subcutaneous injection of hGH to determine bioavailability relative to the standard administration route (FREL%) (Fig. 10). All MMP constructs achieved improved bioavailability compared to hGH alone. Constructs comprising MMP5 or MMP8-cleavable linkers achieved bioavailability of ~3%.

[0403] Comparison of PK profiles of MMP4, MMP5 and MMP8 constructs demonstrated that Chx-based delivery results in hGH serum profiles observed after ~10 min with a peak serum level between 15-60 min after administration for all three constructs, with MMP8 having the highest peak (Fig. 1 1 ).

[0404] These data confirm that matrix metalloproteinase cleavage sequence are suitable for use as cleavable linkers in the present invention.

[0405] Discussion

[0406] Chx266, or a slightly shorter version (Chxig?) can be genetically or chemically coupled to therapeutic cargo. However, systemic delivery of such cargo remained an issue. For efficient systemic delivery following oral delivery, the carrier and cargo must separate, since the Chx will be consumed by receptors that it normally targets on non-polarised cells in the lamina propria.

[0407] While we found that furin-cleavable linkers were effective at enabling this separation, the challenge remained for a Chx-based carrier-cargo containing a furin-cleavable linker to bypass the furin-containing 008866329 38 apical vesicular compartment to reach the basal compartment for efficient secretion of the separated transporter and cargo.

[0408] We found that combining the Chx transporter with the CH2 element of the Fc domain of human IgG 1 in a position-specific manner resulted in efficient entry of the construct into the apical surface of enterocytes and targeting of the basal vesicular compartment while avoiding the furin rich apical compartment. This achieved efficient release of the cargo into systemic circulation. Co-localisation of the Cn2-Chx construct with the Fc receptor-like A protein in the apical region of enterocytes soon after receptor-mediated endocytosis provides a mechanism for this modification from the previously established Chx A^B transcytosis pathway.

[0409] References

[0410] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

[0411] Abramson, A. et al. Oral delivery of systemic monoclonal antibodies, peptides and small molecules using gastric auto-injectors. Nat Biotechnol 40, 103-109 (2022).

[0412] Eckhard, U. et al. Active site specificity profiling of the matrix metalloproteinase family: Proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses. Matrix Biol 49, 37-60 (2016).

[0413] Fay, N.C. et al. A Novel Fusion of IL-10 Engineered to Traffic across Intestinal Epithelium to Treat Colitis. J Immunol 205, 3191 -3204 (2020).

[0414] Hsieh, J.C. et al. Intranasal immunization strategy to impede pilin-mediated binding of Pseudomonas aeruginosa to airway epithelial cells. Infect Immun 73, 7705-7717 (2005).

[0415] Liu, K. et al. GRP75 as a functional element of cholix transcytosis. Tissue Barriers, 2039003 (2022).

[0416] Prasad Awasthi et al. Novel Cholix Toxin Variants, ADP-Ribosylating Toxins in Vibrio cholerae Non- O1 / Non-O139 Strains, and Their Pathogenicity. Infect Immun 81 , 531 -541 (2013). 008866329 39

[0417] Santiago, T. et al. FCRLA is a resident endoplasmic reticulum protein that associates with intracellular Igs, IgM, IgG and IgA. Int Immunol 23, 43-53 (2011 ).

[0418] Taverner, A. et al. Cholix protein domain I functions as a carrier element for efficient apical to basal epithelial transcytosis. Tissue Barriers 8, 1710429 (2020).

[0419] Zhang, J., Field, C.J., Vine, D. & Chen, L. Intestinal uptake and transport of vitamin B12-loaded soy protein nanoparticles. Pharm Res 32, 1288-1303 (2015).

[0420] For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press

[0421] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0422] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0423] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0424] Any section headings used herein are for organisational purposes only and are not to be construed as limiting the subject matter described.

[0425] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. 008866329 40

[0426] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

Claims

008866329 41Claims1. A delivery construct comprising:(i) a transport facilitator comprising an immunoglobulin CH2 domain;(ii) a transporter comprising a cholix domain la; and(Hi) a therapeutic cargo; wherein the transporter is cleavably coupled to the therapeutic cargo and wherein the transport facilitator is N-terminal to the transporter.

2. The delivery construct of claim 1 , wherein the delivery construct comprises, from N to C, the transport facilitator, the transporter and the therapeutic cargo.

3. The delivery construct of claim 1 or claim 2, wherein the transporter is cleavably coupled to the therapeutic cargo via a cleavable linker.

4. The delivery construct of claim 3, wherein the cleavable linker comprises a furin cleavage sequence or a matrix metalloproteinase cleavage sequence.

5. The delivery construct of claim 4, wherein the cleavable linker comprises a furin cleavage sequence, optionally comprising the sequence RHKR (SEQ ID NO: 14).

6. The delivery construct of any one of the preceding claims, wherein the immunoglobulin CH2 domain comprises an IgG CH2 domain.

7. The delivery construct of claim 6, wherein the IgG CH2 domain comprises an IgG 1 CH2 domain.

8. The delivery construct of any one of the preceding claims, wherein the immunoglobulin CH2 domain is a human or humanised immunoglobulin CH2 domain.

9. The delivery construct of any one of the preceding claims, wherein the immunoglobulin CH2 domain comprises a peptide having at least 80% identity to SEQ ID NO: 31 .

10. The delivery construct of any one of the preceding claims, wherein the immunoglobulin CH2 domain comprises SEQ ID NO: 31 .11 . The delivery construct of any one of the preceding claims, wherein the transport facilitator does not comprise any immunoglobulin variable domains.

12. The delivery construct of any one of the preceding claims, wherein the therapeutic cargo comprises a polypeptide or polynucleotide sequence.008866329 4213. The delivery construct of claim 12, wherein the polynucleotide sequence comprises an RNA sequence, such as an siRNA.

14. The delivery construct of any one of the preceding claims, wherein the therapeutic cargo comprises a cytokine, a hormone, a single-chain Fv fragment (scFv), a Fab fragment, a diabody, a minibody or a nanobody.

15. The delivery construct of claim 14, wherein the therapeutic cargo comprises a hormone.

16. The delivery construct of claim 15, wherein the hormone comprises human growth factor hormone or glucagon-like-peptide-1 .

17. The delivery construct of any one of the preceding claims, wherein the transporter comprises SEQ ID NO: 5.

18. The delivery construct of any one of the preceding claims, wherein the transporter comprises SEQ ID NO: 3.

19. The delivery construct of any one of the preceding claims, wherein the transporter comprises SEQ ID NO: 4.

20. A pharmaceutical composition comprising a delivery construct of any one of claims 1 to 19 and a pharmaceutically acceptable carrier.21 . The pharmaceutical composition of claim 20, wherein the pharmaceutical composition is formulated for oral administration.

22. The pharmaceutical composition of claim 20 or claim 21 , wherein the pharmaceutical composition is formulated in a capsule or tablet.

23. The delivery construct of any one of claims 1 to 19 or pharmaceutical composition of any one of claims 20 to 22 for use in a method of treatment.

24. One or more nucleic acid(s) encoding the delivery construct of any one of claims 1 to 19.

25. A vector comprising the nucleic acid(s) of claim 24.

Images