Transcytosing composition

Abstract

The present invention relates to transporter proteins capable of transcytosing the intestinal wall and delivering antibodies to the blood stream.

Description

[0001] Transcytosing composition

[0002] The present invention relates to transporter proteins capable of transcytosing the intestinal wall and delivering antibodies to the blood stream.

[0003] Introduction

[0004] A major function of the epithelial cell monolayer that lines the gastro-intestinal (Gl) tract is to serve as a physical barrier to the environment. This barrier function extends to the molecular level, with free diffusion possible for ions and small molecules but not for larger molecules or protein macromolecules. These large molecules do not partition in and diffuse passively across a cell’s plasma membrane, and they are also too large to diffuse through the tight intercellular interstices, the Tight Junctions (TJs), that are a hallmark of the Gl tract barrier.

[0005] The use of permeation enhancers (PEs) to improve oral delivery of small-sized molecules with drug properties, including peptides, is well established. For small molecules, transcytosis by direct penetration of a cell’s plasma membrane and passive diffusion across the cell is feasible, depending on the physicochemical properties of the molecule. PEs can promote transcytosis of such small molecules, either by modifying the composition and behaviour of the cell membranes, or by complexing to and shielding of these molecules. Another way via which PEs can improve transcytosis of small molecules is to partially open the TJ’s between the epithelial cells. However, in practice, size remains a limitation and larger macromolecules such as proteins generally cannot traverse TJs, although new technologies do offer potential ways forward.

[0006] Rather than relying on passive diffusion, there are instances in nature where active transport processes are used to get proteins and larger complexes across the epithelial barrier in an intact manner. Examples include secretory IgA via the transferrin receptor CD71 in celiac disease (Matysiak-Budnik et al., 2008), Botulinum toxin A via retro-transcytosis in microfold M cells (Ghosal et al., 2018; Matsumura et al., 2015), and HIV1 virion transport via complement receptor 3 (CD11 / CD18) across cervical epithelial cells (Day et al., 2022).

[0007] Efforts have been made to mimic and make use of active transport processes as a means to get drug cargo across epithelial barriers, e.g. the transport of nanocarriers through intestinal organoid monolayers via M cells (Tong et al., 2020). Another approach uses a Cholix protein fragment from the pathogen Vibrio cholerae as a transcytosis carrier, leading to tissue penetration of the molecule in an intact form into the lamina propria (Fay et al., 2020; Liu et al., 2023; Ogura et al., 2021 ; Taverner et al., 2020).

[0008] Trinity Biosystems, Inc have developed a number of systems for the needleless delivery of antibodies. W02007 / 067596 describes the use of a carrier construct comprising a receptor binding domain, a transcytosis domain and antibody binding domain to which an antibody or antibody fragment is non-covalently bound. The antibody or fragment forms part of a fusion protein with a bioactive molecule. W02008 / 021234 describes a delivery construct comprising a receptor binding domain, a transcytosis domain and a particle.

[0009] W02006 / 044205 discloses a delivery construct comprising a receptor binding domain, and a transcytosis domain linked via a cleavable linker to a macromolecule. In all of these cases the transcytosis domain is derived from a bacterial species such as Pseudomonas endotoxin A, botulinum toxin, diphtheria toxin, pertussis toxin, cholera toxin, heat-labile E. Coli enterotoxin, Shiga toxin or Shiga - like toxin.

[0010] These studies indicate that active transport mechanisms in principle hold promise as a means to get antibodies and large protein molecules intact across the epithelial barrier. However, the reported approaches fall well short of the ideal drug delivery goal: ability to deliver antibodies, via an oral route, systemically to the blood, quickly and with large capacity, in an inert manner that does not lead to immunogenic responses against the active carrier molecule. Cholix is not a carrier that can meet these goals: it does not achieve systemic delivery and buildup of cargo in the blood, and it is a pathogen-derived molecule that will elicit an immune response and thus cannot repeatedly be used in a therapeutic setting. The inventors have identified human proteins that can serve as effective transcytosis drivers of antibody cargo in bispecific format, opening the way to generating humanized, non-immunogenic active cargo carriers that - in a universal manner - bring antibody proteins intact across the Gl tract tissue into the blood stream.

[0011] There are a small number of human enzymes and hormones that are normally secreted into the lumen of the Gl tract and which then naturally transcytose into the blood stream. Two hormones belong to this group: intestinal insulin (Bendayan et al., 1990, 1994; Ziv & Bendayan, 2000), as well as gastric leptin (Bendayan et al., 2010; P. Cammisotto & Bendayan, 2012; P. G. Cammisotto et al., 2007.) However, because of their highly potent biological effects and the absence of mutations that inactivate function while retaining ability to bind to their receptor and thus to transcytose (Salum et al., 2021), these two proteins may not be suitable for use as transcytosis drivers in biotherapeutics. The intestinal enzymes pancreatic lipase (Cloutier et al., 2006), bile salt-dependent lipase (Bruneau, Bendayan, et al., 2003; Bruneau, Richard, et al., 2003) and amylase (Cloutier et al., 2006) have also been reported to transcytose and reach the blood stream intact under physiological conditions. The present invention utilises these enzymes as efficient transcytosis drivers of antibody cargo using a bispecific format across Gl tissue. Summary

[0012] The present invention provides a composition comprising, consisting or consisting essentially of a transporter protein domain capable of transcytosing the intestinal wall linked to at least one antibody binding moiety. Preferably the transporter protein domain is a non- bacterial transporter protein domain.

[0013] Preferably, the transporter protein domain is derived from a protein capable of transcytosing the intestinal wall.

[0014] Preferably the protein capable of transcytosing the intestinal wall is selected from pancreatic triglyceride lipase, bile salt dependent lipase (BSDL) and a-amylase or a homolog thereof. More preferably, the protein capable of transcytosing the intestinal wall is selected from pancreatic triglyceride lipase, and a-amylase or a homolog thereof.

[0015] Preferably, the antibody binding moiety binds to the Fc region of the antibody. Preferably, the antibody binding moiety binds to non-variable regions on the light chain of an antibody (the CL region), to non-variable regions in the CH1 region of the heavy chain, or simultaneously to non-variable regions from both the CL and CH1.

[0016] Preferably, the antibody binding moiety comprises a Fc-binding domain or a homolog or derivative thereof. Preferably, the antibody binding moiety is a peptide.

[0017] Preferably the protein capable of transcytosing the intestinal wall is substantially identical to a protein derived from a mammal, more preferably a human. Preferably the protein capable of transcytosing the intestinal wall is endogenous to the subject being treated, i.e. it is derived from the protein naturally expressed by the subject species.

[0018] The invention also provides a fusion protein comprising a transporter protein domain derived from a protein capable of transcytosing the intestinal wall and an antibody binding domain.

[0019] The invention also provides a nucleic acid sequence encoding a fusion protein of the invention.

[0020] An expression vector comprising the nucleic acid sequence of the invention is also provided.

[0021] The invention also provides a cell comprising the expression vector of the invention.

[0022] The present invention also provides the use of composition for transporting an antibody across the intestinal wall.

[0023] The present invention further provides a pharmaceutical composition comprising (i) a protein capable of transcytosing the intestinal wall linked to an antibody binding moiety as described herein and (ii) and antibody. The pharmaceutical composition of the invention can be used in medicine. The pharmaceutical composition of the invention can be used in the treatment of a number of conditions known to be ameliorated by a therapeutic antibody including cancer, autoimmune diseases, and Alzheimer’s Disease. Thus, the invention also provides a method of treating cancer, an autoimmune disease or Alzheimer’s Disease comprising administering to a subject in need thereof a pharmaceutically effective amount of the pharmaceutical composition of the invention.

[0024] Detailed description

[0025] The present invention provides a composition comprising a transporter protein domain capable of transcytosing the intestinal wall linked to an antibody binding moiety.

[0026] The transporter protein domain comprises, consists, consists essentially of, or is derived from a protein capable of transcytosing the intestinal wall, retaining the ability transcytose the intestinal wall. As used herein “transcytosis” is the transport of a macromolecule from one side of a cell (e.g. the apical side of an epithelial cell) to the other side of the cell (i.e. the basolateral side of an epithelial cell) within a membrane bound vesicle.

[0027] As used herein, “capable of transcytosing the intestinal wall” means that a compound, preferably a protein or peptide, is capable of passing through the intestinal lining whilst remaining intact, and not undergoing degradation. Preferably, the ability of the compound, in particular the protein, to transcytose an intestinal wall can be identified by using a llssing Chamber such as that described in the examples. A compound (i.e. a protein) is capable of transcytosing the intestinal wall if it increases the level of a label detected on the serosal side of the chamber following introduction of the label into the mucosal side. For example, if a protein capable of transcytosing the intestinal wall is labelled with a detectable label e.g. FITC, the amount of the label detected on the serosal side of the chamber is increased compared to the label alone.

[0028] The intestinal wall comprises a number of layers, namely the mucosa, submucosa, a muscular layer and serosa or adventitia. A protein capable of transcytosing the intestinal wall can move from the lumen of the gut to the blood stream by passing through the epithelial cells lining the wall. It is thought that the proteins are transported transcytotically through intestinal enterocytes, from the luminal to basolateral side, and subsequently discharged into the intestinal interstitial space. The proteins then transfer across the capillary wall either directly into the bloodstream or indirectly into the lymphatic system which itself drains into the bloodstream. As used herein “intestinal wall” refers to the wall lining the gastrointestinal tract after the stomach. This includes the small intestine and large intestine. The small intestine is made up of the duodenum, jejunum and ileum, while the large intestine is also known as the colon. The intestinal wall preferably forms part of the small intestine. Preferably the protein capable of transcytosing the intestinal wall is capable of transcytosing the wall of the small intestine.

[0029] Preferably the protein capable of transcytosing the intestinal wall is a non-bacterial transporter protein domain. As used herein, a “non-bacterial transporter protein domain “ is a transporter protein domain which is not identical to a protein derived from or produced by a bacterial species, in particular a bacterial toxin. Preferably, the transcytosis domain is not derived from Pseudomonas endotoxin A, botulinum toxin, diphtheria toxin, pertussis toxin, cholera toxin, heat-labile E. Coli enterotoxin, Shiga toxin or Shiga - like toxin. Preferably, the transcytosis domain is not derived from Pseudomonas endotoxin A. The use of a transporter protein derived from a bacteria is not desirable, as it may cause undesirable side effects and adverse immunological reactions.

[0030] Preferably, the transporter protein domain is derived from the same species as the subject to be treated, i.e. a native or endogenous protein. Preferably, the protein capable of transcytosing the intestinal wall is a mammalian protein, more preferably a human protein. If the protein is derived from a non-human source, then the protein may be humanised, so that the protein sequence is modified to increase the similarity to the sequence naturally produced in humans.

[0031] Preferably the protein capable of transcytosing the intestinal wall is not a hormone, such as insulin or gastric leptin. The use of a hormone peptide is not desirable, as it may cause undesirable side effects. The small nature of these peptides also means that they may be more difficult to inactivate so they no longer act as a hormone whilst still retaining the ability of the peptide to transcytose the intestinal wall.

[0032] The transporter protein domain may comprise an inactive form of a native protein capable of transcytosing the intestinal wall protein. The inactive form is functionally inhibited or disabled so it does not retain the usual function of the protein, but does retain the ability to transcytose the intestinal wall. For example, if the protein is an enzyme, the inactive form has reduced or no enzyme activity compared to the native form. The inactive form may have 50% or less enzyme activity compared to the native form, preferably less than 25% activity, less than 10% activity, less than 5% activity. The inactive form of an enzyme preferably has no enzyme activity compared to the native form. Methods of assaying enzyme activity are well known to the person skilled in the art. For example, the activity of pancreatic triglyceride lipase can be measured using the 3H-triolein assay as set out in Lowe (1992). The inactive form may be a mutated version of the native form of the protein. For example, one or more amino acid residues known to be required to form the catalytic pocket can be substituted so that an enzyme is no longer active.

[0033] The transporter protein domain is preferably derived from a protein capable of transcytosing the intestinal wall selected from pancreatic triglyceride lipase, bile salt dependent lipase (BSDL) and a-amylase or a homolog thereof. Preferably the transporter protein domain comprises or consists of an amino acid sequence of any one of SEQ ID No. 2 to 11 or a fragment or homolog thereof. Preferably the transporter protein domain is derived from pancreatic triglyceride lipase.

[0034] “Pancreatic triglyceride lipase”, as used herein refers to Pancreatic triglyceride lipase as well as its related family members Pancreatic triglyceride lipase related protein - 1, Pancreatic triglyceride lipase related protein - 2, and Pancreatic triglyceride lipase related protein - 3, and their homologs.

[0035] The amino acid sequences for these proteins are publicly available and may be encoded by the nucleic acid sequence as set out below:

[0036] PNLIP - NCBI gene: 1056 https: / / www.ncbi. nlm.nih.gov / gene / ?term=1056

[0037] PNLIPRP1 - NCBI gene: 5407 https: / / www.ncbi. nlm.nih.gov / gene / ?term=5407

[0038] PNLIPRP2 - NCBI gene: 5408 https: / / www.ncbi. nlm.nih.gov / gene / ?term=5408

[0039] PNLIPRP3 - NCBI gene: 119548 https: / / www.ncbi.nlm.nih.gov / gene / 119548

[0040] Preferably the native form of the pancreatic triglyceride lipase comprises or consists of the amino acid sequence:

[0041] PNLIP:

[0042] MKEVCYERLGCFSDDSPWSGITERPLHILPWSPKDVNTRFLLYTNENPNNFQEVAADSSSIS GSNFKTNRKTRFIIHGFIDKGEENWLANVCKNLFKVESVNCICVDWKGGSRTGYTQASQNI RIVGAEVAYFVEFLQSAFGYSPSNVHVIGHSLGAHAAGEAGRRTNGTIGRITGLDPAEPCFQ GTPELVRLDPSDAKFVDVIHTDGAPIVPNLGFGMSQVVGHLDFFPNGGVEMPGCKKNILSQI VDIDGIWEGTRDFAACNHLRSYKYYTDSIVNPDGFAGFPCASYNVFTANKCFPCPSGGCPQ MGHYADRYPGKTNDVGQKFYLDTGDASNFARWRYKVSVTLSGKKVTGHILVSLFGNKGNS KQYEIFKGTLKPDSTHSNEFDSDVDVGDLQMVKFIWYNNVINPTLPRVGASKIIVETNVGKQ FNFCSPETVREEVLLTLTPC (SEQ. ID No:2) or a fragment or homolog thereof.

[0043] Preferably the native forms of the pancreatic triglyceride lipase related proteins 1-3 (lacking the leader peptide) comprises or consists of one the following amino acid sequences: PNLIPRP1:

[0044] KEVCYEDLGCFSDTEPWGGTAIRPLKILPWSPEKIGTRFLLYTNENPNNFQILLLSDPSTIEAS NFQMDRKTRFIIHGFIDKGDESWVTDMCKKLFEVEEVNCICVDWKKGSQATYTQAANNVRV VGAQVAQMLDILLTEYSYPPSKVHLIGHSLGAHVAGEAGSKTPGLSRITGLDPVEASFESTP EEVRLDPSDADFVDVIHTDAAPLIPFLGFGTNQQMGHLDFFPNGGESMPGCKKNALSQIVD LDGIWAGTRDFVACNHLRSYKYYLESILNPDGFAAYPCTSYKSFESDKCFPCPDQGCPQMG HYADKFAGRTSEEQQKFFLNTGEASNFARWRYGVSITLSGRTATGQIKVALFGNKGNTHQY SIFRGILKPGSTHSYEFDAKLDVGTIEKVKFLWNNNVINPTLPKVGATKITVQKGEEKTVYNF CSEDTVREDTLLTLTPC (SEQ. ID No:3) or a fragment or homolog thereof.

[0045] PNLIPRP2:

[0046] KEVCYGQLGCFSDEKPWAGTLQRPVKLLPWSPEDIDTRFLLYTNENPNNFQLITGTEPDTIE ASNFQLDRKTRFIIHGFLDKAEDSWPSDMCKKMFEVEKVNCICVDWRHGSRAMYTQAVQN

[0047] IRWGAETAFLIQALSTQLGYSLEDVHVIGHSLGAHTAAEAGRRLGGRVGRITGLDPAGPCF QDEPEEVRLDPSDAVFVDVIHTDSSPIVPSLGFGMSQKVGHLDFFPNGGKEMPGCKKNVL STITDIDGIWEGIGGFVSCNHLRSFEYYSSSVLNPDGFLGYPCASYDEFQESKCFPCPAEGC PKMGHYADQFKGKTSAVEQTFFLNTGESGNFTSWRYKISVTLSGKEKVNGYIRIALYGSNE NSKQYEIFKGSLKPDASHTCAIDVDFNVGKIQKVKFLWNKRGINLSEPKLGASQITVQSGED GTEYNFCSSDTVEENVLQSLYPC (SEQ. ID No:4) or a fragment or homolog thereof.

[0048] PNLIPRP3:

[0049] KEVCYERLGCFKDGLPWTRTFSTELVGLPWSPEKINTRFLLYTIHNPNAYQEISAVNSSTIQA SYFGTDKITRINIAGWKTDGKWQRDMCNVLLQLEDINCINLDWINGSREYIHAVNNLRVVGA EVAYFIDVLMKKFEYSPSKVHLIGHSLGAHLAGEAGSRIPGLGRITGLDPAGPFFHNTPKEV RLDPSDANFVDVIHTNAARILFELGVGTIDACGHLDFYPNGGKHMPGCEDLITPLLKFNFNA YKKEMASFFDCNHARSYQFYAESILNPDAFIAYPCRSYTSFKAGNCFFCSKEGCPTMGHFA DRFHFKNMKTNGSHYFLNTGSLSPFARWRHKLSVKLSGSEVTQGTVFLRVGGAVRKTGEF AIVSGKLEPGMTYTKLIDADVNVGNITSVQFIWKKHLFEDSQNKLGAEMVINTSGKYGYKST FCSQDIMGPNILQNLKPC (SEQ. ID No:5) or a fragment or homolog thereof.

[0050] Pancreatic triglyceride lipase and related proteins can be inactivated, as described by Lowe (1992) by disrupting the catalytic triad (Ser153 / His264 / Asp177, with numbering starting after the leader peptide sequence of PNLIP) by substituting one or more these amino acids. For example, His264 can be substituted with Leucine; Asp177 can be substituted with glutamic acid .alanine or asparagine, and Ser153 can be substituted with alanine, cysteine, glycine, phenylalanine, asparagine, proline, threonine or valine. Preferably the Pancreatic triglyceride lipase or related proteins comprises the following substitutions: His264 to Leucine, Asp177 to asparagine and Ser 153 to alanine.

[0051] “Bile salt dependent lipase” as used herein refers to Bile salt dependent lipase and homologs thereof. Bile salt dependent lipase (BSDL), also known as carboxyl ester lipase (CEL) or cholesterol esterase has been demonstrated to undergo intestinal transcytosis. The human BSDL protein may be encoded by the nucleic acid sequence as set out in NCBI Gene: 1056 https: / / www.ncbi.nlm.nih.gov / gene / 1056

[0052] Preferably the native form of BSDL comprises or consists of the amino acid sequence

[0053] AKLGAVYTEGGFVEGVNKKLGLLGDSVDIFKGIPFAAPTKALENPQPHPGWQGTLKAKNFK KRCLQATITQDSTYGDEDCLYLNIWVPQGRKQVSRDLPVMIWIYGGAFLMGSGHGANFLN NYLYDGEEIATRGNVIVVTFNYRVGPLGFLSTGDANLPGNYGLRDQHMAIAWVKRNIAAFG GDPNNITLFGESAGGASVSLQTLSPYNKGLIRRAISQSGVALSPWVIQKNPLFWAKKVAEKV GCPVGDAARMAQCLKVTDPRALTLAYKVPLAGLEYPMLHYVGFVPVIDGDFIPADPINLYAN AADIDYIAGTNNMDGHIFASIDMPAINKGNKKVTEEDFYKLVSEFTITKGLRGAKTTFDVYTE

[0054] SWAQDPSQENKKKTWDFETDVLFLVPTEIALAQHRANAKSAKTYAYLFSHPSRMPVYPKW

[0055] VGADHADDIQYVFGKPFATPTGYRPQDRTVSKAMIAYWTNFAKTGDPNMGDSAVPTHWE PYTTENSGYLEITKKMGSSSMKRSLRTNFLRYWTLTYLALPTVTDQEATPVPPTGDSEATP VPPTGDSETAPVPPTGDSGAPPVPPTGDSGAPPVPPTGDSGAPPVPPTGDSGAPPVPPTG DSGAPPVPPTGDSGAPPVPPTGDSGAPPVPPTGDSGAPPVPPTGDAGPPPVPPTGDSGA PPVPPTGDSGAPPVTPTGDSETAPVPPTGDSGAPPVPPTGDSEAAPVPPTDDSKEAQMPA VIRF(SEQ. ID No:6) or a fragment or homolog thereof.

[0056] The activity of BSDL can be measured for example using 4-nitrophenyl hexanoate as described by Gjellesvik et al (1992).

[0057] “a-amylase” as used herein refers to alpha amylase enzymes and homologs thereof. This includes salivary amylases such as alpha amylase enzyme AMY1A-1C, pancreatic enzymes such as AMY2A and AMY2B. It also includes the bacterial alpha- amylase type IIA which has been shown to transcytose the intestinal epithelium. The alpha amylase enzymes may be encoded by the nucleic acid sequence as set out below:

[0058] AMY1A - NCBI Gene: 276, https: / / www.ncbi. nlm.nih.gov / gene / ?term=276

[0059] AMY1B - NCBI Gene: 277, https: / / www.ncbi. nlm.nih.gov / gene / ?term=277

[0060] AMY1C - NCBI Gene: 278, https: / / www.ncbi. nlm.nih.gov / gene / ?term=278

[0061] AMY2A - NCBI Gene: 279, https: / / www.ncbi. nlm.nih.gov / gene / ?term=279

[0062] AMY2B - NCBI Gene: 280, https: / / www.ncbi. nlm.nih.gov / gene / ?term=280 Bacterial alpha-amylase type HA is available commercially, for example Sigma Aldrich (A6380), consisting of amylases from various Bacillus licheniformis sources (see product information), including NCIB 6346 (Morgan, F.J., and Priest, F.G., J. Appl. Bacteriol., 50(1), 107-114 (1981)), 44MB82-A (Ivanova, V.N. et al., J. Biotech., 28(2-3), 277-289 (1993)) and MTCC 1483 (Rao, M.D. et al., World J. Microbiol. Biotech., 18 , 547-550 (2002)). The amyS alpha-amylase from Bacillus licheniformis is encoded by the nucleic acid sequence of NCBI gene 66217199, https: / / www.ncbi.nlm.nih.gov / gene / 66217199.

[0063] Preferably the native form of a-amylase comprises or consists of one of the following amino acid sequences:

[0064] AMY1A

[0065] QYSSNTQQGRTSIVHLFEWRWVDIALECERYLAPKGFGGVQVSPPNENVAIHNPFRPWWE RYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMCGNAVSAGTSSTCGSYFN PGSRDFPAVPYSGWDFNDGKCKTGSGDIENYNDATQVRDCRLSGLLDLALGKDYVRSKIA EYMNHLIDIGVAGFRIDASKHMWPGDIKAILDKLHNLNSNWFPEGSKPFIYQEVIDLGGEPIK SSDYFGNGRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFMPSDRALVFVDNHDN QRGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRYFENGKDVNDWVG PPNDNGVTKEVTINPDTTCGNDWVCEHRWRQIRNMVNFRNVVDGQPFTNWYDNGSNQV AFGRGNRGFIVFNNDDWTFSLTLQTGLPAGTYCDVISGDKINGNCTGIKIYVSDDGKAHFSI SNSAEDPFIAIHAESKL (SEQ. ID No:7) or a fragment or homolog thereof.

[0066] AMY1B :

[0067] QYSSNTQQGRTSIVHLFEWRWVDIALECERYLAPKGFGGVQVSPPNENVAIHNPFRPWWE RYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMCGNAVSAGTSSTCGSYFN PGSRDFPAVPYSGWDFNDGKCKTGSGDIENYNDATQVRDCRLSGLLDLALGKDYVRSKIA EYMNHLIDIGVAGFRIDASKHMWPGDIKAILDKLHNLNSNWFPEGSKPFIYQEVIDLGGEPIK SSDYFGNGRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFMPSDRALVFVDNHDN QRGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRYFENGKDVNDWVG PPNDNGVTKEVTINPDTTCGNDWVCEHRWRQIRNMVNFRNVVDGQPFTNWYDNGSNQV AFGRGNRGFIVFNNDDWTFSLTLQTGLPAGTYCDVISGDKINGNCTGIKIYVSDDGKAHFSI SNSAEDPFIAIHAESKL (SEQ. ID No:8) or a fragment or homolog thereof.

[0068] AMY1C :

[0069] QYSSNTQQGRTSIVHLFEWRWVDIALECERYLAPKGFGGVQVSPPNENVAIHNPFRPWWE RYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMCGNAVSAGTSSTCGSYFN PGSRDFPAVPYSGWDFNDGKCKTGSGDIENYNDATQVRDCRLSGLLDLALGKDYVRSKIA EYMNHLIDIGVAGFRIDASKHMWPGDIKAILDKLHNLNSNWFPEGSKPFIYQEVIDLGGEPIK SSDYFGNGRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFMPSDRALVFVDNHDN QRGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRYFENGKDVNDWVG PPNDNGVTKEVTINPDTTCGNDWVCEHRWRQIRNMVNFRNVVDGQPFTNWYDNGSNQV AFGRGNRGFIVFNNDDWTFSLTLQTGLPAGTYCDVISGDKINGNCTGIKIYVSDDGKAHFSI

[0070] SNSAEDPFIAIHAESKL (SEQ. ID No:9) or a fragment or homolog thereof.

[0071] AMY2A

[0072] QYSPNTQQGRTSIVHLFEWRWVDIALECERYLAPKGFGGVQVSPPNENVAIYNPFRPWWE RYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMCGNAVSAGTSSTCGSYFN PGSRDFPAVPYSGWDFNDGKCKTGSGDIENYNDATQVRDCRLTGLLDLALEKDYVRSKIA EYM N H LI DIGVAGFRLDASKHM WPGDI KAI LDKLH N LNSN WFPAGSKPFIYQEVI DLGGEPI K SSDYFGNGRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFVPSDRALVFVDNHDNQ RGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRQFQNGNDVNDWVGP PNNNGVIKEVTINPDTTCGNDWVCEHRWRQIRNMVIFRNVVDGQPFTNWYDNGSNQVAF GRGNRGFIVFNNDDWSFSLTLQTGLPAGTYCDVISGDKINGNCTGIKIYVSDDGKAHFSISN

[0073] SAEDPFIAI HAESKL (SEQ. I D No: 10) or a fragment or homolog thereof.

[0074] AMY2B

[0075] QYSPNTQQGRTSIVHLFEWRWVDIALECERYLAPKGFGGVQVSPPNENVAIHNPFRPWWE RYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMSGNAVSAGTSSTCGSYFN PGSRDFPAVPYSGWDFNDGKCKTGSGDIENYNDATQVRDCRLVGLLDLALEKDYVRSKIA EYM N H LI DIGVAGFRLDASKHM WPGDI KAI LDKLH N LNSN WFPAGSKPFIYQEVI DLGGEPI K SSDYFGNGRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFMPSDRALVFVDNHDN QRGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRQFQNGNDVNDWVG PPNNNGVIKEVTINPDTTCGNDWVCEHRWRQIRNMVNFRNVVDGQPFTNWYDNGSNQVA FGRGNRGFIVFNNDDWTFSLTLQTGLPAGTYCDVISGDKINGNCTGIKIYVSDDGKAHFSIS NSAEDPFIAIHAESKL(SEQ. ID No:11.) or a fragment or homolog thereof.

[0076] The activity of amylase can be measured for example by using 2-chloro-4-nitrophenyl-alpha- maltotrioside as substrate, and monitoring the production of 2-chloro-4-nitrophenol.

[0077] As used herein the term “transporter protein domain” include homologs of the above described native proteins. This includes equivalent proteins in different species with the same activity and function i.e. homologs and orthologs. This also includes fragments of these proteins and their homologs which retain the ability to transcytose the intestinal cell wall. Homologs

[0078] As used herein, the term “homolog” refers to proteins or peptides which include one or more additions, deletions, substitutions or the like, and are encompassed by the present invention. In addition, it may be possible to replace one amino acid with another of similar "type". For instance replacing one hydrophobic amino acid with another. One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of analysis are contemplated in the present invention.

[0079] Desirably, the term "substantial identity" when used in relation to an amino acid sequence indicates that said sequence has a greater degree of identity with the sequence described herein than with prior art amino acid sequences.

[0080] For the purposes of the present invention, an amino acid sequence may be regarded as having “substantial homology” to another amino acid sequence if a significant number of the constituent amino acids exhibit homology when using the one of the algorithms mentioned above. Preferably, the degree of homology is measured over the entire amino acid sequence. For example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or even 99%, in increasing order of preference, of the amino acids, may be identical. Alternatively, for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or even 99%, in increasing order of preference, of the amino acids, may have similarity. Most preferably, homologues or derivatives having at least 90% or even 95% similarity are provided.

[0081] The amino acid residues comprising the proteins or peptides of the invention may be chemically modified. Examples of chemical modifications include those corresponding to post translational modifications for example phosphorylation, acetylation and deamidation Chemical modifications may not correspond to those that may be present in vivo. For example, the N or C terminal ends of the peptide may be modified to improve the stability, bioavailability and or affinity of the peptides. Further examples of non-natural modifications include incorporation of non-encoded a-amino acids, D-forms of proteogenic amino acids, photoreactive cross-linking amino acids, N-methylated amino acids, and p-amino acids, backbone reduction, retroinversion by using d-amino acids, N-terminal methylation and C- terminal amidation and pegylation. Preferably, the transporter protein domain may comprise N-linked glycosylation units, as these may be involved in the endocytosis of the proteins by the luminal epithelial cells or enterocytes. These may be naturally occurring, or added as modified amino acids.

[0082] Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position. Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent to one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent to another inserted amino acid residue.

[0083] Inserted amino acids and replacement amino acids may be naturally occurring amino acids or may be non-naturally occurring amino acids and, for example, may contain a non-natural side chain, and / or be linked together via non-native peptide bonds. Such altered proteins are discussed further in Douat-Casassus et al., J. Med. Chem, 2007 Apr. 5; 50(7): 1598-609 and Hoppes et al., J. Immunol 2014 Nov. 15; 193(10):4803-13 and references therein). If more than one amino acid residue is substituted and / or inserted, the replacement / inserted amino acid residues may be the same as each other or different from one another. Each replacement amino acid may have a different side chain to the amino acid being replaced.

[0084] Amino acid substitutions may be conservative, by which it is meant the substituted amino acid has similar chemical properties to the original amino acid. A skilled person would understand which amino acids share similar chemical properties. For example, the following groups of amino acids share similar chemical properties such as size, charge and polarity: Group 1 Ala, Ser, Thr, Pro, Gly; Group 2 Asp, Asn, Glu, Gin; Group 3 His, Arg, Lys; Group 4 Met, Leu, lie, Vai, Cys; Group 5 Phe Thy Trp.

[0085] Thus, what is important for homologs, derivatives and fragments is that they possess at least a degree of the ability of the native protein from which they are derived. For example the ability to transcytose the intestinal lining for the transporter protein domain and ability to bind the antibody of concern for the antibody binding peptides.

[0086] Antibody binding moiety

[0087] Preferably the antibody binding moiety binds to a region of the antibody so as not to interfere with antigen recognition and / or binding activity of the antibody. If the antibody is a monoclonal antibody, then the antibody binding moiety preferably binds to the constant heavy chain, or Fc region, or non-variable regions CL and / or CH1 . Preferably the antibody binding moiety comprises or consists of an Fc binding domain or homolog thereof which is capable of binding specifically to the Fc region of an antibody. The antibody binding moiety may bind to any class or subclass of antibody, for example any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. lgG1 , I gG2, I gG3, I gG4, I gA1 and lgA2). Preferably the antibody being moiety binds to an IgG antibody.

[0088] The antibody binding moiety preferably reversibly binds to the antibody, so that following transcytosis the antibody is released.

[0089] The term "specifically bind" as used herein means that the antibody binding moiety is selective for the antibody and that this binding can be distinguished from unwanted or nonspecific interactions. The ability of an antibody binding moiety to bind to a specific antibody can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed e.g. on a BIAcore instrument) and traditional binding assays. The extent of binding of an antibody binding moiety to an unrelated (non-antibody) protein other than the antibody of interest is less than about 10% of the binding of the antibody binding moiety to the antibody of interest as measured, e.g., by ELISA. “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., the Fc region of an antibody) and its binding partner (e.g., antibody binding moiety). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g., an antibody binding moiety and an antibody). The affinity of a molecule for its partner can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (kOff and kon, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. The dissociation constant represents the concentration of the antibody at which half of the binding sites on the available antibody binding moiety are occupied. A lower Kd indicates a higher binding affinity between the antibody and antibody binding moiety, while a higher Kd reflects weaker binding. Several methods are available to measure the Kd of an antibody binding moiety, including surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence-based assays. In certain aspects, the antibody binding moiety that binds to the antibody has a dissociation constant (Kd) of < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10 '8M or less, e.g. from 10'8M to 10'13M, e.g., from 10'9M to 10-16M). The binding moiety preferably has a dissociation constant (Kd) between 0.01 pM to 1nM, more preferably 0.5pM to 0.5 nM, 1 pM to 0.1 nM. Alternatively the antibody binding moiety has an IC50 of 30nM to 70,000nM at pH7.4, preferably 100 nM to 1000 nM, more preferably 240 nM to 600 nM . Preferably the ratio of transporter protein to antibody binding moiety within the composition is 1 :1. However, the transporter protein may be linked to more than one antibody binding moiety. For example the transporter protein may be linked to 2, 3, 4 or more antibody binding moieties, which may be the same or different.

[0090] Aptamers

[0091] In one embodiment the antibody binding moiety may be an aptamer. Aptamers are synthetic oligonucleotides (DNA or RNA) that recognize target molecules with high affinity and specificity through a combination of shape complementarity and non-covalent chemical bonds (Blank & Blind, Current Opin. Chem. Biol., 2005, 9:336-342). These artificial ligands are quite easy to obtain in vitro and can be developed to recognise a large variety of different molecule classes which range from mere ions (e.g. Pb2+, Liu & Lu, 2003. J Am Chem Soc., 125, 6642-6643) to nucleotides, small molecules, proteins, viruses, and cells up to whole organisms (Menger et al., 2006. Handbook of Experimental Pharmacology, 359-373). High binding affinity aptamers have been selected through the well-known SELEX method (Ellington & Szostak, 1990. Nature, 346, 818-822) for the detection of low molecular weight molecules like theophyllin (Jenison et al., 1994. Science, 263, 1425-1429), L-arginine (Geiger et al., 1996. Nucl. Acids Res., 24, 1029-1036), moenomycin (Schuerer et al., 2001. Bioorg. Med. Chem., 92, 2557-2563), 17b-estradiol (Kim et al., 2007. Biosens. Bioelectron., 22, 2525-2531) but also for larger molecules like thrombin (thrombin-binding aptamer:5’- GGTTGGTGTGGTTGG-3’) (Baldrich et al., Anal Chem. 2004, 76, 23,7053-63), cholera toxin or HIV-1 tat protein, among others (for review see Tombelli et al., 2007, Biomolec Eng., 24, 191-200). Some of the above mentioned aptamers have been used in ELISA-like assays on microplates or on the surface of biosensor transducers (QCM, SPR). An aptamer-modified AuNP colorimetric system has also been developed for the determination of the protein PDGF in a sandwich-based assay (Huang et al., 2005, 77, 5735-5741). Aptamers capable of binding the Fc regions of the antibody are known in the art. Suitable aptamers include those described in Hu et al (2019).

[0092] Antibody binding peptide

[0093] In an alternative, the antibody binding moiety may be a protein, polypeptide or peptide. In a preferred embodiment the antibody binding moiety comprises or consists of a Fc binding domain or an antibody light chain (LC) binding domain. For example, the Fc binding domain may be derived from a Fc receptor protein or a homolog thereof which retains the ability to bind the Fc region. Suitable Fc Receptors are known in the art and include the IgG receptors (FcyR), high-affinity IgE receptor (FCERI), IgA and IgA / IgM receptors, and neonatal Fc receptor for IgG (FcRn). For example, the antibody LC binding domain may be derived from Protein L sequences.

[0094] In a more preferred embodiment, the antibody binding moiety is an antibody binding peptide.

[0095] The term “antibody binding peptide” as used herein refers to a peptide which specifically binds to an antibody. Generally, “peptides” as used herein contain less than 50 amino acid bases, preferably less than 30 amino acids, more preferably less than 20 amino acids, even more preferably less than 15 amino acids. The term ‘peptides’ also includes the so-called knob antibodies, which are derived from ultralong cow CDRH3 regions, are generally identified via cow immunization strategies, and are generally less than 50 amino acids in length.

[0096] Suitable antibody binding peptides are known in the art, such as those described by Choe et al, (2016) and De Lano et al (2000) . Suitable peptides include Protein A, Protein G, Protein L, Protein Z (derived from B-domain of SpA), Protein LG, Protein LA, Protein AG ,SpA, PAM (peptide with the sequence (RTY)4K2KG) (SEQ. ID No:18), Fc-lll, FcBP-2, FC-III-4C, FcRM, or a peptide with a sequence selected from TWKTSRISIF (SEQ. ID No: 19), FGRLVSSIRY (SEQ. ID No:20), EPIHRDTLTALL (SEQ. ID No:21), APAR (SEQ. ID No:22), HWRGWV (SEQ. ID No:23), HWRGWVC (SEQ. ID No 40), HYFKFD (SEQ. ID No:24), HFRRHL (SEQ. ID No:25), HWCitGWV (SEQ. ID No:26), DAAG (SEQ. ID No:27),D2AAG (SEQ. ID No:28), NKFRGKYK (SEQ. ID No:29), NARKFYKG (SEQ. ID No:30), FYWHCLDE (SEQ. ID No:31), FYCHWALE (SEQ. ID No:32), FYCHTIDE (SEQ. ID No:33), RRGW (SEQ. ID No:34), KHRFNKD (SEQ. ID No:35), cyclo(Na-Ac)S(A)-RWHYFK-Lact-E (SEQ. ID No:37), cyclo(Na-Ac)Dap(A)-RWHYFK-Lact-E (SEQ. ID No:38) and cyclo [Link-M-WFRHYK] (SEQ. ID No:39) or homologs thereof. The homologs preferably have substantial homology as defined herein but retain the ability to specifically bind an antibody. Preferably, the antibody binding peptide comprises, consists or consists essentially of the sequence HWRGWV (SEQ. ID No 23) and / or HWRGWVC (SEQ. ID No 40). Preferably, the antibody binding peptide comprises, consists or consists essentially of the Protein L sequences:

[0097] MNIKFAGKETPETPEEPKEEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYADLLAKVNGEY TADLEDGGNHMNIKFAG (SEQ. ID No.41) or PFVENKEETPETPGTDSEEEVTIKANLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDNGE YTVDVADKGYTLNIKFAG (SEQ.ID No. 42).

[0098] Preferably the antibody binding peptide comprises, consists or consists essentially of the sequence AWHLGELVW (SEQ. ID No. 12). Preferably the antibody binding peptide is FcBP-1 , FcB-2, Fc-I II-4C or Fc-lll. Preferably the antibody binding peptide comprises, consists or consists essentially of the sequence DCAWHLGELVWCT (SEQ. ID No. 13). Preferably the antibody binding peptide is FcB-2, Fc-I I I-4C or Fc-lll.

[0099] The antibody binding peptide may comprise one or more modified amino acids. For example, the antibody binding peptide may comprise one or more D amino acids, which may help to prevent proteolysis and therefore undesirable degradation in the intestinal lumen.

[0100] Antibodies

[0101] The antibody binding moiety specifically binds to an antibody.

[0102] As used herein, the term “antibody” is used interchangeably with “immunoglobulin” and encompasses polyclonal antibodies, monoclonal antibodies, multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity, and contain at least the CH2 portion of at least one heavy chain. Preferably the antibody is a monoclonal antibody. An antibody can be a member of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. lgG1, lgG2, lgG3, lgG4, lgA1 and lgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Preferably, the antibody is an IgG antibody, more preferably an lgG1 or lgG4. The term “antibody” is also intended to include single domain antibodies (sdAb) or nanobodies including VHH fragments and VNAR fragments, which consist of a single monomeric antibody chain, but are still able to selectively bind a specific antigen. The term “antibody” is also intended to include conjugates of the antibody, for example conjugates with polyethylene glycol, PEG.

[0103] Further, except where the context requires otherwise, the term “antibody” should be understood to encompass complete antibodies and antibody fragments comprising an antigen-binding region of the complete antibody and the CH2 domain, including scFv-CH2- CH3 fusion proteins. An antibody can be produced by a hybridoma, or by synthetic means such as recombinant DNA techniques, phage display or yeast display technologies or using transgenic mice, or liquid or solid phase peptide synthesis.

[0104] It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et al., Nature 341:544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab’)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., Science 242:423-426 (1988); Huston et al., PNAS USA 85:5879- 5883 (1988)); (viii) bispecific single chain Fv dimers (PCT / US92 / 09965) and (ix) “diabodies”, multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; P. Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)).

[0105] Preferably the antibody is a known therapeutic antibody, which is capable of treating, ameliorating or alleviating a disease or condition. This includes an antibody which has received regulatory approval, by for example the Food and Drug Administration (FDA) or European Medicines Agency (EMA).

[0106] The transporter protein domain derived from a protein capable of transcytosing the intestinal wall is linked to the antibody binding moiety. The linkage may take any form known to the person skilled in the art. It may comprise a binding pair, such as biotin / streptavidin, wherein one member of each binding pair is attached to the transporter protein domain and antibody binding peptide respectively. The binding pair may comprise, for example, complementary nucleic acid sequences. The linkage may comprise one or more covalent bonds. For example, the linkage may comprise one or more amino acids bases. The linkage may contain one or more affinity tags to aid purification and isolation of the transcytosing domain and / or antibody binding moiety. In a preferred embodiment the transporter protein domain is linked to an antibody binding peptide by one or more amino acids bases so as to form a fusion protein.

[0107] Fusion proteins

[0108] In a second aspect the invention provides a fusion protein comprising a transporter protein domain derived from a protein capable of transcytosing the intestinal wall and an antibody binding domain. The antibody binding domain comprises or consists of an antibody binding peptide as described above.

[0109] The transporter protein domain may be linked to the antibody binding domain via one or more amino acids. The amino acids may be selected to provide an additional function such as an affinity tag to assist in isolating or purifying the fusion protein. The fusion protein may further comprise one or more affinity tags, such as (but not restricted to) the StrepTactinll, His6, Myc, HA, FLAG and, V5 affinity tags. Preferred embodiments and features of the transporter protein domain and the antibody binding domain will be apparent from the above discussion of the preferred embodiments of the composition described above.

[0110] The transporter protein domain and the antibody binding domain can be present in any order. The antibody binding domain may be located N or C terminal of the transporter protein domain. The antibody binding domain may split the sequence of transporter protein domain or vice versa, provided that the function of each domain is retained by the fusion protein.

[0111] Preferably the fusion protein comprises or consists of the amino acid sequence of SEQ ID No:1, SEQ ID No:43, SEQ ID No:44, SEQ ID No:45, SEQ ID No:46 or has substantial homology or identity thereto.

[0112] Nucleic acid sequences

[0113] The invention also provides a nucleic acid sequence encoding a fusion protein of the second aspect.

[0114] Thus, the invention provides:

[0115] (i) a sequence which codes for a fusion protein comprising a transporter protein domain derived from a protein capable of transcytosing the intestinal wall and an antibody binding domain;

[0116] (ii) a sequence which codes for a homolog, or derivative of the fusion protein as described herein;

[0117] (iii) a sequence which is complementary to the sequence in (i) or (ii); and

[0118] (iv) a sequence which has substantial identity with any of those of (i), (ii) or (iii).

[0119] Preferably the nucleic acid sequence (i) encodes the fusion protein of the amino acid sequence of SEQ. ID No:1 , SEQ. ID No:43, SEQ. ID No:44, SEQ. ID No:45 or SEQ. ID No:46; (ii) is complementary to sequences of (i); or (iii) is substantially identical to sequences of (i) or (ii).

[0120] The nucleic acid molecules of the invention may include a plurality of such sequences, and / or fragments. The skilled person will appreciate that the present invention can include variants of the nucleic acid molecules which are described herein. Such variants are encompassed by the present invention. These may occur in nature, for example because of strain variation. For example, additions, substitutions and / or deletions are included. In addition, and particularly when utilising microbial expression systems, one may wish to engineer the nucleic acid sequence by making use of known preferred codon usage in the particular organism being used for expression. Thus, synthetic or non-naturally occurring variants are also included within the scope of the invention.

[0121] When comparing nucleic acid sequences for the purposes of determining the degree of homology or identity one can use programs such as BESTFIT and GAP (both from the Wisconsin Genetics Computer Group (GCG) software package) BESTFIT, for example, compares two sequences and produces an optimal alignment of the most similar segments. GAP enables sequences to be aligned along their whole length and finds the optimal alignment by inserting spaces in either sequence as appropriate. Suitably, in the context of the present invention when discussing identity of nucleic acid sequences, the comparison is made by alignment of the sequences along their whole length.

[0122] Desirably, the term "substantial identity" indicates that said sequence has a greater degree of identity with the sequence described herein than with prior art nucleic acid sequences.

[0123] Preferably, sequences which have substantial identity have at least 50% sequence identity, desirably at least 75% sequence identity and more desirably at least 90 or at least 95% sequence identity with said sequences. In some cases, the sequence identity may be 99% or above.

[0124] The nucleic acid sequence may be codon optimised using known techniques for expression by a host cell.

[0125] The nucleic acid molecule may be in isolated or recombinant form. It may be incorporated into a vector and the vector may be incorporated into a host. Such vectors and suitable hosts form yet further aspects of the present invention.

[0126] Pharmaceutical formulations

[0127] In a further aspect the present invention provides a pharmaceutical composition comprising (i) a transporter protein domain capable of transcytosing the intestinal wall linked to an antibody binding moiety and (ii) and antibody.

[0128] Preferred embodiments and features of the transporter protein domain and the antibody binding moiety will be apparent from the above discussion of the preferred embodiments of the composition described above.

[0129] Preferably the transporter protein domain capable of transcytosing the intestinal wall and the antibody binding peptide are comprised in a fusion protein. Preferred embodiments and features of the fusion protein comprising a transporter protein domain and the antibody binding domain will be apparent from the above discussion of the preferred embodiments of the fusion protein described above.

[0130] Preferred embodiments and features of the antibody will be apparent from the above discussion of the preferred embodiments of the composition described above.

[0131] The pharmaceutical compositions according to the invention are preferably in solid or semisolid form, and preferably they are suitable for oral administration. Such formulations may be prepared by a number of known methods established in the art. For example, the transporter protein linked to an antibody binding peptide, and the antibody may be admixed together, optionally together with other excipients required in the dosage form.

[0132] Preferably the pharmaceutical formulation contains one or more excipients known to stabilise the antibody within the intestinal tract, such as those described in W02020 / 063728 and WO2023 / 152234 (both of which are incorporated herein by reference.) Preferably the pharmaceutical formulation further comprises 5-aminolevulinic acid (5-ALA) and optionally a pH modulator and / or an enzyme inhibitor. Alternatively, the pharmaceutical formulation further comprises one or more di-peptides and optionally an enzyme inhibitor.

[0133] Preferably the enzyme inhibitor is a protease inhibitor, such as for example, aprotinin, Ovomucoid type I l-O (containing ovoinhibitor), Bowman-Birk inhibitor (BBI), or Kunitz trypsin inhibitor for protection of proteins. Preferably the enzyme inhibitor is aprotinin. Aprotinin is a protease inhibitor, which is known to inhibit trypsin and other similar proteases. It is often also referred to as Bovine Pancreatic Trypsin Inhibitor (BPTI). Aprotinin is a 58 amino acid protein that is formed after processing of a 100 amino acid polypeptide that comprises a signal peptide, propeptide domains, and a Kunitz domain. Aprotinin in its mature 58 amino acid form is available commercially and is also sold under the trade name Trasylol®, which is indicated for prophylactic use to reduce blood loss during surgery.

[0134] The aprotinin for use in the invention may comprise the amino acid sequence RPDFCLEPPYTGPCKARMIRYFYNAKAGLCQPFVYGGCRAKRNNFKSSEDCMRTCGGA (SEQ ID NO 14).

[0135] The aprotinin or fragment thereof, or aprotinin analogue or fragment thereof for use in the invention may be prepared recombinantly (for example, in E. coli, mammalian cells or insect cells), synthetically (for example, using standard organic chemistry techniques, such as solution or solid phase peptide synthesis), or it may be a native protein from an animal source, such as a bovine source.

[0136] Further, except where the context requires otherwise, the term “aprotinin” should be understood to encompass aprotinin, analogues of aprotinin, fragments of aprotinin and fragments of analogues of aprotinin. Suitable analogues and fragments for use in the present invention are those which enhance the stability of a protein, such as the transcytosing protein / antibody binding domain and / or antibody in the presence of the body fluids found in the lower gastrointestinal tract, for example the ileum, duodenum and / or the jejunum. Methods for confirming the ability of aprotinin, a fragment of aprotinin, an analogue of aprotinin or a fragment thereof to enhance the stability of the active ingredient (a protein) in the presence of the body fluids found in the lower gastrointestinal tract, such as the ileum and / or the colon are described in the examples in W02020 / 063728. “Enhanced” stability as used herein means that at least 50% of the active ingredient present remains intact after 4 hours in the gastrointestinal fluid. Preferably at least 60%, 70%, 75%, 80%, 90% 95% or more of the transcytosing protein / antibody binding domain and / or antibody remains intact.

[0137] Preferably the di-peptide is selected from diglycine or carnosine (also known as beta-alanyl- L-histidine).

[0138] The pharmaceutical composition may also further comprise a co-protein or peptide that acts to assist the transporter protein domain. A co-protein or peptide increases the efficiency of transcytosis by increasing the amount of antibody that is transported through the intestinal wall, or by reducing the time required for the same amount of antibody to be transported through the intestinal wall as compared to the transcytosis protein alone. For example if the transporter protein domain is derived from a pancreatic triglyceride lipase, then the pharmaceutical composition may further optionally comprise a co-lipase or a functional homolog or fragment thereof.

[0139] The colipase is necessary for efficient dietary fat digestion by the pancreatic triglyceride lipase. The nucleic acid sequence for human co-lipase protein is identified as NCBI Gene 1208 https: / / www.ncbi.nlm.nih.gov / gene / 1208.

[0140] The colipase protein preferably comprises or consists of the amino acid sequence: MEKILILLLVALSVAYAAPGPRGIIINLTLYGIYYKCPCERGLTCEGDKTIVGSITNTNFGICHDA GRSKQ (SEQ ID No. 15). If the transporter protein domain is derived from BSDL, the pharmaceutical composition may further optionally comprise glucose regulated protein 95 (Grp94) or a functional homolog or fragment thereof.

[0141] Grp94 has been shown to be internalised together with BSDL by endocytosis, and is thought to act as a molecular chaperone. The nucleic acid sequence for human grp94 is identified as NCBI Gene 7184 https: / / www.ncbi.nlm.nih.gov / gene / 7184.

[0142] The grp94 protein preferably comprises or consists of following amino acid sequence:

[0143] MRALWVLGLCCVLLTFGSVRADDEVDVDGTVEEDLGKSREGSRTDDEWQREEEAIQLDG LNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENA LSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQ STSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNTLGRGTTITL VLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAV EEEEEEKKPKTKKVEKTVWDWELMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYI HFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKG VVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKYNDTFWKEFGTNIKLGVIE DHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLL KKGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLN WMKDKALKDKIEKAWSQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYAS QKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERML RLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDEDEEMDVGTDEEEETAKESTAEKDEL (SEQ ID No. 16).

[0144] Pharmaceutical compositions in the present invention that are suitable for oral administration may be presented either in the form of tablets, capsules, mini-tablets, pellets, powders, granules, microparticles, nanoparticles or hydrogels.

[0145] Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, tablets, mini-tablets, or pellets, or as powders, granules or crystals. In a solid composition, the minimum diameter of each particle is typically at least 10'4m, usually at least 5 x 10'4m and, preferably at least 10'3m. The maximum diameter is usually no more than 30 mm, typically no more than 20 mm and, preferably, no more than 10 mm. In preferred embodiments, the particle has a diameter from about 0.2 mm to about 15 mm, preferably from about 1 mm to about 4 mm (e.g. for pellets or mini-tablets) or from about 6 mm to about 12 mm (e.g. for certain tablets or capsules). The term "diameter" refers to the largest linear dimension through the particle. Compositions according to the invention may of course contain any further conventional excipients as required such as binders, extenders, disintegrants, diluents and lubricants. Excipients used in solid forms include for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate, calcium sulfate, sorbitol, glucose and / or lactose and / or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art. Suitable binders include starch, gelatine, natural sugars such as glucose or betalactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Disintegrators include without limitation starch, methylcellulose, agar, bentonite, xanthan gum and the like. Fast dissolving diluents include mannitol, lactose, sucrose and / or cyclodextrins. Lubricants, glidants, flavours, colouring agents and stabilizers may also be added for ease of fabrication and use. Lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride.

[0146] A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow, delayed or controlled release of the antibody. Preferred examples of coatings are given below.

[0147] Capsules may have solid, semi-solid or non-solid contents. Exemplary contents for capsules may include suspensions which can contain, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, and methylcellulose as a viscosity enhancer, as well as any of the solid or semi-solid forms above.

[0148] Preferred unit dosage formulations are those containing an effective dose, or an appropriate fraction thereof, of the active ingredient. Release from certain formulations may also be sustained, if the composition contains suitable controlled-release excipients. However, in preferred formulations, release is pulsatile.

[0149] The pharmaceutical composition typically contains an equimolar amount of the antibody binding moiety and the antibody. Where the ratio of antibody binding moiety to the transporter protein domain present is approximately 1:1, the pharmaceutical composition typically contains an equimolar amount of the transporter protein domain I antibody binding moiety composition, and the antibody.

[0150] The compositions according to the invention will typically comprise a therapeutically effective amount of the transporter protein domain I antibody binding moiety composition, which may be from 0.01 wt % to 80 wt %, based on the total weight of the composition. The actual dosage would be determined by the skilled person using common general knowledge. However, by way of example, "low" dose formulations typically comprise no more than 20 wt % of the transcytosis protein / antibody binding moiety composition, and preferably comprise from 1 wt % to 10 wt %, e.g. 5 wt %, of the transporter protein domain I antibody binding moiety composition,. "High" dose formulations typically comprise at least 40 wt % of the transporter protein domain I antibody binding moiety composition, and preferably from 45 wt % to about 75 wt %, e.g. 50 wt % or 60 wt %.

[0151] The transporter protein domain I antibody binding moiety composition is typically present in an amount resulting in a concentration of 0.1-25 mg / ml in the lower gastrointestinal tract. Each dosage form may contain 50-5000mg transporter protein domain I antibody binding moiety composition, typically 100-1000mg; 250-750mg or 300-500mg.

[0152] The compositions according to the invention will typically comprise a therapeutically effective amount of the antibody which may be from 0.01 wt % to 80 wt %, based on the total weight of the composition. The actual dosage would be determined by the skilled person using common general knowledge. However, by way of example, "low" dose formulations typically comprise no more than 20 wt % of the antibody, and preferably comprise from 1 wt % to 10 wt %, e.g. 5 wt %, of the antibody. "High" dose formulations typically comprise at least 40 wt % of the antibody, and preferably from 45 wt % to about 75 wt %, e.g. 50 wt % or 60 wt %.

[0153] The antibody is typically present in an amount resulting in a concentration of 0.1-25 mg / ml in the lower gastrointestinal tract. Each dosage form may contain 50-5000mg antibody, typically 100-1000mg; 250-750mg or 300-500mg.

[0154] Except where the context requires otherwise, throughout this specification and claims, any reference to a pharmaceutical composition in solid or semi-solid form should be understood to include individual solid or semi-solid particles or unit forms which are solid or semi-solid throughout, as well as those having a solid or semi-solid exterior and a non-solid, for example liquid or gel, interior. For example, a capsule may have liquid or gel contents. Delivery to the gastrointestinal tract

[0155] The composition according to the invention is adapted for delayed or selective release of the transporter protein domain / antibody bonding moiety and antibody in the lower gastrointestinal tract, in particular the duodenum, jejunum, and / or ileum suitably following oral administration. This may be accomplished by the use of particular coatings. The compositions of the invention may be delayed release oral (DRO) compositions. The DRO compositions pass through the stomach substantially unaltered and deliver the active ingredient to the gastrointestinal tract, typically the ileum, duodenum and / or jejunum.

[0156] The compositions according to the invention may have an enteric coating. Enteric coatings protect the active ingredients in a composition from attack and degradation in the stomach, but dissolve and release the contents of the dosage form within the intestines, usually due to the change in pH. Suitable enteric coatings are well known in the art. The optimal coating for any particular formulation depends on the exact intended use, and coatings may be tailored to release the active ingredient in a particular region of the intestines, or at a particular time following ingestion. Such a formulation may if desired contain one or more intermediate layers between the active ingredient and the outer enteric coating. In this case, it is possible for a composition of the invention to release a portion of its contents at one particular region of the intestine, and a further portion of its contents in a second region of the intestine, such as the ileum, duodenum and / or jejunum. Preferably, the composition of the present invention is in a solid or semi-solid form which comprises an enteric coating adapted to release the transporter protein domain / antibody binding moiety and antibody in the ileum, duodenum and / or jejunum. Useful enteric coatings are those which remain intact in the low pH environment of the stomach, but readily dissolve when the optimum pH for dissolution is reached. This can vary between pH 3 to 7.5, preferably 5 to 7, depending on the chemical composition of the coating. The thickness of the coating required will depend on the solubility of the coating and the intended site to be treated. Typically, the coating has a thickness of 25 to 200pm, especially 75 to 150 pm.

[0157] The composition of the invention is adapted for release of the active ingredient to the part of the lower gastrointestinal tract where the transcytosis domain transports the antibody through the intestinal lining into the blood stream to provide systemic delivery. Typically, the enteric coating should dissolve in the pH of the jejunum (about pH 5.5), ileum (about pH 6) and / or duodenum (about pH 6) so that the majority of the transporter protein domain / antibody binding moiety and antibody are released at the desired site. Medical applications

[0158] The present invention provides a pharmaceutical composition according to the invention for use in medicine. It also provides a method of treating or preventing a disease or condition in a subject, especially a human subject, which comprises administering to the subject via the lower gastro-intestinal tract, especially the duodenum, ileum and / or the jejunum a pharmaceutical composition which comprises the transporter protein domain / antibody binding moiety and antibody. Preferably, the compositions are adapted for administration via the oral route. The pharmaceutical formulation has application as a portal for entry of an antibody into the systemic circulation by absorption from the gastro-intestinal tract, especially the duodenum, ileum and / or the jejunum, and hence finds utility in the treatment of a wide range of diseases and conditions.

[0159] The pharmaceutical composition may comprise any known therapeutic antibody. This includes including all therapeutic antibodies that contain an Fc domain which have received regulatory approval, for example by the FDA or EMA. Antibody treatments are already commercially available for a number of conditions. In a preferred embodiment the pharmaceutical composition can be used to treat an autoimmune disease, cancer, Alzheimer’s disease, cardiovascular disease, Gram-negative sepsis, ankylosing spondylitis, migraine, haemophilia, Multiple sclerosis, Cold agglutinin disease, Muckle-Wells syndrome, Sickle cell disease, Osteoporosis, Castleman disease, Macular degeneration, Neuromyelitis optica, Thyroid eye disease, Ebola infection, HIV infection, Anthrax infection, treating or preventing Sars- COV2 infection or related disease such as COVID-19, or, any other condition or disease known to be treatable by Fc-containing protein therapies

[0160] Examples of therapeutic antibodies include Zolbetuximab, Odronextamab, Camrelizumab, Serplulimab, Sugemalimab, Pozelimab, Concizumab, Elranatamab, Cosibelimab, Rozanolixizumab, Talquetamab, Epcoritamab, Lebrikizumab, Trastuzumab duocarmazine, Glofitamab, Mirikizumab, Donanemab, Lecanemab, Tislelizumab, Penpulimab, Sintilimab, Toripalimab, Omburtamab, Retifanlimab, Narsoplimab, Teplizumab, Ublituximab, brazikumab, Mirvetuximab soravtansine, Nirsevimab, Tremelimumab, Spesolimab, Teclistamab, Mosunetuzumab, Tixagevimab, cilgavimab, Relatlimab, Tebentafusp, Faricimab, Sutimlimab, Sotrovimab, Regdanvimab, Casirivimab + imdevimab, Tezepelumab, Tisotumab vedotin, Amivantamab, Anifrolumab, Loncastuximab tesirine, Bimekizumab, Tralokinumab, Evinacumab, Aducanumab, Dostarlimab, Ansuvimab, Margetuximab, Naxitamab, Atoltivimab, Maftivimab, Odesivimab-ebgn, Belantamab mafodotin, Tafasitamab, Satralizumab, Inebilizumab, Sacituzumab govitecan, Teprotumumab, Isatuximab, Eptinezumab, [fam]-trastuzumab deruxtecan, Enfortumab vedotin, Crizanlizumab, Brolucizumab, Polatuzumab vedotin, Risankizumab, Romosozumab, Caplacizumab, Ravulizumab, Emapalumab, Cemiplimab, Fremanezumab, Moxetumomab pasudotox, Galcanezumab, Lanadelumab, Mogamulizumab, Erenumab, Tildrakizumab, Ibalizumab, Burosumab, Durvalumab, Emicizumab, Benralizumab, Ocrelizumab, Guselkumab, Inotuzumab, ozogamicin, Sarilumab, Dupilumab, Avelumab, Brodalumab, Atezolizumab, Bezlotoxumab, Olaratumab, Reslizumab, Obiltoxaximab, Ixekizumab, Daratumumab, Elotuzumab, Necitumumab, Idarucizumab, Alirocumab, Mepolizumab, Evolocumab, Dinutuximab, Secukinumab, Nivolumab, Blinatumomab, Pembrolizumab, Ramucirumab, Vedolizumab, Siltuximab, Obinutuzumab, Ado-trastuzumab emtansine, Raxibacumab, Pertuzumab, Brentuximab vedotin, Belimumab, Ipilimumab, Denosumab, Tocilizumab, Ofatumumab, Canakinumab, Golimumab, Ustekinumab, Certolizumab pegol, Catumaxomab, Eculizumab, Ranibizumab, Panitumumab, Natalizumab, Bevacizumab, Cetuximab, Efalizumab, Omalizumab, Tositumomab-1131, Ibritumomab tiuxetan, Adalimumab, Ozoralizumab, Alemtuzumab, Gemtuzumab, ozogamicin, Trastuzumab, Infliximab, Palivizumab, Basiliximab, Daclizumab, Rituximab, Abciximab, Edrecolomab, Nebacumab, Nimotuzumab, Itolizumab (Alzumab), RabiMabs (Twinrab) TM, Rmab (RabiShield), Sintilimab (Tyvyt), Toripalimab (Tuoyi), Camrelizumab, Tislelizumab, Disitamab vedotin (Aidixi), Penpulimab, Zimberelimab, Netakimab, Prolgolimab (Forteca), Olokizumab (Artlegia), Levilimab (llsira) , Cetuximab saratolacan sodium, Pabinafusp alfa (IZCARO), Sotrovimab, Muromonab-CD3, dupilumab (Dupixent), alirocumab, Telisotuzumab vedotin, Nipocalimab, Bentracimab, Datopotamab deruxtecan, Zenocutuzumab , Nemolizumab, Zanidatamab, Linvoseltamab, Axatilimab, Patritumab deruxtecan, Tarlatamab, Marstacimab, Garadacimab, Vilobelimab, Crovalimab and evolocumab.

[0161] Autoimmune conditions include rheumatoid arthritis, psoriasis, gout, recurrent pericarditis, hiradentis suppurativa, polyarticular juvenile idiopathic arthritis, giant cell arteritis, neuromyelitis optical spectrum disorder, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease including Crohn's disease and ulcerative colitis, and allergic asthma. The pharmaceutical composition may also be used to prevent acute rejection of transplanted organs such as kidneys. Suitable monoclonal antibodies for treating autoimmune conditions include infliximab, adalimumab, ozoralizumab, golimumab, certolizumab, canakinumab, sirukumab, olokizumab, clazakizumab, siltuximab, tocilizumab, sarilumab, dupilumab (Dupixent), vobarilizumab, ixekizumab, secukinumab, brodalumab, bimekizumab, ustekinumab, guselkumab, risankizumab, tildrakizumab, mirikizumab, ianalumab, belimumab, inebilizumab, Netakimab, Olokizumab, Ixekizumab, Vedolizumab, Spesolimab, Itolizumab, Efalizumab, brazikumab, Benralizumab, Mepolizumab, Tralokinumab, Tezepelumab, Lebrikizumab, iscalimab, ublituximab , ofatumumab, ocrelizumab, rituximab, Axatilimab and anifrolumab.

[0162] Cancers that can be treated with monoclonal antibodies include chronic lymphocytic leukaemia, non-Hodgkin lymphoma, large B cell lymphoma, .multiple myeloma, adenocarcinoma, endometrial cancer, ovarian cancer, cervical cancer, neuroblastoma, breast cancer, stomach cancer, skin cancer, melanoma, liver cancer, urothelial cancer, bladder cancer, small cell lung cancer, non-small cell lung cancer, bowel cancer and head and neck cancer. Suitable monoclonal antibodies for treating cancer include rituximab (Mabthera), bevacizumab(Avastin), Dostarlimab, Moxetumomab, Margetuximab, Naxitamab, Sacituzumab govitecan, Enfortumab vedotin, Durvalumab, Atezolizumab, Avelumab, Necitumumab, Nivolumab, Ramucirumab, Pembrolizumab, Pertuzumab, Edrecolomab, Disitamab vedotin (Aidixi), Prolgolimab, Penpulimab, Zimberelimab, racotumomab, Mirvetuximab, Serplulimab, Sugemalimab, Sintilimab, Tremelimumab, Relatlimab, Loncastuximab, Tisotumab, Mosunetuzumab, Teclistamab, Retifanlimab, Omburtamab, Toripalimab, Glofitamab, Epcoritamab, Talquetamab, Cosibelimab, Elranatamab, Camrelizumab, Odronextamab, Zolbetuximab, panitumumab (Vectibix), blinatumomab (Blincyto), brentuximab, cetuximab (Erbitux), Telisotuzumab vedotin, Datopotamab deruxtecan, Zenocutuzumab, Zanidatamab, Linvoseltamab, Patritumab deruxtecan, Tarlatamab, and trastuzumab (herceptin).

[0163] Suitable monoclonal antibodies for treating Alzheimer’s disease include aducanumab, donanemab and lecanemab.

[0164] Suitable monoclonal antibodies for treating or reducing the risk of cardiovascular disease include anti-PCSK9 monoclonal antibodies, for example alirocumab and evolocumab.

[0165] Suitable monoclonal antibodies for treating or preventing COVID 19 include Levilimab, Sotrovimab, Regdanvimab, Tixagevimab, cilgavimab, Casirivimab, Vilobelimab and imdevimab.

[0166] The following non-limiting Examples illustrate the invention, and refer to following figures:

[0167] Figure 1 IIITACC promotes IgG transmission across pig small intestine tissue.

[0168] Figure 1A. Experimental set-up of the llssing Chamber experiment. Figure 1B. SDS-PAGE of the protein preps that were used in the experiment. The same relative amounts of each prep on gel is also used in the llssing Chamber experiment.

[0169] Figure 1C. Evaluation of FITC-IgG presence in the serosal chamber at 1 hr and 2 hrs after administering in the mucosal chamber.

[0170] Figure 2. lUTACC’s ability to transport IgG across Gl tissue depends on its IgG binding site.

[0171] Figure 2A. Protein preps were made for wildtype IIITACC and a mutant IUTACC that lacks its engineered IgG binding site. Relative protein amounts reflect the relative protein amounts used in the llssing Chamber experiment.

[0172] Figure 2B. Evaluation of FITC-IgG presence in the serosal chamber at 1 hr and 2 hrs after administering in the mucosal chamber. The graph shows combined data from 3 experiments done in duplicate. In this and subsequent experiments, a variation in procedure was used that led to higher transport values for FITC-IgG (see Materials and Methods: rather than adding the various proteins directly to the 1 ,5ml KBR buffer in the Ussing chamber to start the experiment, the proteins were pre-incubated in a smaller (300pl) volume at 37°C for 15 mins before adding them to the Ussing Chamber to promote reaching binding equilibrium prior to the start of the Ussing experiment).

[0173] Figure 3. IUTACC requires active vesicular transport to promote transport of FITC-IgG across the Gl tissue. Chlorpromazine was dissolved directly into the KBR buffer of the mucosal Ussing chamber compartment at 6 pg / ml. Tissue was incubated for 15 mins, prior to addition of the FITC-IgG and IUTACC proteins. FITC-IgG levels were measured in the serosal chamber after 1 and 2 hrs of incubation.

[0174] Figure 4. IUTACC works only with small intestine - not colon - tissue.

[0175] Figure 4A. The Ussing Chamber experiment was performed using pig colon tissue, rather than small intestine tissue. One of the values (encircled) was considered to be an outlier, as determined by the Grubbs’ test / ESD Extreme Studentized Deviate method in relation to the other values in the data set.

[0176] Figure 4B. Plotting of the data in Figure 3A, excluding the outlier value. The graph shows combined data from 2 experiments done in triplicate. A protocol variation was used to maximise IUTACC transport activity as seen for small intestine tissue, e.g. in Figure 2 (see Materials and Methods).

[0177] Figure 5. IUTACC works with fresh (non-frozen) duodenal Gl tissue. Fresh Gl tract tissue, dissected from the duodenum at 30 cm posterior to the stomach, was used to set up an llssing Chamber experiment.

[0178] Figure 6. In-vivo evaluation of transcytosis activity.

[0179] Figure 6A shows the SDS-PAGE analysis (reducing conditions: +DTT) of the formulations used for the in-vivo study.

[0180] Figure 6B provides a schematic depiction of the design of the in-vivo experiment

[0181] Figure 6C shows the antibody levels detected in blood at various time points during once- daily intraduodenal administration of the antibody.

[0182] Figure 7. The use of alpha-amylase and pancreatic lipase-related protein as transcytosis carriers.

[0183] Figure 7A shows the SDS-PAGE analysis under reducing and non-reducing conditions of a- amylase 2A construct,

[0184] Figure 7B shows the ability of the a- amylase 2A construct to transcytose the antibody Infliximab using the llssing chamber.

[0185] Figure 7C shows SDS-PAGE analysis under reducing conditions of pancreatic lipase related protein 2 construct (UTAC-20), and the ability of this construct to transcytose the antibody Infliximab using the Ussing chamber.

[0186] Materials and Background

[0187] A fusion protein comprising the sequence of human pancreatic triacylglycerol lipase (PNLIP) linked to the Fell I antibody peptide was designed and designated “IUTACC” (‘ / ntract Universal Transcytosis Active Cargo Carrier’).

[0188] Constructs:

[0189] IUTACC constructs were cloned in the bacterial expression vector pET-21a(+). The amino acid sequence of the open reading frame is shown in Table 1.

[0190] IIITACC expression, purification and refolding:

[0191] IIITACC bacterial expression in E. coir. The pET21a(+) bacterial expression vectors containing the IIITACC open reading frames (Table 1) were freshly transformed into BL21 / DE3 chemically competent bacterial cells and plated on LB-AMP (100pg / ml) plates. Colonies were picked and used to inoculate liquid LB-AMP (100pg / ml) cultures. These cultures were grown at 37°C in baffled flasks, subject to vigorous shaking at 275 rpm. At optical density ODeoo = 0.6, IPTG was added to 1 mM final concentration, and induction of protein expression was achieved by ongoing incubation at 37°C for another 4 hours with continued vigorous shaking at 275 rpm. Cells were spun down at 5000 rpm for 25 mins in a MegaFuge16R (TX-400 rotor) Thermo Scientific / Heraeus centrifuge, washed 1x by resuspension in 30ml of PBS per 500ml-culture pellet, spun down again as above, and stored as dry pellets at -80°C until further processing.

[0192] Purification of IIITACC from bacterial inclusion bodies: IPTG-induced cell pellets (from 500ml cultures) were thawed in a water bath at 37°C, resuspended in B-PER (78243, Thermo Scientific) bacterial protein extraction reagent supplemented with 1.5pl benzonase (E1014, Millipore, 250U / ul), and rotated for 30 mins at room temperature to achieve cell lysis. The cell lysates were centrifuged at 5000 rpm for 25 mins in a MegaFuge16R (TX-400 rotor, Thermo Scientific / Heraeus) centrifuge, and pellets were washed 4x in 30 ml of 20mM Tris- HCI [pH8], 150 mM NaCI, 1 mM ethylenediaminetetraacetic acid (EDTA) [pH8], 0.5% Triton X-100. In the first of these four washes, 1 l of benzonase (E1014, Millipore, 250U / ul) was added to the 30ml wash volume and, after resuspension of the pellet, the mixture was rotated at room temperature (around 20°C) for 30 mins to promote washing and degradation of remaining DNA / RNA species. After washing, the pellets were resuspended and dissolved in 30 ml of 20 mM Tris-HCI [pH8], 150 mM NaCI, 1 mM EDTA [pH8], 8M urea, 10 mM dithiothreitol (DTT). To promote solubilization of the inclusion bodies, the solution was placed for 30 mins in a water bath at 37°C. The solution was clarified by centrifugation (6 mins at 5000 rpm in a MegaFuge16R (TX-400 rotor) Thermo Scientific / Heraeus centrifuge). Imidazole was added to the supernatant, to a final concentration of 5 mM. Next, 1 ml slurry of Ni++NTA affinity resin beads (H5037, His-Select HF Nickel Affinity Gel, Millipore) was added to the protein-urea solution, followed by rotation at room temperature for 1 hr. Resin beads were harvested by centrifugation (6 mins at 4000 rpm in above-mentioned centrifuge), and washed 4x in urea buffer. Next, resin-bound IIITACC protein was eluted using 20 mM Tris-HCI [pH8], 150 mM NaCI, 1 mM EDTA [pH8], 8 M urea, 10 mM DTT, 250 mM imidazole.

[0193] Refolding of urea-denatured IIITACC protein: In order to refold the IIITACC constructs, to a large extent the protocol that was described for refolding human pancreatic triacylglycerol lipase was followed (Kawaguchi et al., 2018). The affinity-purified, eluted IIITACC protein - still denatured in 8M urea - was placed in a dialysis cassette (Slide-a-Lyzer, MWCO 3500 Da, Thermo Scientific) and refolded using a sequence of successive dialysis incubations with diminishing urea concentrations, in order to slowly remove the urea and promote correct refolding. The refolding buffer for each dialysis incubation consisted of 10 mM Tris-HCI [pH8], 150 mM NaCI, 10% glycerol, 2.5 mM CaCh, 1 mM L-cysteine, 0.1 mM cystine, 400 mM L-arginine, and - in addition - was supplemented with either 6M, 4M, 2M, 1M or no urea. Each dialysis incubation lasted for a minimum of two hours up to overnight. After the final dialysis step, the IIITACC preparation was retrieved from the dialysis cassette, clarified by centrifugation, aliquoted, and frozen at -80°C.

[0194] Evaluation of lUTACC effect on IgG transcytosis in vitro using an Ussing Chamber set-up:

[0195] Remainder pig small intestine and large intestine tissue was obtained according to ethical guidelines and approval from the ethics review board, from control pigs sacrificed for unrelated ongoing research projects (Royal Veterinary College, London). Tissue was couriered on wet ice immediately after sacrifice, dissected in the laboratory, and frozen in aliquots as soon as possible at -80°C. For each experiment, a new aliquot was taken from the -80 °C freezer. The Ussing Chamber experiments were performed using a Warner Instruments 6-chamber vertical Navicyte system (66-0032), allowing for the tissue to be kept at 37°C during the experiment and continually to be perfused with an 90% 02 / 10% CO2 carbogen gas mixture. The chamber aperture of the chambers was 0.29 cm2. Experiments were performed in Krebs-Bicarbonate Ringer (KBR) buffer (PBS, 2 mM CaCh, 1 mM MgCh, 10 mM D-glucose). Set up was standard according to manufacturer’s protocol, and performed essentially as described elsewhere (Thomson et al., 2019). To measure IgG transcytosis, FITC-IgG was purchased from Sigma / Merck (F9636) and subjected to gel filtration and IgG fractionation on a Superdex200 column (GE Healthcare) in order to remove all free, non-conjugated FITC. 20pg / ml FITC-IgG was added to the apical chamber, by itself or with 20pg / ml IIITACC protein. Samples were taken from the basal (serosal) side, and analyzed for the presence of FITC fluorescence signal as an indication of IgG transmission across the mounted tissue sample. Quantification of signal was done using standard curve measurements. In the experiment of Figure 1 , the various proteins were added directly to the 1 ,5ml KBR buffer in the llssing chamber to start the experiment. A protocol variation was introduced for the experiments of Figure 2 onwards: the FITC-IgG and IIITACC proteins were pre-incubated separately in a smaller (300pl) volume at 37°C for 15 mins prior to adding them to the llssing Chamber, in order to promote reaching full binding equilibrium and to have the maximum amount of FITC-IgG pre-bound to the IUTACC protein at the start of the Ussing experiment. This protocol variation led to higher values of FITC-IgG reaching the serosal chamber.

[0196] IUTACC promotes IgG transmission across pig small intestine tissue.

[0197] IUTACC protein was expressed, purified and refolded as described above, and tested in vitro for ability to enhance IgG transmission across small intestine tissue. Pig small intestine, from a gut-healthy animal, was used in an Ussing Chamber set-up. FITC-labeled (and gelfiltration fractionated and purified) IgG was used as a biomarker readout.

[0198] The experimental set-up is depicted in Figure 1A. A small fragment of pig small intestine is clamped over the aperture between the apical / mucosal and basal / serosal chambers of the Ussing Chamber set-up, with the luminal side of the tissue facing the apical / mucosal chamber. Tissue viability is maintained as best as possible with temperature control at 37°C, the presence of glucose and salts in the KBR buffer, and continuous perfusion of carbogen gas bubbling through the KBR buffer in both compartments of the Ussing chambers. At the start of the experiment 20pg / ml FITC-IgG is added to the 1 ,5ml KBR buffer in the mucosal chamber, either alone or along with an equal mass of IUTACC protein. Samples taken from the serosal chamber at 1 hrs and 2 hrs were analyzed for penetration of FITC-IgG through the small intestine tissue. Figure 1B shows the protein preparations that were used. The gel loading reflects the relative amounts of the two preparations used in the experiment. FITC- IgG, as a disulfide-stabilized antibody of 150 kDa size, falls apart under reducing gel conditions into its heavy and light chain sub-components. IUTACC has a size of ~ 55 kDa. This preparation also contains an apparent disulfide stabilized IIITACC dimer species. Figure 1C shows that adding IIITACC alongside FITC-IgG to the mucosal chamber increases the amount of FITC-IgG that is found on the serosal side after 2 hrs of incubation. lUTACC’s ability to transport IgG across Gl tissue depends on its IgG binding site.

[0199] In order to understand how IIITACC facilitates IgG transport, a mutant IIITACC was engineered which lacks its engineered IgG binding site (see Materials and Methods). Equivalent protein preparations were generated for both the IIITACC construct and the IgG- binding impaired mutant IIITACC (Figure 2A). Comparing the effect of wildtype and mutant IIITACC protein on FITC-IgG transport from mucosal to serosal chamber revealed that the mutant which lacked the engineered IgG binding site had severely reduced ability to transport FITC-IgG across the intestinal tissue. Thus, lUTACC’s molecular interaction with IgG is necessary for its ability to promote IgG transport.

[0200] IUTACC requires active vesicular transport to promote transport of FITC-IgG across the Gl tissue. lUTACC’s ability to enhance FITC-IgG transport at very low concentrations (roughly equimolar to the FITC-IgG biomarker) and in a manner that depends on its IgG binding site suggests that IUTACC acts via an active transport mechanism. To test this, inhibitors of vesicular intracellular transport were tested. The first step in retrograde vesicular transport is endocytosis. A cell has various ways of endocytosing cargo, either via various dynamin- dependent and dynamin-independent pathway or caveolae. Many small-molecule inhibitors of endocytosis exist, but their mechanism of action is often less clear as these molecules often impact various levels of transport. A well-studied inhibitor of endocytosis is chlorpromazine, which is believed to inhibit dynamin I GTPase function and thus interferes with the various dynamin-dependent endocytosis pathways. Chlorpromazine has also been reported to partially inhibit retrograde transport of leptin in a Caco-2 / 15 epithelial barrier tissue culture monolayer system. A short pre-incubation of the Gl tissue with chlorpromazine in the Ussing set-up partially interfered with lUTACC’s ability to transport FITC-IgG to the serosal chamber side (Figure 3). Thus, the data indicate that IUTACC uses active endocytosis and dynamin-dependent pathways to transport IgG across the Gl tissue.

[0201] Tissue specificity: IUTACC works only with small intestine - not colon - tissue.

[0202] In order to further understand the specificity of lUTACC’s action, colon tissue rather than small intestine tissue was tested (Figure 4A, 4B). IUTACC did not promote FITC-IgG transport with this tissue. This finding raises the possibility that the mechanism via which IUTACC works involves a receptor or transport component with tissue-restricted expression. IUTACC works with fresh (non-frozen) duodenal Gl tissue in an Ussing Chamber setup. lUTACC’s ability to transfer FITC-IgG across fresh, i.e. non-frozen, tissue from the pig duodenal tract was tested, with the experiment being performed shortly after the animal’s sacrifice. Figure 5 shows that IUTACC is able to transport FITC-IgG across fresh tissue, and its action is thus not dependent on the presence of microscopic damage to the tissue as a result of a freeze-thaw cycle. This data is as close to an in vivo experiment as possible, and also validates the use of the Ussing Chamber setup with frozen pig tissue as a screening assay for IUTACC development research.

[0203] In-vivo evaluation of the ability of bispecific molecules to transport antibodies from the mouse Gl lumen across the Gl epithelial barrier into the blood stream.

[0204] The ability of two bispecific fusion proteins to transport the antibody Infliximab was tested in vivo. The bispecific fusion proteins had the following sequences:

[0205] UTAC-7

[0206] MKEVCYERLGCFSDDSPWSGITERPLHILPWSPKDVNTRFLLYTNENPNNFQEVAADSSSIS GSNFKTNRKTRFIIHGFIDKGEENWLANVCKNLFKVESVNCICVDWKGGSRTGYTQASQNI RIVGAEVAYFVEFLQSAFGYSPSNVHVIGHSLGAHAAGEAGRRTNGTIGRITGLDPAEPCFQ GTPELVRLDPSDAKFVDVIHTDGAPIVPNLGFGMSQVVGHLDFFPNGGVEMPGCKKNILSQI VDIDGIWEGTRDFAACNHLRSYKYYTDSIVNPDGFAGFPCASYNVFTANKCFPCPSGGCPQ MGHYADRYPGKTNDVGQKFYLDTGDASNFARWRYKVSVTLSGKKVTGHILVSLFGNKGNS KQYEIFKGTLKPDSTHSNEFDSDVDVGDLQMVKFIWYNNVINPTLPRVGASKIIVETNVGKQ

[0207] FNFCSPETVREEVLLTLTPCSAWSHPQFEKGGGSHH H H HHGGGGSH VRGI VC* (SEQ. I D NO: 43)

[0208] And

[0209] UTAC-15

[0210] MKEVCYERLGCFSDDSPWSGITERPLHILPWSPKDVNTRFLLYTNENPNNFQEVAADSSSIS GSNFKTNRKTRFIIHGFIDKGEENWLANVCKNLFKVESVNCICVDWKGGSRTGYTQASQNI RIVGAEVAYFVEFLQSAFGYSPSNVHVIGHSLGAHAAGEAGRRTNGTIGRITGLDPAEPCFQ GTPELVRLDPSDAKFVDVIHTDGAPIVPNLGFGMSQVVGHLDFFPNGGVEMPGCKKNILSQI VDIDGIWEGTRDFAACNHLRSYKYYTDSIVNPDGFAGFPCASYNVFTANKCFPCPSGGCPQ MGHYADRYPGKTNDVGQKFYLDTGDASNFARWRYKVSVTLSGKKVTGHILVSLFGNKGNS KQYEIFKGTLKPDSTHSNEFDSDVDVGDLQMVKFIWYNNVINPTLPRVGASKIIVETNVGKQ FNFCSPETVREEVLLTLTPCSAWSH PQFEKGGGSH H H H H HGGGGSHI4 / RG WCGGGGS GGGGSGGGGSHWRGWVG* (SEQ.ID NO: 44)

[0211] In the amino acid sequences of the bispecific fusion polypeptide constructs depicted above: the underlined sequence is the that of the human pancreatic triacylglycerol lipase (PNLIP) gene minus its N-terminal 16 amino acid leader peptide. This sequence is fused to a linker containing (in bold) the StrepTactinll and His6 affinity tags respectively, followed (underlined and in italics) by either one or two copies of the immunoglobulin Fc-binding peptide(s).

[0212] Infliximab antibody and aprotinin (a component of Intract’s Soteria protease inhibition formulation) were co-formulated with equal amounts of purified bispecific fusion protein (UTAC-7 or UTAC-15), in such a way as to permit 20mg / kg dosing in mice (each dose contains 0.5 mg Infliximab and 0.5 mg aprotinin, with or without 0.5 mg protein (UTAC-7 or UTAC-15) per 25 gr mouse, in a final dosing volume of 100ul). All protein components were stable when co-formulated at 5mg / ml concentrations, as judged by clear solutions, ability to undergo freeze-thaw cycles, and no observable degradation either by size exclusion chromatography or SDS-PAGE analysis. Figure 6 panel A shows the SDS-PAGE analysis (reducing conditions: +DTT) of the formulations used for this in-vivo study.

[0213] Figure 6B provides a schematic depiction of the design of the in-vivo experiment: 12 mice underwent surgery for duodenal cannulation, followed by 10 days of recovery. On day 0, once-daily (QD) intraduodenal dosing for six consecutive days started for the three experimental arms (each arm consisting of 4 mice). The mice in these arms received either infliximab / aprotinin alone, infliximab / aprotinin+UTAC-7 or lnfliximab / aprotinin+UTAC-15. Blood samples were taken from each mouse on Day 0 (1 hr after dosing), Day 2 (2 hrs after dosing), Day 4 (2 hrs after dosing) and Day 5 (5 hrs after dosing). Blood samples were then analysed for the presence of infliximab using ELISA. The ELISA method that was used has been described (Awad et al., ‘3D printed infliximab suppositories for rectal biologic delivery’, International Journal of Pharmaceutics: X 5 (2023) 100176, https: / / doi.org / 10.1016 / jJjpx.2023.100176).

[0214] As shown in Figure 6C, Infliximab levels increase and build-up in the blood following QD dosing for 6 consecutive days. UTAC-7 and UTAC-15 both promote very rapid transport of Infliximab to the blood as compared to infliximab alone (see first time point: Day 0, 1 hr after first dosing). In addition, when building up infliximab levels in the blood by sustained QD dosing (and taking advantage of the long half-life of antibodies), co-formulation with UTAC-7 (and to a lesser effect UTAC-15) also appears beneficial over the longer term with a trend of higher steady-state levels of infliximab in the blood compared to control infliximab alone. UTAC-7 and UTAC-15 contain the same IgFc binding peptide, but differ in format design (UTAC-7 contains one copy of the HWRGWV (SEQ. ID No: 23) antibody-binding peptide, whereas UTAC-15 carries has two copies of this peptide in tandem; see SEQ. ID No: 43 and SEQ. ID No: 44). Three of the four blood samples for the infliximab-alone control arm at the Day 0, 1 hr time point are below detection limit (orange dots).

[0215] Alternative transporter constructs that contain either human alpha amylase or pancreatic triacylglycerol lipase-related proteins as the transcytosis-driving components

[0216] Constructs were generated and tested that contain either human alpha amylase or pancreatic triacylglycerol lipase-related proteins as the transcytosis-driving components.

[0217] A fusion construct containing the (full-length) human pancreatic alpha amylase gene AMY2A linked to an IgFc binding peptide was produced. The amino acid sequence of the construct is provided below:

[0218] Human alpha-amylase (AMY2A) transporter construct (for mammalian expression):

[0219] MKFFLLLFT / GFCI / I / AQYSPNTQQGRTSIVHLFEWRVWDIALECERYLAPKGFGGVQVSPPN ENVAIYNPFRPWWERYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMCGNA VSAGTSSTCGSYFNPGSRDFPAVPYSGWDFNDGKCKTGSGDIENYNDATQVRDCRLTGLL DLALEKDYVRSKIAEYMNHLIDIGVAGFRLDASKHMWPGDIKAILDKLHNLNSNWFPAGSKP FIYQEVIDLGGEPIKSSDYFGNGRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFVP SDRALVFVDNHDNQRGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRQ FQNGNDVNDWVGPPNNNGVIKEVTINPDTTCGNDWVCEHRWRQIRNMVIFRNVVDGQPF TNWYDNGSNQVAFGRGNRGFIVFNNDDWSFSLTLQTGLPAGTYCDVISGDKINGNCTGIKI YVSDDGKAHFSISNSAEDPFIAIHAESKLGGGSHH H H HHGGGGSDCAWHLGELVWCT* (SEQ ID.NO: 45)

[0220] In the amino acid sequences above: the underlined sequence is the that of the human pancreatic alpha amylase (AMY2A) gene with leader peptide in italics. This sequence is fused to a linker containing (in bold) His6 affinity tag, followed (underlined and in italics) by the immunoglobulin Fc-binding peptide.

[0221] The fusion protein was expressed in HEK293 mammalian cells, purified, and analyzed on SDS-PAGE under reducing and non-reducing conditions (Figure 7A).

[0222] Using fresh rat small-intestine tissue in an Ussing set-up, infliximab was added to the apical chambers either alone, pre-incubated with the alpha-amylase (AMY2A) transporter construct. The level of infliximab reaching the basal compartment was assessed with an ELISA readout using TNFa-captured material. The ELISA method that was used has been described (Awad et al., ‘3D printed infliximab suppositories for rectal biologic delivery’, International Journal of Pharmaceutics: https: / / doi.Org / 10.1016 / i.iipx.2023.100176).)

[0223] As shown in Figure 7B the construct containing alpha amylase enhanced the transport of infliximab across the rat Gl epithelium, indicating that alpha amylases do work in as a part of a transporter protein in a transcytosis setting.

[0224] A further transporter fusion construct containing the (full-length) human pancreatic lipase related protein 2 (PNLIPRP2) gene, fused to an IgFc binding peptide was generated. The amino acid sequence of the construct is provided below:

[0225] UTAC-20 construct (lntract-20), containing the full-length human PNLIPRP2 gene sequence minus leader sequence (for bacterial expression):

[0226] MKEVCYGQLGCFSDEKPWAGTLQRPVKLLPWSPEDIDTRFLLYTNENPNNFQLITGTEPDTI EASNFQLDRKTRFIIHGFLDKAEDSWPSDMCKKMFEVEKVNCICVDWRHGSRAMYTQAVQ NIRVVGAETAFLIQALSTQLGYSLEDVHVIGHSLGAHTAAEAGRRLGGRVGRITGLDPAGPC FQDEPEEVRLDPSDAVFVDVIHTDSSPIVPSLGFGMSQKVGHLDFFPNGGKEMPGCKKNVL STITDIDGIWEGIGGFVSCNHLRSFEYYSSSVLNPDGFLGYPCASYDEFQESKCFPCPAEGC PKMGHYADQFKGKTSAVEQTFFLNTGESGNFTSWRYKISVTLSGKEKVNGYIRIALYGSNE NSKQYEIFKGSLKPDASHTCAIDVDFNVGKIQKVKFLWNKRGINLSEPKLGASQITVQSGED

[0227] GTEYNFCSSDTVEENVLQSLYPCSAWSH PQFEKGGGSH H H H HHGGGGSH VRGI VC* (SEQ. ID NO:46)

[0228] In the amino acid sequences above: the underlined sequence is the that of the human PNLIPRP2 gene sequence, minus the leader sequence (PNLIPRP218-469). This sequence is fused to a linker containing (in bold) streptactinll affinity tag and His6 affinity tag , followed (underlined and in italics) by the immunoglobulin Fc-binding peptide.

[0229] This UTAC-20 construct was produced, purified and refolded in E.coli, in the same way as was done for IUTACC fusion proteins that carry the PNLIP pancreatic lipase gene. The preparation was analyzed on SDS-PAGE under reducing conditions (Figure 7C left panel).

[0230] The PNLIPRP2 construct was tested in an Ussing setting, using pig small intestine Gl tissue, with FITC-labelled human IgG as readout. Similar to PNLIP constructs described above, PNLIPRP2 also can increase transcytosis of IgG (Figure 7C right panel). Bibliography

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Claims

CLAIMS1. A composition comprising a non- bacterial transporter protein domain capable of transcytosing the intestinal wall linked to an antibody binding moiety.

2. A composition of claim 1 wherein the transporter protein domain comprises an inactivated form of a protein capable of transcytosing the intestinal wall.

3. A composition of any preceding claim wherein the transporter protein domain is derived from a protein selected from pancreatic lipase, bile salt dependent lipase (BSDL) / carboxyl ester lipase (CEL) / cholesterol esterase and a-amylase or a homologue thereof, preferably pancreatic lipase.

4. A composition of any preceding claim wherein the antibody binding moiety comprises a Fc-binding domain or an antibody light chain (LC) binding domain or a homolog or derivative thereof.

5. A composition of any preceding claim wherein the antibody binding moiety comprises an antibody binding peptide.

6. A composition of claim 5 wherein the antibody binding peptide is selected from Protein A, Protein G, Protein L, Protein Z (derived from B-domain of SpA), Protein LG, Protein LA, Protein AG ,SpA, PAM (peptide with the sequence (RTY)4K2KG) (SEQ. ID No:18), Fc-lll, FcBP-2, FC-III-4C, FcRM, or a peptide with a sequence selected from TWKTSRISIF (SEQ. ID No:19), FGRLVSSIRY (SEQ. ID No:20), EPIHRDTLTALL (SEQ. ID No:21), APAR (SEQ. ID No:22), HWRGWV (SEQ. ID No:23), HWRGWVC (SEQ. ID No 40), HYFKFD (SEQ. ID No:24), HFRRHL (SEQ. ID No:25), HWCitGWV (SEQ. ID No:26), DAAG (SEQ. ID NO:27),D2AAG (SEQ. ID No:28), NKFRGKYK (SEQ. ID No:29), NARKFYKG (SEQ. ID No:30), FYWHCLDE (SEQ. ID No:31), FYCHWALE (SEQ. ID No:32), FYCHTIDE (SEQ. ID No:33), RRGW (SEQ. ID No:34), KHRFNKD (SEQ. ID No:35), cyclo(Na-Ac)S(A)- RWHYFK-Lact-E (SEQ. ID No:37), cyclo(Na-Ac)Dap(A)-RWHYFK-Lact-E (SEQ. ID No:38) and cyclo [Link-M-WFRHYK] (SEQ. ID No:39) or homologs thereof, preferably HWRGWV (SEQ. ID No 23) and / or HWRGWVC (SEQ. ID No 40). .

7. A composition of claim 5 or claim 6 wherein the antibody binding peptide comprises the sequence AWHLGELVW (SEQ. ID No. 12.)8. A composition of any one of claims 5 to 7 wherein the antibody binding peptide comprises the sequence DCAWHLGELVWCT (SEQ. ID No. 13.)9. A composition of any preceding claim wherein the antibody binding moiety is linked via a covalent bond to the transporter protein domain.

10. A composition of any one of claims 5 to 9 wherein the transporter protein domain and the antibody binding peptide form a fusion protein.

11. A composition of any preceding claim further comprising an antibody.

12. A composition of claim 11 wherein the composition is a pharmaceutical composition.

13. A pharmaceutical composition of claim 11 further comprising one more excipients.

14. A pharmaceutical composition of claim 11 or claim 12 further comprising one or more of an enzyme inhibitor, an amino acid, or a dipeptide.

15. A pharmaceutical composition of claim 13 wherein the enzyme inhibitor is aprotinin.

16. A pharmaceutical composition of claim 13 or claim 14 wherein the dipeptide is carnosine or diglycine.

17. A pharmaceutical composition of any one of claims 11 to 15 further comprising a coprotein.

18. A pharmaceutical composition of any one of claims 11 to 16 for oral delivery.

19. A composition of any one of claims 11 to 18 for use in medicine.

20. A composition of any one of claims 11 to 19 for use in the treatment of cancer, an autoimmune disease or Alzheimer’s disease.

21. A composition of claim 20 wherein the autoimmune disease is selected from rheumatoid arthritis, Crohn's disease and ulcerative colitis, and allergic asthma.

22. A fusion protein comprising a transporter protein domain capable of transcytosing the intestinal wall and an antibody binding domain.

23. A fusion protein of claim 22 wherein transporter protein domain comprises an inactivated form of a protein capable of transcytosing the intestinal wall.

24. A fusion protein of claim 22 or claim 23 wherein the transporter protein domain is derived from a protein selected from pancreatic lipase, bile salt dependent lipase (BSDL) and a-amylase or a homologue thereof.

25. A fusion protein of any one of claims 22 to 23 wherein the antibody binding domain is located C or N terminally to the transporter protein domain.

26. A fusion protein of any one of claims 22 to 25 wherein antibody binding domain is selected from Protein A, Protein G, Protein L, Protein Z (derived from B-domain of SpA), Protein LG, Protein LA, Protein AG ,SpA, PAM (peptide with the sequence (RTY)4K2KG)(SEQ. ID No:18), Fc-lll, FcBP-2, FC-III-4C, FcRM, or a peptide with a sequence selected from TWKTSRISIF (SEQ. ID No:19), FGRLVSSIRY (SEQ. ID No:20), EPIHRDTLTALL (SEQ. ID No:21), APAR (SEQ. ID No:22), HWRGWV (SEQ. ID No:23), HWRGWVC (SEQ. ID No 40), HYFKFD (SEQ. ID No:24), HFRRHL (SEQ. ID No:25), HWCitGWV (SEQ. ID No:26), DAAG (SEQ. ID No:27),D2AAG (SEQ. ID No:28), NKFRGKYK (SEQ. ID No:29), NARKFYKG (SEQ. ID No:30), FYWHCLDE (SEQ. ID No:31), FYCHWALE (SEQ. ID No:32), FYCHTIDE (SEQ. ID No:33), RRGW (SEQ. ID No:34), KHRFNKD (SEQ. ID No:35), cyclo(Na-Ac)S(A)-RWHYFK-Lact-E (SEQ. ID No:37), cyclo(Na-Ac)Dap(A)-RWHYFK-Lact-E (SEQ. ID No:38) and cyclo [Link-M-WFRHYK] (SEQ. ID No:39) or homologs thereof, preferably HWRGWV (SEQ. ID No 23) and / or HWRGWVC (SEQ. ID No 40).

27. A fusion protein of any one of claims 22 to 25 wherein the antibody binding peptide comprises the sequence AWHLGELVW (SEQ. ID No. 12).

28. A fusion protein of any one of claims 22 to 26 wherein the fusion protein has the sequence of SEQ ID No. 1 , SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46 or a homologue with at least 60% homology.

29. A nucleic acid sequence which:(i) encodes a fusion protein as claimed in any one of claims 22 to 28;(ii) encodes a homologue, or derivative of the fusion protein as claimed in any one of claims 22 to 28;(iii) has a sequence which is complementary to the sequence in (i) or (ii); or(iv) has a sequence which has substantial identity with any of those of (i), (ii) or (iii).

30. An expression vector comprising the nucleic acid sequence of claim 29.31 . A cell comprising the expression vector of claim 30.

32. A pharmaceutical composition comprising (i) transporter protein domain capable of transcytosing the intestinal wall linked to an antibody binding peptide and (ii) an antibody.

Images