Endocytic transport compositions

Abstract

The present invention relates to transporters capable of transcytosis across the gut wall and delivery of antibodies to the bloodstream.

Description

[0001] This invention relates to transport proteins capable of transintestinal endocytosis and delivery of antibodies into the bloodstream. Background Technology

[0002] A primary function of the epithelial cell monolayer lining the gastrointestinal (GI) tract is to act as a physical barrier to the environment. This barrier function extends to the molecular level; ions and small molecules can diffuse freely, but larger molecules or protein macromolecules cannot. These macromolecules cannot be distributed and passively diffuse across the cell membrane, and they are also too large to diffuse through tight junctions (TJs), which are a hallmark of the GI tract barrier.

[0003] The use of penetration enhancers (PEs) to improve the oral delivery of small molecules, including peptides, with pharmaceutical properties has a well-established research foundation. For small molecules, endocytic transport via direct penetration of the cell membrane and passive diffusion across cells are feasible, depending on the physicochemical properties of the molecule. PEs can facilitate endocytic transport of such small molecules by altering the composition and behavior of the cell membrane, or by forming complexes with and shielding these molecules. Another way PEs improve endocytic transport of small molecules is by partially opening the transepithelial junction (TJ) between epithelial cells. However, in practice, while new technologies do offer potential solutions, size remains a limiting factor, and larger macromolecules such as proteins often cannot cross the TJ.

[0004] Several examples exist in nature where active transport processes are used to transport proteins and larger complexes across the epithelial barrier intact, rather than relying on passive diffusion. Examples include the transport of secretory IgA in celiac disease via the transferrin receptor CD71 (Matysiak-Budnik et al., 2008), the retrograde endocytosis of botulinum toxin A via microfolded M cells (Ghosal et al., 2018; Matsumura et al., 2015), and the transport of HIV-1 viral particles across cervical epithelial cells via complement receptor 3 (CD11 / CD18) (Day et al., 2022). Existing research has focused on mimicking and utilizing active transport processes as a means of enabling drug cargo to cross the epithelial barrier, for example, by transporting nanocarriers across the intestinal organoid monolayer via M cells (Tong et al., 2020). Another approach utilizes the Cholix protein fragment from the pathogen Vibrio cholerae as an endocytic transporter, enabling the molecule to penetrate the tissue and enter the lamina propria intact (Fay et al., 2020; Liu et al., 2023; Ogura et al., 2021; Taverner et al., 2020).

[0005] Trinity Biosystems has developed several systems for needle-free antibody delivery. WO2007 / 067596 describes an application of a vector construct comprising a receptor-binding domain, an endocytic transport domain, and an antibody-binding domain, wherein an antibody or antibody fragment binds non-covalently to the antibody-binding domain. The antibody or fragment, together with a bioactive molecule, constitutes part of a fusion protein. WO2008 / 021234 describes a delivery construct comprising a receptor-binding domain, an endocytic transport domain, and particles. WO2006 / 044205 discloses a delivery construct comprising a receptor-binding domain and an endocytic transport domain linked to a macromolecule via a cleavable linker. In all of the above cases, the endocytic transport domain is derived from bacterial species such as Pseudomonas endotoxin A, botulinum toxin, diphtheria toxin, pertussis toxin, cholera toxin, heat-labile Escherichia coli enterotoxin, Shiga toxin, or Shiga-like toxin.

[0006] These studies demonstrate that active transport mechanisms hold promise in principle as a means of enabling antibodies and large protein molecules to cross the epithelial barrier intact. However, reported methods fall far short of the ideal drug delivery goal: the rapid, high-volume, systemic delivery of antibodies into the bloodstream via oral administration in an inert manner, without triggering an immunogenic response against the active carrier molecule. Cholix is ​​not a carrier capable of achieving this goal: it cannot achieve systemic delivery and accumulation of the cargo in the bloodstream, and its pathogen-derived molecules would trigger an immune response, thus limiting its reusability in therapeutic settings. The inventors have identified a human protein that can serve as an effective endocytic transport driver for antibody cargo in a bispecific form, paving the way for the generation of humanized, non-immunogenic active cargo carriers—carriers capable of transporting antibody proteins intact across GI tissues into the bloodstream in a universal manner.

[0007] Small amounts of human enzymes and hormones are normally secreted into the GI tract lumen, where they are subsequently transported into the bloodstream via endocytosis. Two of these hormones belong to this group: intestinal insulin (Bendayan et al., 1990, 1994; Ziv & Bendayan, 2000) and leptin (Bendayan et al., 2010; P. Cammisotto & Bendayan, 2012; PG Cammisotto et al., 2007). However, due to their highly potent biological effects and the lack of mutations that would inactivate their function while preserving their receptor-binding ability (and thus their endocytosis transport ability) (Salum et al., 2021), these two proteins may not be suitable as endocytosis transport drivers in biotherapeutic agents. Intestinal enzymes such as 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 undergo endocytosis under physiological conditions and reach the bloodstream intact. This invention utilizes these enzymes as highly efficient drivers of endocytosis to propel antibody cargo across GI tissues in a bispecific manner. Summary of the Invention

[0008] This invention provides a composition comprising, consisting of, or substantially consisting of a transporter domain capable of trans-intestinal endocytosis, said transporter domain being linked to at least one antibody-binding moiety. Preferably, said transporter domain is a non-bacterial transporter domain.

[0009] Preferably, the transporter protein domain is derived from a protein capable of transintestinal endocytosis.

[0010] Preferably, the protein capable of transintestinal endocytosis is selected from pancreatic triglyceride lipase, bile salt-dependent lipase (BSDL), and α-amylase, or their homologs. More preferably, the protein capable of transintestinal endocytosis is selected from pancreatic triglyceride lipase, α-amylase, or their homologs.

[0011] Preferably, the antibody-binding portion binds to the Fc region of the antibody. Preferably, the antibody-binding portion binds to a non-variable region on the antibody light chain (CL region), or to a non-variable region in the heavy chain CH1 region, or simultaneously to the non-variable regions of both CL and CH1.

[0012] Preferably, the antibody-binding moiety comprises an Fc-binding domain or a homologue or derivative thereof. Preferably, the antibody-binding moiety is a peptide.

[0013] Preferably, the protein capable of transintestinal endocytosis is substantially identical to proteins derived from mammals (more preferably humans). Preferably, the protein capable of transintestinal endocytosis is endogenous to the treated subject, i.e., it is derived from proteins naturally expressed by the subject's species.

[0014] The present invention also provides a fusion protein comprising a transporter domain derived from a protein capable of transintestinal endocytosis and an antibody-binding domain.

[0015] The present invention also provides a nucleic acid sequence encoding the fusion protein of the present invention.

[0016] The present invention also provides an expression vector containing the nucleic acid sequence of the present invention.

[0017] The present invention also provides cells comprising the expression vector of the present invention.

[0018] The present invention also provides the use of the composition for transporting antibodies across the intestinal wall.

[0019] The present invention further provides a pharmaceutical composition comprising (i) a protein capable of transintestinal endocytosis and transported via an antibody-binding portion as described herein, and (ii) an antibody.

[0020] The pharmaceutical compositions of the present invention can be used in the medical field. The pharmaceutical compositions of the present invention can be used to treat a variety of conditions known to be improveable by therapeutic antibodies, including cancer, autoimmune diseases, and Alzheimer's disease. Therefore, the present invention also provides a method for treating cancer, autoimmune diseases, or Alzheimer's disease, the method comprising administering a pharmaceutically effective amount of the pharmaceutical composition of the present invention to a subject in need. Detailed Implementation

[0021] The present invention provides a composition comprising a transporter domain capable of transintestinal endocytosis, the transporter domain being linked to an antibody-binding portion.

[0022] The transporter domain comprises, is composed of, is substantially composed of, or is derived from the following: a protein capable of trans-intestinal endocytosis and retaining the ability to trans-intestinal endocytosis. As used herein, “endocytosis” refers to the process by which macromolecules are transported within membrane-bound vesicles from one side of a cell (e.g., the apical side of an epithelial cell) to the other side (i.e., the basal side of an epithelial cell).

[0023] As used herein, "capable of transintestinal endocytosis" means that a compound (preferably a protein or peptide) can cross the intestinal wall intact without degradation. Preferably, the ability of said compound (especially a protein) to transintestinal endocytosis can be identified by using a using chamber as described in the examples. If a compound (i.e., a protein) increases the level of the marker detected on the serosal side of the chamber after a marker is introduced onto the mucosal side of the chamber, then the compound is capable of transintestinal endocytosis. For example, if a protein capable of transintestinal endocytosis is labeled with a detectable marker (such as FITC), the amount of marker detected on the serosal side of the chamber increases compared to using the marker alone.

[0024] The intestinal wall consists of multiple layers: the mucosa, submucosa, muscularis propria, and serosa or adventitia. Proteins capable of trans-intestinal endocytosis can be transported from the intestinal lumen to the bloodstream by passing through the epithelial cells lining the intestinal wall. It is currently believed that these proteins are transported from the luminal side to the basal side via endocytosis across the intestinal epithelial cells, and are subsequently released into the intestinal interstitial space. These proteins then cross the capillary walls, directly entering the bloodstream or indirectly entering the lymphatic system, which ultimately merges into the bloodstream.

[0025] As used herein, "intestinal wall" refers to the wall lining the gastrointestinal tract behind the stomach. This includes the small intestine and the large intestine. The small intestine consists of the duodenum, jejunum, and ileum, while the large intestine is also called the colon. The intestinal wall preferably forms part of the small intestine. Preferably, proteins capable of trans-intestinal endocytosis are capable of trans-intestinal endocytosis within the small intestinal wall.

[0026] Preferably, the protein capable of transintestinal endocytosis is a non-bacterial transporter domain. As used herein, a "non-bacterial transporter domain" refers to a transporter domain that is not entirely identical to proteins derived from or produced by bacterial species (especially bacterial toxins). Preferably, the endocytotic transporter domain is not derived from Pseudomonas aeruginosa endotoxin A, botulinum toxin, diphtheria toxin, pertussis toxin, cholera toxin, heat-labile Escherichia coli enterotoxin, Shiga toxin, or Shiga-like toxin. Preferably, the endocytotic transporter domain is not derived from Pseudomonas aeruginosa endotoxin A. The use of bacterial-derived transporters is undesirable because they may cause adverse side effects and adverse immune responses.

[0027] Preferably, the transporter protein domain is derived from the same species as the subject of treatment, i.e., a natural or endogenous protein. Preferably, the protein capable of transintestinal endocytosis is a mammalian protein, more preferably a human protein. If the protein is derived from a non-human source, it can be humanized to modify the protein sequence to increase its similarity to naturally occurring human sequences.

[0028] Preferably, the protein capable of transintestinal endocytosis is not a hormone, such as insulin or leptin. Using hormonal peptides is undesirable because they can cause adverse side effects. These peptides are also relatively small, meaning that preserving their ability to be transintestinally endocytosed while depriving them of their hormonal function may be more challenging.

[0029] The transporter domain may contain an inactive form of the native protein capable of transintestinal endocytosis. The inactive form is functionally inhibited or lost, thus no longer retaining the protein's usual function, but still retaining the ability to perform transintestinal endocytosis. 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%, less than 10%, or less than 5%. Preferably, the inactive form of the enzyme has no enzyme activity compared to the native form. Methods for detecting enzyme activity are well known to those skilled in the art. For example, the activity of pancreatic triglyceride lipase can be determined using the 3H-trioleylglycerol assay described in Lowe (1992).

[0030] The inactive form can be a mutated version of the native protein. For example, one or more amino acid residues known to be essential for the formation of the catalytic pocket can be substituted, rendering the enzyme inactive.

[0031] The transporter domain is preferably derived from a protein capable of transintestinal endocytosis, selected from pancreatic triglyceride lipase, bile salt-dependent lipase (BSDL), and α-amylase, or homologs thereof. Preferably, the transporter domain comprises, or consists of, the amino acid sequence of any one of SEQ ID No. 2 to 11, a fragment thereof, or a homolog thereof. Preferably, the transporter domain is derived from pancreatic triglyceride lipase.

[0032] As used in this article, "pancreatic triglyceride lipase" refers to pancreatic triglyceride lipase and its related family members pancreatic triglyceride lipase-associated protein-1, pancreatic triglyceride lipase-associated protein-2, and pancreatic triglyceride lipase-associated protein-3, as well as their homologs.

[0033] The amino acid sequences of these proteins are publicly available and can be encoded by nucleic acid sequences as shown below: PNLIP – NCBI Gene: 1056 https: / / www.ncbi.nlm.nih.gov / gene / ?term=1056 PNLIPRP1 – NCBI Gene: 5407 https: / / www.ncbi.nlm.nih.gov / gene / ?term=5407 PNLIPRP2 – NCBI Gene: 5408 https: / / www.ncbi.nlm.nih.gov / gene / ?term=5408 PNLIPRP3 – NCBI Gene: 119548 https: / / www.ncbi.nlm.nih.gov / gene / 119548 Preferably, the natural form of pancreatic triglyceride lipase comprises, or is composed of, the following amino acid sequence: PNLIP: MKEVCYERLGCFSDDSPWSGITERPLHILPWSPKDVNTRFLLYTNENPNNFQEVAADSSSISGSNFKTNRKTRFIIHGFIDKGEENWLANVCKNLFKVESVNCICVDWKGGSR TGYTQASQNIRIVGAEVAYFVEFLQSAFGYSPSNVHVIGHSLGAHAAGEAGRRTNGTIGRITGLDPAEPCFQGTPELVRLDPSDAKFVDVIHTDGAPIVPNLGFGMSQVVGHLD FFPNGGVEMPGCKKNILSQIVDIDGIWEGTRDFAACNHLRSYKYYTDSIVNPDGFAGFPCASYNVFTANKCFPCPSGGCPQMGHYADRYPGKTNDVGQKFYLDTGDASNFARWR YKVSVTLSGKKVTGHILVSLFGNKGNSKQYEIFKGTLKPDSTHSNEFDSDVDVGDLQMVKFIWYNNVINPTLPRVGASKIIVETNVGKQFNFCSPETVREEVLLTLTPC (SEQ. ID No: 2) or fragments or homologues thereof.

[0034] Preferably, the native form of pancreatic triglyceride lipase-associated proteins 1-3 (lacking a leader peptide) contains, or is composed of, one of the following amino acid sequences: PNLIPRP1: KEVCYEDLGCFSDTEPWGGTAIRPLKILPWSPEKIGTRFLLYTNENPNNFQILLLSDPSTIEASNFQMDRKTRFIIHGFIDKGDESWVTDMCKKLFEVEEVNCICVDWKKGSQATYTQAANNVRVVGAQVAQMLDILLTEYSYPPSKVHLIGHSLGAHVAGEAGSKTPGLSRITGLDPVEASFESTPEEVRLDPSDADFVDVIHTDAAPLIPFLGFGTNQQMGHLDFFPNGGESMPGCKKNALSQIVDLDGIWAGTRDFVACNHLRSYKYYLESILNPDGFAAYPCTSYKSFESDKCFPCPDQGCPQMGHYADKFAGRTSEEQQKFFLNTGEASNFARWRYGVSITLSGRTATGQIKVALFGNKGNTHQYSIFRGILKPGSTHSYEFDAKLDVGTIEKVKFLWNNNVINPTLPKVGATKITVQKGEEKTVYNFCSEDTVREDTLLTLTPC (SEQ.ID No: 3) or a fragment or homolog thereof.

[0035] PNLIPRP2: KEVCYGQLGCFSDEKPWAGTLQRPVKLLPWSPEDIDTRFLLYTNENPNNFQLITGTEPDTIEASNFQLDRKTRFIIHGFLDKAEDSWPSDMCKKMFEVEKVNCICVDWRHGSRAMYTQAVQNIRVVGAETAFLIQALSTQLGYSLEDVHVIGHSLGAHTAAEAGRRLGGRVGRITGLDPAGPCFQDEPEEVRLDPSDAVFVDVIHTDSSPIVPSLGFGMSQKVGHLDFFPNGGKEMPGCKKNVLSTITDIDGIWEGIGGFVSCNHLRSFEYYSSSVLNPDGFLGYPCASYDEFQESKCFPCPAEGCPKMGHYADQFKGKTSAVEQTFFLNTGESGNFTSWRYKISVTLSGKEKVNGYIRIALYGSNENSKQYEIFKGSLKPDASHTCAIDVDFNVGKIQKVKFLWNKRGINLSEPKLGASQITVQSGEDGTEYNFCSSDTVEENVLQSLYPC (SEQ.ID No: 4) or a fragment or homolog thereof.

[0036] PNLIPRP3: KEVCYERLGCFKDGLPWTRTFSTELVGLPWSPEKINTRFLLYTIHNPNAYQEISAVNSSTIQASYFGTDKITRINIAGWKTDGKWQRDMCNVLLQLEDINCINLDWINGSREY IHAVNNLRVVGAEVAYFIDVLMKKFEYSPSKVHLIGHSLGAHLAGEAGSRIPGLGRITGLDPAGPFFHNTPKEVRLDPSDANFVDVIHTNAARILFELGVGTIDACGHLDFYPN GGKHMPGCEDLITPLLKFNFNAYKKEMASFFDCNHARSYQFYAESILNPDAFIAYPCRSYTSFKAGNCFFCSKEGCPTMGHFADRFHFKNMKTNGSHYFLNTGSLSPFARWRHK LSVKLSGSEVTQGTVFLRVGGAVRKTGEFAIVSGKLEPGMTYTKLIDADVNVGNITSVQFIWKKHLFEDSQNKLGAEMVINTSGKYGYKSTFCSQDIMGPNILQNLKPC (SEQ. ID No: 5) or fragments or homologues thereof.

[0037] Pancreatic triglyceride lipase and related proteins can be inactivated as described by Lowe (1992), i.e., by substituting one or more amino acids in the catalytic triad (Ser153 / His264 / Asp177, numbered starting from the leader peptide sequence of PNLIP). 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, pancreatic triglyceride lipase or related proteins contain the following substitutions: His264 is replaced with leucine, Asp177 with asparagine, and Ser153 with alanine.

[0038] As used in this article, "bile salt-dependent lipase" refers to bile salt-dependent lipase and its homologs. Bile salt-dependent lipase (BSDL), also known as carboxy ester lipase (CEL) or cholesterol esterase, has been shown to undergo endocytic transport in the intestine. Human BSDL protein is encoded by the nucleic acid sequence listed in NCBI Gene:1056 (https: / / www.ncbi.nlm.nih.gov / gene / 1056).

[0039] Preferably, the natural form of BSDL contains, or consists of, the following amino acid sequence: (SEQ. ID No: 6) or fragments or homologs thereof.

[0040] The activity of BSDL can be determined, for example, using 4-nitrophenylhexanoate according to the method described by Gjellesvik et al. (1992).

[0041] As used herein, “α-amylase” refers to α-amylases and their homologs. This includes salivary amylases, such as α-amylase AMY1A-1C, and pancreatic enzymes, such as AMY2A and AMY2B. It also includes bacterial α-amylase type IIA, which has been shown to undergo endocytosis in the intestinal epithelium. α-amylases can be encoded by the following nucleic acid sequences: AMY1A - NCBI gene: 276, https: / / www.ncbi.nlm.nih.gov / gene / ?term=276 AMY1B - NCBI gene: 277, https: / / www.ncbi.nlm.nih.gov / gene / ?term=277 AMY1C - NCBI gene: 278, https: / / www.ncbi.nlm.nih.gov / gene / ?term=278 AMY2A - NCBI gene: 279, https: / / www.ncbi.nlm.nih.gov / gene / ?term=279 AMY2B - NCBI gene: 280, https: / / www.ncbi.nlm.nih.gov / gene / ?term=280 Bacterial α-amylase type IIA is available commercially, such as Sigma Aldrich (A6380), which consists of amylases from various Bacillus licheniformis sources (see product information), including NCIB 6346 (Morgan, FJ, and Priest, FG, J. Appl. Bacteriol., 50(1), 107-114 (1981)), 44MB82-A (Ivanova, VN et al., J. Biotech., 28(2-3), 277-289 (1993)) and MTCC 1483 (Rao, MD et al., World J. Microbiol. Biotech., 18, 547-550 (2002)). The amyS α-amylase from Bacillus licheniformis is encoded by the nucleic acid sequence of NCBI gene 66217199, https: / / www.ncbi.nlm.nih.gov / gene / 66217199.

[0042] Preferably, the natural form of α-amylase contains, or is composed of, one of the following amino acid sequences: AMY1A QYSSNTQQGRTSIVHLFEWRWVDIALECERYLAPKGFGGVQVSPPNENVAIHNPFRPWWERYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMCGNAVSAGTSSTCGSYFNPGSRDFPAVPYSGWDFNDGKCKTGSGDIENYNDATQVRDCRLSGLLDLALGKDYVRSKIAEYMNHLIDIGVAGFRIDASKHMWPGDIKAILDKLHNLNSNWFPEGSKPFIYQEVIDLGGEPIKSSDYFGNGRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFMPSDRALVFVDNHDNQRGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRYFENGKDVNDWVGPPNDNGVTKEVTINPDTTCGNDWVCEHRWRQIRNMVNFRNVVDGQPFTNWYDNGSNQVAFGRGNRGFIVFNNDDWTFSLTLQTGLPAGTYCDVISGDKINGNCTGIKIYVSDDGKAHFSISNSAEDPFIAIHAESKL (SEQ. ID No: 7) or a fragment or homolog thereof.

[0043] AMY1B: QYSSNTQQGRTSIVHLFEWRWVDIALECERYLAPKGFGGVQVSPPNENVAIHNPFRPWWERYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMCGNAVSAGTSSTCGSYFNPGSRDFPAVPYSGWDFNDGKCKTGSGDIENYNDATQVRDCRLSGLLDLALGKDYVRSKIAEYMNHLIDIGVAGFRIDASKHMWPGDIKAILDKLHNLNSNWFPEGSKPFIYQEVIDLGGEPIKSSDYFGNGRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFMPSDRALVFVDNHDNQRGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRYFENGKDVNDWVGPPNDNGVTKEVTINPDTTCGNDWVCEHRWRQIRNMVNFRNVVDGQPFTNWYDNGSNQVAFGRGNRGFIVFNNDDWTFSLTLQTGLPAGTYCDVISGDKINGNCTGIKIYVSDDGKAHFSISNSAEDPFIAIHAESKL (SEQ. ID No: 8) or a fragment or homolog thereof.

[0044] AMY1C: QYSSNTQQGRTSIVHLFEWRWVDIALECERYLAPKGFGGVQVSPPNENVAIHNPFRPWWERYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMCGNAVSAGTSSTCGSYFNPGSRDFPAVPYSGWDFNDGKCKTGSGDIENYNDATQVRDCRLSGLLDLALGKDYVRSKIAEYMNHLIDIGVAGFRIDASKHMWPGDIKAILDKLHNLNSNWFPEGSKPFIYQEVIDLGGEPIKSSDYFGNGRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFMPSDRALVFVDNHDNQRGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRYFENGKDVNDWVGPPNDNGVTKEVTINPDTTCGNDWVCEHRWRQIRNMVNFRNVVDGQPFTNWYDNGSNQVAFGRGNRGFIVFNNDDWTFSLTLQTGLPAGTYCDVISGDKINGNCTGIKIYVSDDGKAHFSISNSAEDPFIAIHAESKL (SEQ. ID No: 9) or a fragment or homolog thereof.

[0045] AMY2A QYSPNTQQGRTSIVHLFEWRWVDIALECERYLAPKGFGGVQVSPPNENVAIYNPFRPWWERYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMCGNAVSAGTSSTCGSYFNPGSRDFPAVPYSGWDFNDGKCKTGSGDIENYNDATQVRDCRLTGLLDLALEKDYVRSKIAEYMNHLIDIGVAGFRLDASKHMWPGDIKAILDKLHNLNSNWFPAGSKPFIYQEVIDLGGEPIKSSDYFGNGRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFVPSDRALVFVDNHDNQRGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRQFQNGNDVNDWVGPPNNNGVIKEVTINPDTTCGNDWVCEHRWRQIRNMVIFRNVVDGQPFTNWYDNGSNQVAFGRGNRGFIVFNNDDWSFSLTLQTGLPAGTYCDVISGDKINGNCTGIKIYVSDDGKAHFSISNSAEDPFIAIHAESKL (SEQ. ID No: 10), or a fragment or homolog thereof.

[0046] AMY2B QYSPNTQQGRTSIVHLFEWRWVDIALECERYLAPKGFGGVQVSPPNENVAIHNPFRPWWERYQPVSYKLCTRSGNEDEFRNMVTRCNNVGVRIYVDAVINHMSGNAVSAGTSSTCGSYFNPGSRD FPAVPYSGDFNDGKCKTGSGDIENYNDATQVRDCRLVGLLDLALEKDYVRSKIAEYMNHLIDIGVAGFRLDASKHMWPGDIKAILDKLHNLNSNWFPAGSKPFIYQEVIDLGGEPIKSSSDYFGN GRVTEFKYGAKLGTVIRKWNGEKMSYLKNWGEGWGFMPSDRALVFVDNHDNQRGHGAGGASILTFWDARLYKMAVGFMLAHPYGFTRVMSSYRWPRQFQNGNDVNDWVGPPNNNGVIKEVTINPD TTCGNDWVCEHRWRQIRNMVNFRNVVDGQPFTNWYDNGSNQVAFGRGNRGFIVFNNDDWTTFSLTLQTGLPAGTYCDVISGDKINGNCTGIKIYVSDDGKAHFSISNSAEDPFIAIHAESKL(SEQ. ID No: 11.) or fragments or homologs thereof.

[0047] Amylase activity can be determined, for example, by using 2-chloro-4-nitrophenyl-α-maltotriose as a substrate and monitoring the formation of 2-chloro-4-nitrophenol.

[0048] As used herein, the term "transporter protein domain" includes homologs of the aforementioned native proteins. This includes equivalent proteins with the same activity and function in different species, i.e., homologs and orthologs. It also includes fragments of these proteins and their homologs that retain the ability to be transported across the intestinal cell wall.

[0049] homologs As used herein, the term "homology" refers to a protein or peptide containing one or more additions, deletions, substitutions, or similar variations, all of which are covered by this invention. Furthermore, one amino acid can be substituted for another amino acid of a similar "type." For example, one hydrophobic amino acid can be substituted for another hydrophobic amino acid. Amino acid sequences can be compared using programs such as CLUSTAL. This program compares amino acid sequences and finds the optimal alignment by appropriately inserting gaps in either sequence. The amino acid identity or similarity (identity plus conservation of amino acid type) of the optimal alignment can be calculated. Programs such as BLASTx will align the longest similar sequence fragments and assign a score to the degree of match. Thus, a comparison result can be obtained where multiple similar regions are found, each with a different score. This invention covers both types of analysis described above.

[0050] Ideally, when the term "substantial identity" is used for an amino acid sequence, it means that the sequence is more identical to the sequence described herein than to prior art amino acid sequences.

[0051] For the purposes of this invention, if a substantial number of amino acids constituting an amino acid sequence exhibit homology when one of the above algorithms is used, then the amino acid sequence can be considered to have "substantial homology" with another amino acid sequence. Preferably, the degree of homology is measured across the entire amino acid sequence. For example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 99% of the amino acids may be identical in increasing order of preference. Alternatively, for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 99% of the amino acids may have similarity in increasing order of preference. Most preferably, homologs or derivatives having at least 90% or even 95% similarity are provided.

[0052] The amino acid residues constituting the proteins or peptides of this invention can be chemically modified. Examples of chemical modifications include those corresponding to post-translational modifications, such as phosphorylation, acetylation, and deamidation. Chemical modifications may not correspond to modifications that may exist in vivo. For example, the N-terminus or C-terminus of the peptide can be modified to improve peptide stability, bioavailability, and / or affinity. Further examples of non-natural modifications include: incorporation of non-coding α-amino acids, D-form of protein amino acids, photoreactive cross-linked amino acids, N-methylated and β-amino acids, backbone reduction, retroinversion using d-amino acids, N-terminal methylation and C-terminal amidation, and polyethylene glycolation. Preferably, the transporter domain may contain N-linked glycosylation units, as these units can participate in the endocytosis of proteins by luminal epithelial cells or intestinal epithelial cells. These units may be naturally occurring or added as modifying amino acids.

[0053] Amino acid substitution refers to replacing an amino acid residue at the same position with a substitute amino acid residue. The inserted amino acid residue can be inserted at any position, and can be partially or completely adjacent to each other, or none of the inserted amino acid residues can be adjacent to another inserted amino acid residue.

[0054] The inserted and substituted amino acids can be naturally occurring or non-natural, for example, they may contain non-natural side chains and / or be interconnected by non-natural peptide bonds. Proteins with such alterations are further discussed 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 their references. If more than one amino acid residue is substituted and / or inserted, the substituted / inserted amino acid residues may be the same as or different from each other. Each substituted amino acid may have a different side chain than the substituted amino acid.

[0055] Amino acid substitutions can be conserved, meaning the substituted amino acid has similar chemical properties to the original amino acid. Those skilled in the art will understand which amino acids have similar chemical properties. For example, the following groups of amino acids have similar chemical properties in terms of size, charge, and polarity: Group 1: Ala, Ser, Thr, Pro, Gly; Group 2: Asp, Asn, Glu, Gln; Group 3: His, Arg, Lys; Group 4: Met, Leu, Ile, Val, Cys; Group 5: Phe, Tyr, Trp.

[0056] Therefore, for homologs, derivatives, and fragments, it is important that they possess at least some degree of the ability of the natural protein from which they originate. For example, transporter domains have the ability to cross the intestinal wall for endocytosis, and antibody-binding peptides have the ability to bind to relevant antibodies.

[0057] Antibody binding portion Preferably, the antibody-binding moiety binds to a region of the antibody without interfering with the antibody's antigen recognition and / or binding activity. If the antibody is a monoclonal antibody, the antibody-binding moiety preferably binds to a constant heavy chain, or an Fc region, or a non-variable region CL and / or CH1. Preferably, the antibody-binding moiety includes an Fc-binding domain or a homolog thereof capable of specifically binding to the Fc region of the antibody. The antibody-binding moiety can bind to any class or subclass of antibody, such as any of the five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Preferably, the antibody-binding moiety binds to an IgG antibody.

[0058] Preferably, the antibody-binding portion reversibly binds to the antibody, thereby releasing the antibody after endocytosis and transport.

[0059] As used herein, the term "specific binding" refers to the selectivity of the antibody-binding moiety for the antibody, and that this binding is distinguishable from unwanted or nonspecific interactions. The ability of an antibody-binding moiety to bind to a specific antibody can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques (e.g., analysis on BIAcore instruments) and conventional binding assays. The degree to which an antibody-binding moiety binds to unrelated (non-antibody) proteins (rather than the target antibody) (e.g., as measured by ELISA) is less than approximately 10% of the degree to which the antibody-binding moiety binds to the target antibody. "Affinity" refers to the strength of the sum of all 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., the antibody-binding moiety). Unless otherwise stated, as used herein, "binding affinity" refers to intrinsic binding affinity, reflecting a 1:1 interaction between members of a binding pair (e.g., between the antibody-binding moiety and the antibody). The affinity of a molecule for its partner is typically expressed as a dissociation constant (Kd), i.e., the dissociation rate constant and the binding rate constant (k, k, k) respectively. off and k onThe ratio of the rate constants to the antibody-binding moiety Kd is given by the antibody-binding moiety Kd. Therefore, equivalent affinity can include different rate constants, as long as the ratio of the rate constants remains constant. 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 the antibody-binding moiety, while a higher Kd reflects a weaker binding. Several methods can be used to measure the Kd of the antibody-binding moiety, including surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence-based assays. In some respects, the dissociation constant (Kd) of the antibody-binding moiety is <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 nM). -8 M or lower, for example from 10 -8 M to 10 -13 M, for example, from 10 -9 M to 10 -16 The binding moiety preferably has a dissociation constant (Kd) between 0.01 µM and 1 nM, more preferably 0.5 µM to 0.5 nM or 1 µM to 0.1 nM. Alternatively, the antibody binding moiety has an IC50 value at pH 7.4. 50 The range is from 30 nM to 70,000 nM, preferably from 100 nM to 1,000 nM, and more preferably from 240 nM to 600 nM.

[0060] Preferably, the ratio of transporter protein to antibody-binding moiety in 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.

[0061] fit In one embodiment, the antibody-binding moiety may be an aptamer. An aptamer is a synthetic oligonucleotide (DNA or RNA) that recognizes a target molecule 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 readily available in vitro and can be developed to recognize a wide variety of molecules, ranging from simple ions (e.g., Pb2+, Liu & Lu, 2003. J Am Chem Soc., 125, 6642-6643) to nucleotides, small molecules, proteins, viruses, and cells, and even entire organisms (Menger et al., 2006. Handbook of Experimental Pharmacology, 359-373). High-binding-affinity aptamers have been screened using the well-known SELEX method (Ellington & Szostak, 1990. Nature, 346, 818-822) for the detection of low molecular weight molecules, such as theophylline (Jenison et al., 1994. Science, 263, 1425-1429), L-arginine (Geiger et al., 1996. Nucl. Acids Res., 24, 1029-1036), monomycin (Schuerer et al., 2001. Bioorg. Med. Chem., 92, 2557-2563), and 17β-estradiol (Kim et al., 2007. Biosens. Bioelectron., 22, ). 2525-2531), and larger molecules such as thrombin (thrombin-binding aptamer: 5'-GGTTGGTGTGGTTGG-3') (Baldrich et al., Anal Chem. 2004, 76, 23, 7053-63), cholera toxin, or HIV-1 tat protein (see Tombelli et al., 2007, Biomolec Eng., 24, 191-200 for a review). Some of the aforementioned aptamers have been used in ELISA-like assays on microplates or on the surface of biosensor transducers (QCM, SPR). An aptamer-modified gold nanoparticle (AuNP) colorimetric system has also been developed for the determination of protein PDGF in sandwich assays (Huang et al., 2005, 77, 5735-5741). Aptamers capable of binding to the Fc region of antibodies are known in the art. Suitable aptamers include those described in Hu et al. (2019).

[0062] Antibody-binding peptide In another embodiment, the antibody-binding moiety may be a protein, polypeptide, or peptide. In a preferred embodiment, the antibody-binding moiety comprises an Fc-binding domain or an antibody light chain (LC)-binding domain, or is composed of the latter. For example, the Fc-binding domain may be derived from an Fc receptor protein or its homolog that retains the ability to bind to the Fc region. Suitable Fc receptors are known in the art, including IgG receptors (FcγR), high-affinity IgE receptors (FcεRI), IgA and IgA / IgM receptors, and neonatal Fc receptors for IgG (FcRn). For example, the antibody LC-binding domain may be derived from a protein L sequence.

[0063] In a more preferred embodiment, the antibody-binding portion is an antibody-binding peptide.

[0064] As used herein, the term "antibody-binding peptide" refers to a peptide that specifically binds to an antibody. Typically, as used herein, a "peptide" contains fewer than 50 amino acid bases, preferably fewer than 30 amino acids, more preferably fewer than 20 amino acids, and even more preferably fewer than 15 amino acids. The term "peptide" also includes so-called button antibodies, which are derived from the extra-long bovine CDRH3 region, typically identified by bovine immunization strategies, and are generally less than 50 amino acids in length.

[0065] 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 the B domain of SpA), protein LG, protein LA, protein AG, SpA, PAM (a peptide with the sequence (RTY)4K2KG) (SEQ. ID No:18), Fc-III, FcBP-2, FC-III-4C, FcRM, or peptides having sequences selected from the following: 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:28). 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), cyclic (Nα-Ac)S(A)-RWHYFK-Lact-E (SEQ ID No:37), cyclic (Nα-Ac)Dap(A)-RWHYFK-Lact-E (SEQ ID No:38), and cyclic [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 antibodies. Preferably, the antibody-binding peptide comprises, consists of, or substantially comprises the sequence HWRGWV (SEQ. ID No: 23) or HWRGWVC (SEQ. ID No: 40). Preferably, the antibody-binding peptide comprises, consists of, or substantially comprises the protein L sequence: MNIKFAGKETPETPEEPKEEVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYADLLAKVNGEYTADLEDGGNHMNIKFAG (SEQ. ID No. 41) or PFVENKEETPETPGTDSEEEVTIKANLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDNGEYTVDVADKGYTLNIKFAG (SEQ. ID No. 42).

[0066] Preferably, the antibody-binding peptide comprises, consists of, or is substantially composed of the sequence AWHLGELVW (SEQ. ID No. 12). Preferably, the antibody-binding peptide is FcBP-1, FcB-2, Fc-III-4C, or Fc-III. Preferably, the antibody-binding peptide comprises, consists of, or is substantially composed of the sequence DCAWHLGELVWCT (SEQ. ID No. 13). Preferably, the antibody-binding peptide is FcB-2, Fc-III-4C, or Fc-III.

[0067] The antibody-binding peptide may contain one or more modified amino acids. For example, the antibody-binding peptide may contain one or more D-amino acids, which helps prevent proteolysis and thus prevents unwanted degradation in the intestinal lumen.

[0068] Antibody The antibody-binding portion specifically binds to the antibody.

[0069] As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably to encompass polyclonal antibodies, monoclonal antibodies, multispecific antibodies (such as bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecule containing an antigen recognition site, provided that the antibody exhibits the desired biological activity and contains at least the CH2 portion of at least one heavy chain. Preferably, the antibody is a monoclonal antibody. Antibodies can be members of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), classified according to the characteristics of their respective heavy chain constant domains, referred to as α, δ, ε, γ, and μ, respectively. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Preferably, the antibody is an IgG antibody, more preferably IgG1 or IgG4. The term "antibody" is also intended to include single-domain antibodies (sdAbs) or nanobodies, including V... H H fragment and V NAR An antibody is a fragment composed of a single monomeric antibody chain that can still selectively bind to a specific antigen. The term "antibody" is also intended to include antibody conjugates, such as those with polyethylene glycol (PEG).

[0070] Furthermore, unless the context otherwise requires, the term "antibody" should be understood to encompass both complete antibodies and antibody fragments containing the antigen-binding region and CH2 domain of the complete antibody, including scFv-CH2-CH3 fusion proteins. Antibodies can be produced by hybridomas, or by synthetic means such as recombinant DNA technology, phage display or yeast display technology, or using transgenic mice, or by liquid-phase or solid-phase peptide synthesis.

[0071] Fragments of intact antibodies have been shown to function in binding antigens. Examples of binding fragments include: (i) Fab fragments consisting of VL, VH, CL, and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of the VH domain (Ward, ES et al., Nature 341:544-546 (1989)); (v) isolated CDR regions; (vi) F(ab')2 fragments, which are bivalent fragments containing two linked Fab fragments; and (vii) single-chain Fv molecules (scFv) in which the VH and VL domains are linked by a peptide linker, enabling the two domains to associate and 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 dimer (PCT / US92 / 09965); and (ix) “dimeric antibody”, a multivalent or multispecific fragment constructed by gene fusion (WO94 / 13804; P. Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)).

[0072] Preferably, the antibody is a known therapeutic antibody capable of treating, improving, or alleviating a disease or condition. This includes antibodies that have been approved by regulatory agencies, such as those approved by the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA).

[0073] A transporter domain derived from a protein capable of trans-intestinal endocytosis is linked to the antibody-binding moiety. The link can take any form known to those skilled in the art. It may comprise binding pairs, such as biotin / streptavidin, wherein one member of each binding pair is linked to the transporter domain and the antibody-binding peptide, respectively. The binding pairs may comprise, for example, complementary nucleic acid sequences. The link may comprise one or more covalent bonds. For example, the link may comprise one or more amino acid units. The link may contain one or more affinity tags to assist in the purification and separation of the endocytotic transporter domain and / or the antibody-binding moiety. In a preferred embodiment, the transporter domain is linked to the antibody-binding peptide via one or more amino acid units to form a fusion protein.

[0074] Fusion protein In a second aspect, the present invention provides a fusion protein comprising a transporter domain, derived from a protein, capable of trans-intestinal endocytosis, and an antibody-binding domain. The antibody-binding domain comprises or is composed of an antibody-binding peptide as described above.

[0075] The transporter domain can be linked to an antibody-binding domain via one or more amino acids. These amino acids can be selected to provide additional functionality, such as affinity tags to aid in the isolation or purification of the fusion protein. The fusion protein may further include one or more affinity tags, such as (but not limited to) StrepTactinII, His6, Myc, HA, FLAG, and V5 affinity tags.

[0076] Preferred embodiments and features of the transporter protein domain and antibody-binding domain will become clear from the above discussion of preferred embodiments of the described composition.

[0077] The transporter domain and antibody-binding domain can exist in any order. The antibody-binding domain can be located at the N-terminus or C-terminus of the transporter domain. The antibody-binding domain can cleave the sequence of the transporter domain, and vice versa, provided that the fusion protein retains the function of each domain.

[0078] Preferably, the fusion protein comprises or is composed of the amino acid sequences of SEQ ID No:1, SEQ ID No:43, SEQ ID No:44, SEQ ID No:45, and SEQ ID No:46, or has substantial homology or identity with them.

[0079] Nucleic acid sequence The present invention also provides a nucleic acid sequence encoding a fusion protein of the second aspect.

[0080] Therefore, the present invention provides: (i) A sequence encoding a fusion protein comprising a transporter domain and an antibody-binding domain derived from a protein capable of transintestinal endocytosis. (ii) A sequence encoding a homolog or derivative of the fusion protein as described herein; (iii) A sequence complementary to the sequence in (i) or (ii); and (iv) A sequence that is substantially identical with any of the sequences in (i), (ii) or (iii).

[0081] Preferably, the nucleic acid sequence (i) encodes a fusion protein whose amino acid sequence is 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 the sequence of (i); or (iii) is substantially the same as the sequence of (i) or (ii).

[0082] The nucleic acid molecules of this invention may comprise multiple such sequences and / or fragments. Those skilled in the art will understand that this invention may include variants of the nucleic acid molecules described herein. Such variants are included within the scope of this invention. These variants may be naturally occurring, for example, arising from strain variation. Examples include additions, substitutions, and / or deletions. Furthermore, particularly when utilizing microbial expression systems, it may be desirable to modify nucleic acid sequences by utilizing preferred codon usages known in the specific organism used for expression. Therefore, synthetic or non-natural variants are also included within the scope of this invention.

[0083] When comparing nucleic acid sequences to determine the degree of homology or identity, programs such as BESTFIT and GAP (both from the Wisconsin Genetics Computing Group (GCG) software package) can be used. For example, BESTFIT compares two sequences and generates the optimal alignment for the most similar fragments. GAP is capable of aligning along full-length pairs of sequences and finds the optimal alignment by appropriately inserting gaps in either sequence. Suitably, in the context of this invention, when discussing the identity of nucleic acid sequences, the comparison is performed by aligning along full-length pairs of sequences.

[0084] Ideally, the term "substantial identity" means that the sequence is more identical to the sequence described herein than to prior art nucleic acid sequences.

[0085] Preferably, the sequence having substantial identity has at least 50% sequence identity with the sequence, ideally at least 75% sequence identity, and more ideally at least 90% or at least 95% sequence identity. In some cases, the sequence identity may be 99% or more.

[0086] Known techniques can be used to codon optimize nucleic acid sequences for expression in host cells.

[0087] Nucleic acid molecules can be in isolated or recombinant form. They can be integrated into a vector, and the vector can be integrated into a host. Such vectors and suitable hosts constitute a further aspect of the invention.

[0088] pharmaceutical preparations In a further aspect, the present invention provides a pharmaceutical composition comprising (i) a transporter domain capable of transintestinal endocytosis, said domain being linked to an antibody-binding moiety, and (ii) an antibody.

[0089] Preferred embodiments and features of the transporter protein domain and antibody-binding moiety will become clear from the above discussion of preferred embodiments of the described composition.

[0090] Preferably, the transporter domains and antibody-binding peptides capable of transintestinal endocytosis are included in the fusion protein.

[0091] Preferred embodiments and features of the fusion protein comprising a transporter domain and an antibody-binding domain will become clear from the above discussion of preferred embodiments of the fusion protein.

[0092] Preferred embodiments and characteristics of the antibody will become clear from the above discussion of preferred embodiments of the composition.

[0093] The pharmaceutical compositions according to the invention are preferably in solid or semi-solid form and are preferably suitable for oral administration. Such formulations can be prepared by a variety of known methods established in the art. For example, the transporter protein linked to the antibody-binding peptide and the antibody can be mixed together, optionally with other excipients required in the dosage form.

[0094] Preferably, the pharmaceutical formulation contains one or more excipients known to stabilize antibodies in the gut, such as those described in WO2020 / 063728 and WO2023 / 152234 (both incorporated herein by reference). Preferably, the pharmaceutical formulation further contains 5-aminolevulinic acid (5-ALA), and optionally a pH adjuster and / or an enzyme inhibitor. Alternatively, the pharmaceutical formulation may further contain one or more dipeptides, and optionally an enzyme inhibitor.

[0095] Preferably, the enzyme inhibitor is a protease inhibitor, such as aprotinin, ovomucoid II-O (containing an ovum inhibitor), Bowman-Birk inhibitor (BBI), or Kunitz trypsin inhibitor, for protein protection. Preferably, the enzyme inhibitor is aprotinin. Aprotinin is a protease inhibitor known to inhibit trypsin and other similar proteases. It is also commonly referred to as bovine pancreatic trypsin inhibitor (BPTI). Aprotinin is a 58-amino acid protein formed by processing a 100-amino acid polypeptide containing a signal peptide, a propeptide domain, and a Kunitz domain. Mature 58-amino acid form of aprotinin is commercially available under the trade name Trasylol. ® This product is intended for preventative use to reduce blood loss during surgery.

[0096] The aprotinin used in this invention may contain an amino acid sequence: RPDFCLEPPYTGPCKARMIRYFYNAKAGLCQPFVYGGCRAKRNNFKSSEDCMRTCGGA (SEQ ID NO: 14).

[0097] The aprotinin or fragment thereof used in this invention, or aprotinin analogues or fragments thereof, may be prepared by recombinant means (e.g., in Escherichia coli, mammalian cells, or insect cells), by synthetic means (e.g., using standard organic chemical techniques, such as solution-phase or solid-phase peptide synthesis), or may be a natural protein derived from an animal source (e.g., bovine).

[0098] Furthermore, unless the context otherwise requires, the term "aprotinin" should be understood to encompass aprotinin, aprotinin analogs, fragments of aprotinin, and fragments of aprotinin analogs. Analogs and fragments suitable for use in this invention are those capable of enhancing the stability of proteins (such as endocytic transporter / antibody binding domains and / or antibodies) in the presence of bodily fluids contained in the lower digestive tract (e.g., ileum, duodenum, and / or jejunum). Methods for confirming the ability of aprotinin, fragments of aprotinin, aprotinin analogs, or fragments thereof to enhance the stability of the active ingredient (protein) in the presence of bodily fluids contained in the lower digestive tract (e.g., ileum and / or colon) have been described in the examples of WO2020 / 063728. As used herein, "enhanced" stability means that at least 50% of the active ingredient remains intact after 4 hours in gastrointestinal fluids. Preferably, at least 60%, 70%, 75%, 80%, 90%, 95%, or more of the endocytic transporter / antibody binding domains and / or antibodies remain intact.

[0099] Preferably, the dipeptide is selected from bisglycine peptide or carnosine (also known as β-alanyl-L-histidine).

[0100] The pharmaceutical composition may further comprise a co-protein or peptide that assists the transporter domain in its function. The co-protein or peptide improves the efficiency of endocytic transport by increasing the amount of antibody transported across the intestinal wall, or by reducing the time required to transport the same amount of antibody compared to using an endocytic transporter alone. For example, if the transporter domain is derived from pancreatic triglyceride lipase, the pharmaceutical composition may optionally further comprise a co-lipase or a functional homolog or fragment thereof.

[0101] Colipase is essential for the efficient digestion of dietary fats by pancreatic triglyceride lipase. The nucleic acid sequence of human colipase protein has been identified as NCBI Gene 1208 https: / / www.ncbi.nlm.nih.gov / gene / 1208.

[0102] Colipase proteins preferably comprise or consist of the following amino acid sequence: MEKILILLLVALSVAYAAPGPRGIIINLTLYGIYYKCPCERGLTCEGDKTIVGSITNTNFGICHDAGRSKQ (SEQ ID No. 15).

[0103] If the transporter domain is derived from BSDL, the pharmaceutical composition may further optionally include glucose-regulated protein 95 (Grp94) or a functional homolog or fragment thereof.

[0104] Grp94 has been shown to be internalized along with BSDL via endocytosis and is believed to act as a molecular chaperone. The nucleic acid sequence of human grp94 has been identified as NCBI Gene 7184 https: / / www.ncbi.nlm.nih.gov / gene / 7184.

[0105] The grp94 protein preferably comprises or consists of the following amino acid sequence: The pharmaceutical compositions suitable for oral administration in this invention may be presented in the form of tablets, capsules, microplates, micropellets, powders, granules, microparticles, nanoparticles or hydrogels.

[0106] The compositions of this invention suitable for oral administration can be presented in discrete unit form such as capsules, tablets, microcapsules or granules, or in powder, granule or crystal form. In solid compositions, the minimum diameter of each particle is typically at least 10 mm. - 4 m, typically at least 5 x 10 -4 m, preferably at least 10 -3The maximum diameter is typically no more than 30 mm, typically no more than 20 mm, and preferably no more than 10 mm. In a preferred embodiment, the diameter of the particles is from about 0.2 mm to about 15 mm, preferably from about 1 mm to about 4 mm (e.g., for granules or microtablets) or from about 6 mm to about 12 mm (e.g., for certain tablets or capsules). The term "diameter" refers to the maximum linear dimension through the particles.

[0107] The compositions according to the invention may, of course, contain any other conventional excipients as needed, such as binders, fillers, disintegrants, diluents, and lubricants. Excipients used in solid dosage forms include, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate, calcium sulfate, sorbitol, glucose and / or lactose and / or other excipients, binders, fillers, disintegrants, diluents, and lubricants known in the art. Suitable binders include starch, gelatin, natural sugars such as glucose or β-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, astragalus gum or sodium alginate, carboxymethyl cellulose, polyethylene glycol, waxes, etc. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, etc. Instant diluents include mannitol, lactose, sucrose, and / or cyclodextrin. For ease of manufacture and use, lubricants, flow aids, flavoring agents, colorants, and stabilizers may also be added. Lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride.

[0108] Tablets can be prepared by compression or molding, optionally with one or more excipients. Compressed tablets can be prepared by compressing the active ingredient in a free-flowing form (such as powder or granules) in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, surfactant, or dispersant. Molded tablets can be prepared by molding a mixture of powdered compounds wetted with an inert liquid diluent in a suitable machine. Tablets can be optionally coated or scored and can be formulated into dosage forms that provide a slow, delayed, or controlled release of antibodies. Preferred examples of coating are given below.

[0109] The capsule may contain solid, semi-solid, or non-solid contents. Exemplary forms of capsule contents may include suspensions containing, for example, microcrystalline cellulose for imparting filler volume, alginate or sodium alginate as a suspending agent, and methylcellulose as a thickener, as well as any of the aforementioned solid or semi-solid forms.

[0110] Preferred unit-dose formulations are those containing an effective dose or an appropriate amount of the active ingredient. If the composition contains suitable controlled-release excipients, the release of some formulations may also be continuous. However, in preferred formulations, the release is pulsatile.

[0111] Pharmaceutical compositions typically contain equimolar amounts of an antibody-binding moiety and an antibody. When the ratio of the antibody-binding moiety to the present transporter domain is approximately 1:1, the pharmaceutical composition typically contains equimolar amounts of a transporter domain / antibody-binding moiety composition and an antibody.

[0112] The compositions according to the invention will generally comprise a therapeutically effective amount of the transporter domain / 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 will be determined by those skilled in the art using common knowledge. However, for example, a “low” dose formulation typically comprises no more than 20 wt% of the endocytic transporter domain / antibody binding moiety composition, and preferably 1 wt% to 10 wt%, such as 5 wt%, of the transporter domain / antibody binding moiety composition. A “high” dose formulation typically comprises at least 40 wt% of the transporter domain / antibody binding moiety composition, preferably 45 wt% to about 75 wt%, such as 50 wt% or 60 wt%.

[0113] The transporter domain / antibody binding moiety composition is typically present in an amount that allows it to be in the lower digestive tract at a concentration of 0.1-25 mg / ml. Each dosage form may contain 50-5000 mg of the transporter domain / antibody binding moiety composition, typically 100-1000 mg; 250-750 mg or 300-500 mg.

[0114] The compositions according to the invention will generally contain a therapeutically effective amount of antibody, which can be from 0.01 wt% to 80 wt% based on the total weight of the composition. The actual dosage will be determined by those skilled in the art using common knowledge. However, for example, a “low” dosage formulation typically contains no more than 20 wt% antibody, and preferably from 1 wt% to 10 wt% antibody, such as 5 wt% antibody. A “high” dosage formulation typically contains at least 40 wt% antibody, preferably from 45 wt% to about 75 wt%, such as 50 wt% or 60 wt%.

[0115] The antibody is typically present in amounts that produce concentrations of 0.1–25 mg / ml in the lower digestive tract. Each dosage form may contain 50–5000 mg of antibody, typically 100–1000 mg; 250–750 mg; or 300–500 mg.

[0116] Unless the context otherwise requires, 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 units that are solid or semi-solid in general, as well as those having a solid or semi-solid exterior and a non-solid interior (e.g., liquid or gel). For example, capsules may contain liquid or gel contents.

[0117] Delivery to the gastrointestinal tract The compositions according to the invention are suitable for delayed or selective release of transporter domains / antibody-binding moieties and antibodies in the lower digestive tract, particularly the duodenum, jejunum, and / or ileum, preferably after oral administration. This can be achieved by using specific coatings. The compositions of the invention can be oral delayed-release (DRO) compositions. DRO compositions pass through the stomach substantially unaltered and deliver the active ingredient to the gastrointestinal tract, typically the ileum, duodenum, and / or jejunum.

[0118] The compositions according to the invention may have an enteric coating. An enteric coating protects the active ingredient in the composition from erosion and degradation in the stomach, but dissolves and releases the contents of the dosage form in the intestine, typically due to pH changes. Suitable enteric coatings are well known in the art. The optimal coating for any particular formulation depends on the specific intended use, and the coating may be tailored to release the active ingredient in a specific region of the intestine or at a specific time after ingestion. If desired, such formulations may contain one or more intermediate layers between the active ingredient and the outer enteric coating. In this case, the compositions of the invention may release a portion of their contents in a specific region of the intestine and another portion in a second region of the intestine (such as the ileum, duodenum, and / or jejunum). Preferably, the compositions of the invention are in solid or semi-solid form, comprising an enteric coating suitable for releasing transporter domains / antibody-binding portions and antibodies in the ileum, duodenum, and / or jejunum. Useful enteric coatings are those that remain intact in the low pH environment of the stomach but readily dissolve when the optimal dissolving pH is reached. This can vary between pH 3 and 7.5, preferably 5 to 7, depending on the chemical composition of the coating. The desired coating thickness will depend on the coating's solubility and the intended treatment site. Typically, the coating thickness is 25 to 200 µm, especially 75 to 150 µm.

[0119] The compositions of the present invention are suitable for releasing the active ingredient to the appropriate site in the lower digestive tract, where the endocytic transport domain transports the antibody across the intestinal wall into the bloodstream, thereby achieving systemic delivery. Typically, the enteric coating should dissolve at pH conditions in the jejunum (approximately pH 5.5), ileum (approximately pH 6), and / or duodenum (approximately pH 6) to allow most of the transporter domains / antibody-binding portions and the antibody to be released at the desired site.

[0120] Medical applications This invention provides pharmaceutical compositions according to the invention for medical use. The invention also provides a method for treating or preventing diseases or conditions in a subject (especially a human subject), the method comprising administering a pharmaceutical composition comprising a transporter domain / antibody binding moiety and an antibody to the subject via the lower gastrointestinal tract (especially the duodenum, ileum, and / or jejunum). Preferably, the composition is suitable for oral administration. The pharmaceutical formulation can serve as an entry point for antibody absorption into the systemic circulation via the gastrointestinal tract (especially the duodenum, ileum, and / or jejunum), and therefore can be used to treat a variety of diseases and conditions.

[0121] The pharmaceutical composition may comprise any known therapeutic antibody. This includes all therapeutic antibodies containing an Fc domain that have been approved by a regulatory agency (e.g., the FDA or EMA). Antibody therapy has been commercially available for the treatment of a variety of conditions. In a preferred embodiment, the pharmaceutical composition may be used to treat autoimmune diseases, cancer, Alzheimer's disease, cardiovascular disease, Gram-negative sepsis, ankylosing spondylitis, migraine, hemophilia, multiple sclerosis, cold agglutinin disease, Mueller-Weiss syndrome, sickle cell disease, osteoporosis, Castleman's disease, macular degeneration, neuromyelitis optica, thyroid eye disease, Ebola virus infection, HIV infection, anthrax infection, treatment or prevention of SARS-CoV-2 infection or related diseases (such as COVID-19), or any other condition or disease known to be treatable with Fc protein-containing therapy.

[0122] Examples of therapeutic antibodies include zoltoximab, onituzumab, camrelizumab, slulimab, sugemalimab, parzemarab, concymarab, enatuzumab, cosilimumab, rozeliximab, taquitumumab, ekorituzumab, leprazole, trastuzumab, docarmazine, glimetuzumab, millizumab, donananetumab, lencanemab, tislelizumab, penaplimumab, sintilimab, toripalimab, umbutuzumab, retilimumab, nasolprimab, toripalimab, urorituzumab, bracucumab, mivetoxumab, nisevimab, tesimemab, sparsolimab, teritolumab, mutuzumab, tesaxagvirimab, cigavimab, and liralimumab. Terbenzafoxitin, Faciizumab, Suttelimumab, Sotopelimab, Ridamilimab, Caciziramumab + Idvimab, Tazepilimumab, Tesotropumab, Vitin, Evantolimumab, Aniluromab, Locartolimumab, Ticillin, Bimecrolimus, Tralogizumab, Everanalimumab, Aducanumab, Dotalimumab, Ansuvirumab, Magitoximab, Naxi Tadalafil, Atotetemizumab, Mavtemizumab, Odescivirimab, Belantamarab, Mofostatin, Tafazitum, Saturizumab, Inelizumab, Goxatozumab, Teptozumab, Ixatoxici, Epranetuzumab, Detrastuzumab, Enfotozumab, Vitin, Klexicitumab, Broluruzumab, Polotuzumab, Vitin, Resalitumab Romozumab, Caprazumab, Lavlizumab, Imaparumab, Cimiprizumab, Fremanetumab, Moxiduzumab, Pastoxin, Garcinia galenazumab, Lanaruzumab, Moglizumab, Erenumab, Tedraluzumab, Ibalizumab, Brossoumab, Duvalilumab, Emexazol, Benalizumab, Ofamumab Guseluzumab, Ogaituzumab, Saliruzumab, Dupreruzumab, Averuzumab, Brodatumumab, Atezolizumab, Belotuzumab, Olamumab, Relizumab, Obituximab, Ixizumab, Daretuzumab, Erotozumab, Nexitozumab, Idacilzumab, Alicizumab, Mepleribumab, Iloxizumab Antibiotics, Denutoximab, Secukinumab, Nivolumab, Bonatumab, Pembrolizumab, Ramucirumab, Verdelizumab, Secutoximab, Oxetuzumab, Trastuzumab emtansine, Laxixumab, Pertuzumab, Vibrentuximab, Belimumab, Ipilimumab, Denosumab, Tocilizumab, Ofamumab, Canatumab, Golime Monoclonal antibodies, ustekinumab, pecelizumab, caputoxumab, eculizumab, ranibizumab, panitumumab, natezumab, bevacizumab, cetuximab, efalizumab, omalizumab, tosimozumab-I131, teimozumab-teuktan, adalimumab, ozolizumab, alemtuzumab, gemtuzumab-olzomicin, trastuzumab,Infliximab, Pallizumab, Baliximab, Dalizumab, Rituximab, Acyclovir, Ezotrozole, Nebakumab, Nimotuzumab, Irizumab (Alzumab), Twinrab™ (RabiShield), Sintilimab (Tyvyt), Toripalimab (Tuoyi), Camrelizumab, Tislelizumab, Vidictetuzumab (Aidixi), Peampalimab, Sepalilimab, Nitachymab, Progolimab (Forteca), Ologen (Ar The following are listed: tlegia, livilimumab (Ilsira), cetuximab (Saratoca sodium), perbinafos α (IZCARO), sotopimab, morocumab-CD3, dupilumab (Dapoxetine), aliximumab, tesocumab (Vidodine), nipocalimab, bombordimab, datopromab (druttecan), zanokutumab, nemolizumab, zanidatatumab, linvostatumab, asartelimumab, paretuximab (druttecan), talatumab, masstatin, galadaximab, verobelemab, kovarimib, and evolocumab.

[0123] Autoimmune diseases include rheumatoid arthritis, psoriasis, gout, relapsing pericarditis, hidradenitis suppurativa, polyarticular juvenile idiopathic arthritis, giant cell arteritis, neuromyelitis optica spectrum disorders, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), and allergic asthma. This drug composition can also be used to prevent acute rejection of transplanted organs, such as kidneys. Monoclonal antibodies suitable for treating autoimmune diseases include: infliximab, adalimumab, ozolizumab, golimumab, cetrozumab, canatumab, cilukumab, ollocizumab, clazazumab, secucizumab, tocilizumab, salilumab, dupilumab (Dapoxetine), vobalizumab, ixekizumab, secucizumab, brodatumab, bismuth subcitrate, ustekinumab, gusejinumab, resalizumab, and tebuconazole. Dazumab, Miligizumab, Inaruzumab, Belimumab, Inebizumab, Nittacizumab, Ologenumab, Ikizumab, Vedolzumab, Pesolizumab, Irizumab, Efalizumab, Brekumab, Benalizumab, Meperizumab, Trilocizumab, Terzepluzumab, Leblizumab, Icalizumab, Utoxizumab, Ofamumab, Orezumab, Rituximab, Asastelizumab, and Anifrolumab.

[0124] Cancers that can be treated with monoclonal antibodies include: chronic lymphocytic leukemia, non-Hodgkin's lymphoma, large B-cell lymphoma, multiple myeloma, adenocarcinoma, endometrial cancer, ovarian cancer, cervical cancer, neuroblastoma, breast cancer, gastric cancer, skin cancer, melanoma, liver cancer, urothelial carcinoma, bladder cancer, small cell lung cancer, non-small cell lung cancer, colorectal cancer, and head and neck cancer. Monoclonal antibodies suitable for cancer treatment include: rituximab (Rituxan), bevacizumab (Avastin), dotalimumab, mosetumumab, magitoximab, nalcetuzumab, goxatoximab, enrofloxacin, durvalumab, atezolizumab, averulimumab, nexitoximab, nivolumab, ramucirumab, pembrolizumab, pertuzumab, ezetimibe, vidicticatum (Aidixi), progolimab, penaprilimab, cepalimumab, latomumab, mirtuzumab, sluglimab, sugelimab, sintilimab, trimelimab, and more. Limousinumab, rontoxicumab, tesutoxicumab, motuzumab, teritolumab, riverimab, octotoxicumab, toripalimab, gefituzumab, ipatuzumab, taquitoxicumab, cosimoxicumab, enatoxicumab, camrelizumab, onituzumab, zotoxicumab, panitumab (Vibish), bonnetuzumab (Blincyto), bentoxicumab, cetuximab (Erbitux), tesutoxicumab (Vidotrine), datopromab (druticon), zanokutuzumab, zanidatatumab, linvostatumab, parituzumab (druticon), talatuzumab, and trastuzumab (Herceptin).

[0125] Monoclonal antibodies suitable for the treatment of Alzheimer's disease include: adukanumab, donatumab, and lencanemab.

[0126] Monoclonal antibodies that are suitable for treating or reducing the risk of cardiovascular disease include anti-PCSK9 monoclonal antibodies, such as aliximumab and evolocumab.

[0127] Monoclonal antibodies available for the treatment or prevention of COVID-19 include: rivelimab, sotopimab, regorambimab, texazab, cigavenumab, cascilimumab, verobemumab, and imdevimab.

[0128] The following non-limiting embodiments illustrate the present invention, with reference to the following figures: Figure 1 IUTACC promotes the transport of IgG across the small intestine of pigs.

[0129] Figure 1 A. The experimental setup for the Jules chamber experiment.

[0130] Figure 1B. SDS-PAGE analysis of the protein preparations used in the experiment. The relative amounts of each preparation on the gel were the same as those used in the Uss chamber experiment.

[0131] Figure 1 C. Assessment of the presence of FITC-IgG in serous compartments at 1 hour and 2 hours after administration to the mucosal compartments.

[0132] Figure 2 The ability of IUTACC to transport IgG across GI tissues depends on its IgG binding sites.

[0133] Figure 2 A. Preparation of protein products of wild-type IUTACC and mutant IUTACC lacking engineered IgG binding sites. The relative protein amounts reflect the relative protein amounts used in the Uss chamber experiments.

[0134] Figure 2 B. Assessment of FITC-IgG presence in serous compartments at 1 and 2 hours post-administration in mucosal compartments. The figure shows pooled data from three experiments (in duplicate). In this and subsequent experiments, a modified procedure was used to achieve higher FITC-IgG transport values ​​(see Materials and Methods: Unlike adding each protein directly to the Usse compartment with 1.5 ml KBR buffer to start the experiment, this experiment involved pre-incubating the protein in a smaller volume (300 µl) at 37°C for 15 minutes before adding it to the Usse compartment to promote binding equilibrium before the start of the Usse experiment).

[0135] Figure 3 IUTACC requires active vesicle transport to facilitate the transport of FITC-IgG across GI tissues. Chlorpromazine was dissolved directly in KBR buffer in the mucosal Uuse compartment to a final concentration of 6 µg / ml. After tissue incubation for 15 minutes, FITC-IgG and IUTACC protein were added. FITC-IgG levels in the serous compartment were measured after 1 hour and 2 hours of incubation.

[0136] Figure 4 IUTACC is only applicable to small intestinal tissue, not colon tissue.

[0137] Figure 4 A. The Yuss chamber experiment used porcine colon tissue instead of small intestine tissue. One of the values ​​(circled) was identified as an outlier relative to other values ​​in the dataset according to the Grubbs test / ESD extreme studentization bias method.

[0138] Figure 4 B. Figure 3The data in Figure A is plotted after excluding outliers. This figure shows the combined data from two experiments (in triplicate). A variant of the protocol was used to maximize IUTACC transport activity, as shown in the small intestinal tissue, for example... Figure 2 (See Materials and Methods).

[0139] Figure 5 IUTACC is suitable for fresh (non-frozen) duodenal GI tissue.

[0140] The Yuss chamber experiment was established using fresh GI tract tissue obtained from the dissection of the duodenum 30 cm behind the stomach.

[0141] Figure 6. In vivo assessment of endocytic transport activity.

[0142] Figure 6A The SDS-PAGE analysis of the formulation used in in vivo studies is shown (reduction conditions: +DTT).

[0143] Figure 6B A schematic diagram of the in vivo experimental design is provided.

[0144] Figure 6C The results show the antibody levels detected in the blood at different time points during once-daily intraduodenal administration of the antibody.

[0145] Figure 7. Application of α-amylase and pancreatic lipase-related proteins as endocytic transport vectors.

[0146] Figure 7A The SDS-PAGE analysis of the α-amylase 2A construct under reducing and non-reducing conditions is shown.

[0147] Figure 7B The ability of the α-amylase 2A construct to transport the antibody infliximab via endocytosis using the Yuss chamber was demonstrated.

[0148] Figure 7C The results show the SDS-PAGE analysis of the pancreatic lipase-associated protein 2 construct (UTAC-20) under reducing conditions, as well as the ability of this construct to perform endocytic transport of the antibody infliximab using the Uss chamber.

[0149] Materials and Background A fusion protein containing a human pancreatic triglyceride lipase (PNLIP) sequence linked to an FcIII antibody peptide was designed and named "IUTACC" (Intract Universal Active Substance Endocytotic Transporter). I ntract U universal T ranscytosis A ctive Cargo C arrier')).

[0150] Builder: The IUTACC construct was cloned into the bacterial expression vector pET-21a(+). The amino acid sequences of the open reading frames are shown in Table 1.

[0151] Expression, purification, and refolding of IUTACC: IUTACC bacterial expression in Escherichia coli The pET21a(+) bacterial expression vector containing the IUTACC open reading frame (Table 1) was freshly transformed into BL21 / DE3 chemicompetent bacterial cells and plated on LB-AMP (100 µg / ml) plates. Colonies were picked and inoculated into liquid LB-AMP (100 µg / ml) culture. The cultures were incubated at 37°C in baffled flasks with vigorous shaking at 275 rpm. When the optical density OD... 600 At a concentration of 0.6, IPTG was added to a final concentration of 1 mM, and the cells were cultured at 37°C for 4 hours with vigorous shaking at 275 rpm to induce protein expression. Cells were collected by centrifugation at 5000 rpm for 25 minutes in a MegaFuge16R (TX-400 rotor) Therm Scientific / Heraeus centrifuge. Each 500 ml culture pellet was resuspended and washed once with 30 ml PBS, then centrifuged again under the above conditions. The dried pellet was stored at -80°C until further processing.

[0152] Purification of IUTACC from bacterial inclusion bodiesIPTG-induced cell pellet (from 500 ml of culture) was thawed in a 37°C water bath and resuspended in B-PER (78243, Thermo Scientific) bacterial protein extraction reagent supplemented with 1.5 µl benzonase nuclease (E1014, Millipore, 250 U / µl). Cell lysis was achieved by rotating the pellet at room temperature for 30 minutes. The cell lysate was centrifuged at 5000 rpm for 25 minutes in a MegaFuge 16R (TX-400 rotor, Thermo Scientific / Heraeus) centrifuge. The pellet was washed four times with 30 ml of 20 mM Tris-HCl [pH 8], 150 mM NaCl, 1 mM EDTA [pH 8], and 0.5% Triton X-100. In the first of the four washes, 1 µl of benzonase nuclease (E1014, Millipore, 250 U / µl) was added to 30 ml of wash buffer. After resuspending the precipitate, the mixture was rotated at room temperature (approximately 20°C) for 30 minutes to promote washing and degradation of residual DNA / RNA. After washing, the precipitate was resuspended and dissolved in 30 ml of 20 mM Tris-HCl [pH 8], 150 mM NaCl, 1 mM EDTA [pH 8], 8 M urea, and 10 mM dithiothreitol (DTT). To promote the dissolution of inclusion bodies, the solution was placed in a 37°C water bath for 30 minutes. The solution was clarified by centrifugation (5000 rpm for 6 minutes in a MegaFuge 16R (TX-400 rotor) Thermo Scientific / Heraeus centrifuge). Imidazole was added to the supernatant to a final concentration of 5 mM. Subsequently, 1 ml of Ni was added to the protein-urea solution. ++ NTA affinity resin bead slurry (H5037, His-Select HF Nickel Affinity Gel, Millipore) was rotated at room temperature for 1 hour. Resin beads were collected by centrifugation (4000 rpm for 6 minutes in the centrifuge described above) and washed four times in urea buffer. Subsequently, the resin-bound IUTACC protein was eluted using 20 mM Tris-HCl [pH 8], 150 mM NaCl, 1 mM EDTA [pH 8], 8 M urea, 10 mM MTT, and 250 mM imidazole.

[0153] renaturation of urea-denatured IUTACC proteinTo refold the IUTACC construct, the protocol described for refolding human pancreatic triglyceride lipase was largely followed (Kawaguchi et al., 2018). Affinity-purified, eluted IUTACC protein—still denatured in 8M urea—was placed in a dialysis chamber (Slide-a-Lyzer, MWCO3500 Da, Thermo Scientific) and refolded by continuous dialysis incubation using gradually decreasing urea concentrations to slowly remove urea and promote proper refolding. Each dialysis incubation used a refolding buffer consisting of 10 mM Tris-HCl [pH 8], 150 mM NaCl, 10% glycerol, 2.5 mM CaCl2, 1 mM L-cysteine, 0.1 mM cysteine, and 400 mM L-arginine, supplemented with 6 M, 4 M, 2 M, or 1 M urea, or without urea. Each dialysis incubation lasted a minimum of two hours and a maximum of overnight. After the final dialysis step is completed, the IUTACC preparation is removed from the dialysis box, clarified by centrifugation, aliquoted, and stored frozen at -80°C.

[0154] In vitro assessment of the effect of IUTACC on IgG endocytic transport using the Yuss chamber device: Remaining porcine small and large intestine tissues were obtained from control pigs euthanized for unrelated research projects (Royal Veterinary College, London, UK), in accordance with ethical guidelines and with the approval of the ethics review committee. Tissues were immediately transported by express wet ice after euthanasia, dissected in the laboratory, and frozen as quickly as possible in aliquots at -80°C. New aliquots were removed from the -80°C freezer for each experiment. Uss chamber experiments were conducted using a Warner Instruments 6-chamber vertical Navicyte system (66-0032), which maintains tissues at 37°C during experiments and continuously circulates a carbonaceous gas mixture of 90% O2 / 10% CO2. The chamber pore size was 0.29 cm. 2Experiments were performed in Krebs-Bicarbonate Ringer (KBR) buffer (PBS, 2 mM CaCl2, 1 mM MgCl2, 10 mM D-glucose). Setup was performed according to the manufacturer's standard protocol and largely followed the methods described in other literature (Thomson et al., 2019). To measure IgG endocytic transport, FITC-IgG was purchased from Sigma / Merck (F9636) and subjected to gel filtration and IgG fractionation on a Superdex 200 column (GE Healthcare) to remove all free, uncoupled FITC. 20 µg / ml FITC-IgG was added to the top chamber alone or together with 20 µg / ml IUTACC protein. Samples were taken from the substrate (serosa side), and the presence of FITC fluorescence signal was analyzed as an indicator of IgG transit through the mounted tissue sample. Signal quantification was performed using a standard curve measurement method. Figure 1 In the experiment, various proteins were directly added to 1.5 ml of KBR buffer in the Uss chamber to start the experiment. From Figure 2 The experiment introduced a protocol change: before adding FITC-IgG and IUTACC proteins to the Uss chamber, they were pre-incubated separately in a smaller volume (300 µl) at 37°C for 15 minutes to promote complete binding equilibrium and maximize the amount of FITC-IgG pre-bound to IUTACC protein at the start of the Uss experiment. This protocol change resulted in a higher FITC-IgG level reaching the serous side chamber.

[0155] IUTACC promotes the transport of IgG across the pig small intestine. IUTACC protein was expressed, purified, and refolded as described above, and its ability to enhance IgG transport across small intestinal tissue was tested in vitro. Experiments were conducted in a Uss chamber apparatus using porcine small intestine from healthy intestinal animals. FITC-labeled IgG (which was then separated and purified by gel filtration fractionation) was used as a biomarker for reading.

[0156] Experimental setup such as Figure 1 As shown in Figure A, a small piece of porcine small intestine was clamped at the opening between the apical / mucosal cavity and the basal / serosa side cavity of the Yous chamber apparatus, with the luminal surface of the tissue facing the apical / mucosal cavity. Tissue viability was maintained as much as possible by controlling the temperature to 37°C, the presence of glucose and salt in the KBR buffer, and continuous bubbling of carbon-containing gas into the KBR buffer in both compartments of the Yous chamber. At the start of the experiment, 20 µg / ml FITC-IgG was added to 1.5 ml of KBR buffer in the mucosal cavity, either alone or with an equal mass of IUTACC protein. Samples were taken from the serosa side cavity at 1 hour and 2 hours to analyze the permeability of FITC-IgG through the small intestinal tissue. Figure 1 B shows the protein preparations used. The gel loading volume reflects the relative amounts of the two preparations used in the experiment. FITC-IgG, a disulfide-stabilized antibody of size 150 kDa, decomposes into its heavy and light chain subfractions under reducing gel conditions. IUTACC is approximately 55 kDa in size. This preparation also contains a significant number of disulfide-stabilized IUTACC dimers. Figure 1 C showed that adding IUTACC together with FITC-IgG to the mucosal compartment increased the amount of FITC-IgG found on the serosal side after 2 hours of incubation.

[0157] The ability of IUTACC to transport IgG across GI tissues depends on its IgG binding sites.

[0158] To understand how IUTACC promotes IgG transport, a mutant IUTACC lacking its engineered IgG binding site was constructed (see Materials and Methods). Equal amounts of protein preparations were generated from the IUTACC construct and the IgG-impaired mutant IUTACC, respectively. Figure 2 A). Comparing the effects of wild-type and mutant IUTACC proteins on the transport of FITC-IgG from the mucosal compartment to the serosal compartment, it was found that mutants lacking engineered IgG binding sites had a significantly reduced ability to transport FITC-IgG across intestinal tissue. Therefore, the molecular interaction between IUTACC and IgG is necessary for IUTACC to promote IgG transport.

[0159] IUTACC requires active vesicle transport to facilitate the transGI tissue transport of FITC-IgG. IUTACC enhances FITC-IgG transport at extremely low concentrations (approximately equimolar to the FITC-IgG biomarker), and this enhancement depends on its IgG binding site, suggesting that IUTACC functions through an active transport mechanism. To validate this, inhibitors of intracellular vesicle transport were tested. The first step in retrograde vesicle transport is endocytosis. Cells have multiple ways of endocytizing cargo, which can occur through various dynein-dependent, dynein-independent, or caveolae pathways. Many small-molecule inhibitors of endocytosis exist, but their mechanisms of action are often unclear because these molecules typically affect multiple levels of transport. Chlorpromazine is a well-studied inhibitor of endocytosis, believed to interfere with various dynein-dependent endocytic pathways by inhibiting dynein I GTPase function. Chlorpromazine has also been reported to partially inhibit retrograde transport of leptin in the Caco-2 / 15 epithelial barrier tissue culture monolayer system. In the Us chamber device, brief pre-incubation of GI tissue with chlorpromazine partially interfered with the ability of IUTACC to transport FITC-IgG into the serous side chamber. Figure 3 Therefore, the data indicate that IUTACC utilizes active endocytosis and a dynein-dependent pathway to transport IgG across GI tissues.

[0160] Tissue specificity: IUTACC works only in the small intestine, not in the colon.

[0161] To further understand the specificity of IUTACC action, colon tissue was used instead of small intestinal tissue for testing. Figure 4 (A, 4B). IUTACC failed to promote the transport of FITC-IgG in this tissue. This finding suggests that the mechanism by which IUTACC functions may involve receptors or transport components with tissue-restricted expression.

[0162] IUTACC can be used on fresh (unfrozen) GI tissue in a Us chamber device.

[0163] Shortly after the animal sacrifice, the ability of IUTACC to transport FITC-IgG across fresh (i.e., unfrozen) tissue of the porcine duodenum was tested. Figure 5 The results show that IUTACC can transport FITC-IgG across fresh tissue, therefore its effect does not depend on the presence of tissue microscopic damage caused by freeze-thaw cycles. This data is as close as possible to in vivo experiments and also verifies the feasibility of using the Uss chamber device of frozen porcine tissue as a screening detection method for IUTACC research.

[0164] In vivo assessment of the ability of bispecific molecules to transport antibodies from the GI lumen of mice across the GI epithelial barrier into the bloodstream. The ability of the bispecific fusion protein transport antibody infliximab to transport two bispecific fusion proteins was tested in vivo. These two bispecific fusion proteins have the following sequences: UTAC-7 as well as UTAC-15 In the amino acid sequence of the above-mentioned bispecific fusion protein polypeptide construct: the underlined sequence is the sequence after removing the N-terminal 16 amino acid leader peptide of the human pancreatic triglyceride lipase (PNLIP) gene. This sequence is fused with linkers containing (bold) StrepTactinII and His6 affinity tags, respectively, and then ( Underline and italics () is one or two copies of the immunoglobulin Fc binding peptide.

[0165] Infliximab antibody and aprotinin (a component of Intract's Soteria protease inhibitor) were co-formulated with an equal volume of purified bispecific fusion protein (UTAC-7 or UTAC-15) to achieve a 20 mg / kg dosing dose in mice (each dose contained 0.5 mg of infliximab and 0.5 mg of aprotinin, with or without 0.5 mg of protein (UTAC-7 or UTAC-15) per 25 gr mouse, for a final dosing volume of 100 μl). All protein components remained stable when co-formulated at a concentration of 5 mg / ml, as determined by clear solutions, ability to withstand freeze-thaw cycles, and no degradation observed by size exclusion chromatography or SDS-PAGE analysis. Figure 6, subplot A, shows the SDS-PAGE analysis results of the formulation used in the in vivo study (reduction conditions: +DTT).

[0166] Figure 6BA schematic diagram of the in vivo experimental design is provided: Twelve mice underwent duodenal cannulation and subsequently recovered for 10 days. On day 0, three experimental groups (four mice per group) began intraduodenal administration once daily (QD) for six consecutive days. Mice in each group received infliximab / aprotinin alone, infliximab / aprotinin + UTAC-7, or infliximab / aprotinin + UTAC-15, respectively. Blood samples were collected from each mouse on day 0 (1 hour after administration), day 2 (2 hours after administration), day 4 (2 hours after administration), and day 5 (5 hours after administration). The presence of infliximab in the blood samples was then analyzed using ELISA. The ELISA method used has been described in the literature (Awad et al., '3D printedinfliximab suppositories for rectal biologic delivery', International Journal of Pharmaceutics: X 5 (2023) 100176, https: / / doi.org / 10.1016 / j.ijpx.2023.100176).

[0167] like Figure 6C As shown, after 6 consecutive days of once-daily (QD) dosing, infliximab levels in the blood increased and accumulated. Compared with infliximab alone, both UTAC-7 and UTAC-15 promoted the rapid transport of infliximab into the bloodstream (see first time point: Day 0, 1 hour after the first dose). Furthermore, when infliximab levels accumulated in the blood through continuous QD dosing (taking advantage of the antibody's long half-life), the combination with UTAC-7 (UTAC-15 was slightly less effective) also showed advantages in the long term, with a trend toward higher steady-state levels of infliximab in the blood compared to the control group using infliximab alone. UTAC-7 and UTAC-15 contain the same IgFc-binding peptide, but differ in their formulation (UTAC-7 contains one copy of the HWRGWV (SEQ. ID No: 23) antibody-binding peptide, while UTAC-15 carries two copies of this peptide in tandem; see SEQ. ID NO: 43 and SEQ. ID NO: 44). At day 0, 1 hour after administration, three out of four blood samples from the infliximab-only control group were below the limit of detection (orange dots).

[0168] Alternative transport constructs containing human α-amylase or pancreatic triglyceride lipase-related proteins as endocytic transport-driven components. Constructs containing human α-amylase or pancreatic triglyceride lipase-related proteins as endocytic transport-driven components were generated and tested.

[0169] A fusion protein construct containing the full-length human pancreatic α-amylase gene AMY2A linked to an IgFc-binding peptide was prepared. The amino acid sequence of this construct is shown below: Human α-amylase (AMY2A) transporter construct (for mammalian expression): In the above amino acid sequence: the underlined sequence is the human pancreatic α-amylase (AMY2A) gene sequence, in which... italics Part of it is a leader peptide. This sequence is fused to a linker containing a (bold) His6 affinity tag, followed by ( Underline and italics () is an immunoglobulin Fc-binding peptide.

[0170] The fusion protein was expressed and purified in HEK293 mammalian cells, and SDS-PAGE analysis was performed under both reducing and non-reducing conditions. Figure 7A ).

[0171] Using fresh rat small intestine tissue in the Us device, infliximab, alone or with an α-amylase (AMY2) transport construct, was added to the apical chamber. Infliximab levels reaching the basal chamber were assessed using ELISA readings of the TNFα-capture material. The ELISA method used is described in the literature (Awad et al., '3D printedinfliximab suppositories for rectal biologic delivery', International Journal of Pharmaceutics: https: / / doi.org / 10.1016 / j.ijpx.2023.100176).

[0172] like Figure 7B As shown, the α-amylase-containing construct enhanced the trans-GI epithelial transport of infliximab across rats, indicating that α-amylase does indeed play a role in the endocytic transport system.

[0173] Further, a transport fusion protein construct was generated, containing the (full-length) human pancreatic lipase-associated protein 2 (PNLIPRP2) gene fused to an IgFc-binding peptide. The amino acid sequence of this construct is shown below: The UTAC-20 construct (lntract-20) contains the full-length human PNLIPRP2 gene sequence minus the leader sequence (for bacterial expression): In the above amino acid sequence: the underlined sequence is the human PNLIPRP2 gene sequence minus the leader sequence (PNLIPRP2).18-469 This sequence is fused to a linker containing a (bold) streptavidin II affinity tag and a His6 affinity tag, subsequently ( Underline and italics () is an immunoglobulin Fc-binding peptide.

[0174] The UTAC-20 construct was produced, purified, and refolded in *E. coli* in the same manner as the IUTACC fusion protein carrying the PNLIP pancreatic lipase gene. SDS-PAGE analysis of the preparation was performed under reducing conditions. Figure 7C (Left split diagram).

[0175] The PNLIPRP2 construct was tested using porcine small intestinal GI tissue in a Us device, with FITC-labeled human IgG as the reading. Similar to the aforementioned PNLIP construct, PNLIPRP2 also enhanced IgG endocytic transport. Figure 7C (Right-side diagram).

[0176] References Bendayan, M., Ziv, E., Ben-Sasson, R., Bar-On, H., & Kidron, M. (1990). Morpho-cytochemical and biochemical evidence for insulin absorption by therat ileal epithelium. Diabetologia , 33 (4). https: / / doi.org / 10.1007 / BF00404796 Bendayan, M., Ziv, E., Gingras, D., Ben-Sasson, R., Bar-On, H., & Kidron, M. (1994). Biochemical and morpho-cytochemical evidence for theintestinal absorption of insulin in control and diabetic rats. Comparison between the effectiveness of duodenal and colon mucosa. Diabetologia , 37 (2).https: / / doi.org / 10.1007 / s001250050081 Bruneau, N., Bendayan, M., Gingras, D., Ghitescu, L., Levy, E.,&Lombardo, D. (2003). Circulating bile salt-dependent lipase originates fromthe pancreas via intestinal transcytosis. Gastroenterology , 124 (2). https: / / doi.org / 10.1053 / gast.2003.50051 Bruneau, N., Richard, S., Silvy, F., Verine, A.,&Lombardo, D. 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Claims

1. A composition comprising a nonbacterial transporter domain capable of transcellular endocytosis across the intestinal wall, said transporter domain being linked to an antibody-binding portion.

2. The composition of claim 1, wherein the transporter domain comprises an inactivated form of a protein capable of transintestinal endocytosis.

3. The composition according to any of the preceding claims, wherein the transporter domain is derived from a protein selected from: pancreatic lipase, bile salt-dependent lipase (BSDL) / carboxy ester lipase (CEL) / cholesterol esterase and α-amylase, or homologs thereof, preferably pancreatic lipase.

4. The composition of any of the preceding claims, wherein the antibody-binding portion comprises an Fc-binding domain or an antibody light chain (LC)-binding domain or a homologue or derivative thereof.

5. The composition of any of the preceding claims, wherein the antibody-binding portion comprises an antibody-binding peptide.

6. The composition of claim 5, wherein the antibody-binding peptide is selected from protein A, protein G, protein L, protein Z (derived from the B domain of SpA), protein LG, protein LA, protein AG, SpA, PAM (a peptide having the sequence (RTY)4K2KG) (SEQ. ID No: 18), Fc-III, FcBP-2, FC-III-4C, FcRM, or peptides having sequences 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). 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), cyclic (Nα-Ac)S(A)-RWHYFK-Lact-E (SEQ. ID No:37), cyclic (Nα-Ac)Dap(A)-RWHYFK-Lact-E (SEQ. ID No:38), and cyclic [link-M-WFRHYK] (SEQ. ID No:39) or their homologs, preferably HWRGWV (SEQ. ID No:23) and / or HWRGWVC (SEQ. ID No:40).

7. The composition of claim 5 or claim 6, wherein the antibody-binding peptide comprises the sequence AWHLGELVW (SEQ. ID No. 12).

8. The composition of any one of claims 5 to 7, wherein the antibody-binding peptide comprises the sequence DCAWHLGELVWCT (SEQ. ID No. 13).

9. The composition of any of the preceding claims, wherein the antibody-binding portion is covalently linked to the transporter domain.

10. The composition of any one of claims 5 to 9, wherein the transporter domain forms a fusion protein with the antibody-binding peptide.

11. The composition according to any of the preceding claims, further comprising an antibody.

12. The composition of claim 11, wherein the composition is a pharmaceutical composition.

13. The pharmaceutical composition of claim 11, further comprising one or more excipients.

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

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

16. The pharmaceutical composition of claim 13 or claim 14, wherein the dipeptide is carnosine or diglycinin.

17. The pharmaceutical composition according to any one of claims 11 to 15, further comprising a coprotein.

18. The pharmaceutical composition according to any one of claims 11 to 16, for oral delivery.

19. The composition according to any one of claims 11 to 18, for use in medicine.

20. The composition of any one of claims 11 to 19, for use in the treatment of cancer, autoimmune diseases, or Alzheimer's disease.

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

22. A fusion protein comprising a transporter domain and an antibody-binding domain capable of transintestinal endocytosis.

23. The fusion protein of claim 22, wherein the transporter domain comprises an inactivated form of a protein capable of transintestinal endocytosis.

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

25. The fusion protein of any one of claims 22 to 23, wherein the antibody-binding domain is located at the C-terminus or N-terminus of the transporter domain.

26. The fusion protein according to any one of claims 22 to 25, wherein the antibody-binding domain is selected from protein A, protein G, protein L, protein Z (B domain derived from SpA), protein LG, protein LA, protein AG, SpA, PAM (a peptide having the sequence (RTY) 4K2KG) (SEQ. ID No: 18), Fc-III, FcBP-2, FC-III-4C, FcRM, or a peptide having 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: 40), ... HYFKFD (SEQ. ID No: 24), HFRRHL (SEQ. ID No: 25), HWCitGWV (SEQ. ID No: 26), DAAG (SEQ. ID No: 40), HYFKFD (SEQ. ID No: 24), HFRRHL (SEQ. ID No: 25), HWCitGW 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), cyclic (Nα-Ac)S(A)-RWHYFK-Lact-E (SEQ. ID No:37), cyclic (Nα-Ac)Dap(A)-RWHYFK-Lact-E (SEQ. ID No:38), and cyclic [link-M-WFRHYK] (SEQ. ID No:39) or their homologs, preferably HWRGWV (SEQ. ID No:23) and / or HWRGWVC (SEQ. ID No:40).

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

28. The fusion protein according to any one of claims 22 to 26, wherein the fusion protein has the sequences of SEQ ID No. 1, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46 or sequences of homologs having at least 60% homology.

29. A nucleic acid sequence, wherein: (i) Encoding the fusion protein as described in any one of claims 22 to 28; (ii) Encoding a homolog or derivative of the fusion protein as described in any one of claims 22 to 28; (iii) A sequence that is complementary to the sequence in (i) or (ii); or (iv) Having a sequence that is substantially identical to any one of (i), (ii) or (iii).

30. An expression vector comprising the nucleic acid sequence as described in claim 29.

31. A cell comprising the expression vector as described in claim 30.

32. A pharmaceutical composition comprising (i) a transporter domain linked to an antibody-binding peptide and capable of transintestinal endocytosis, and (ii) an antibody.

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