Compositions and methods for oral administration

Abstract

SOLUTION: Compositions and methods are disclosed for targeted delivery of therapeutic polypeptides and protein-based therapeutics across the gastrointestinal lining. In one embodiment, provided is a polypeptide construct comprising: (a) a first polypeptide, the first polypeptide comprising an amino acid sequence at least 80% identical to an amino acid sequence selected from any one of SEQ ID NOS: 1-40; and (b) a second polypeptide, the second polypeptide being heterologous to the first polypeptide. In one embodiment, the heterologous polypeptide is a therapeutic polypeptide.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 288,579, filed December 11, 2021, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Traditionally, small molecule or low molecular weight drugs have been orally administered. On the other hand, other drugs such as peptides and proteins are often unstable, have large molecular weights, and / or are polar, and have poor permeability through biological membranes, so oral administration does not provide significant therapeutic effects. When such large molecular weight drugs are administered orally, many of them undergo proteolysis in the gastrointestinal tract, making it difficult for them to be transferred into body fluids. For this reason, therapeutic polypeptides and proteins are mainly administered by injection or infusion, which is significantly less convenient and more expensive and burdensome than oral administration.

[0003] Gastric and intestinal proteolytic enzymes break down and inactivate biologics and polypeptide-based therapeutics before they can be absorbed into the bloodstream. Polypeptides that survive proteolysis by gastric proteases (which typically have an acidic pH) are also acted upon by small intestinal proteases and enzymes secreted by the pancreas (which typically have a neutral to basic pH). A particular challenge in the oral administration of polypeptides is due to their relatively large molecular size and the charge distribution they carry. This relatively large molecular size and charge distribution make it difficult for polypeptides to penetrate the mucus of the intestinal wall and to pass into the blood.

[0004] Oral administration of therapeutic polypeptides has two main challenges: a) degradation of the polypeptides by proteolytic enzymes in the stomach and intestine, and b) poor absorption, i.e., difficulty in transporting the polypeptides to the bottom of the intestinal tract and releasing them into the bloodstream. Improving the efficacy of oral administration, i.e., increasing the bioavailability of oral biologics and polypeptide-based drugs, is an unmet medical need. Summary of the Invention

[0005] Disclosed herein are compositions and methods for the targeted delivery of therapeutic polypeptides and protein-based therapeutics across the gastrointestinal lining.

[0006] In one aspect, provided is a polypeptide construct comprising: (a) a first polypeptide, the first polypeptide comprising an amino acid sequence at least 80% identical to an amino acid sequence selected from any one of SEQ ID NOS: 1-40; and (b) a second polypeptide, the second polypeptide being heterologous to the first polypeptide. In one aspect, the heterologous polypeptide is a therapeutic polypeptide.

[0007] In another embodiment, the first polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 1-40. In another embodiment, the first polypeptide comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 1-40.

[0008] In another embodiment, the first polypeptide comprises an amino acid sequence that is at least 98% identical to an amino acid sequence selected from any one of SEQ ID NOs: 1-40. In another embodiment, the first polypeptide comprises an amino acid sequence that is at least 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 1-40. In another embodiment, the first polypeptide comprises an amino acid sequence that comprises an amino acid sequence selected from any one of SEQ ID NOs: 1-40.

[0009] In another embodiment, a pharmaceutical composition for targeted delivery across the gastrointestinal lining following oral administration to a subject comprises a therapeutically effective amount of a polypeptide construct comprising a polypeptide having at least 80% sequence identity to one or more of SEQ ID NOs: 1-40, wherein said polypeptide is linked to a heterologous polypeptide.

[0010] In another embodiment, the composition for targeted delivery across the gastrointestinal lining following oral administration of the composition to a subject further comprises one or more of a pharma- ceutically acceptable additive, excipient, stabilizer, permeability enhancer, or protease inhibitor.

[0011] In another aspect, disclosed herein is a polypeptide construct suitable for targeted delivery of a heterologous polypeptide across the gastrointestinal lining of a subject.

[0012] In another embodiment, a targeted delivery system is configured comprising a heterologous polypeptide and a means for transporting said heterologous polypeptide across said gastrointestinal lining of a subject, said heterologous polypeptide being a therapeutic polypeptide, said means for transporting comprising providing a polypeptide having at least 80% sequence identity to a polypeptide according to SEQ ID NOs: 1-40, and conjugating said polypeptide to said heterologous polypeptide.

[0013] In another embodiment, a polypeptide construct comprises a polypeptide linked to a heterologous polypeptide by a linker, said linker being an amide bond formed between an alkyl-modified peptide on said polypeptide and an azide-modified peptide on said heterologous polypeptide.

[0014] The modular nature of the disclosed compositions and methods for targeted drug delivery provides an advantageous means for oral formulation of polypeptide-based therapeutics. [Brief description of the drawings]

[0015] [Figure 1A] 1 shows an overview of a targeted drug delivery system for delivering a composition comprising a polypeptide construct comprising: (a) a first polypeptide (denoted in the figure as "peptide transporter"), the first polypeptide comprising an amino acid sequence at least 80% identical to an amino acid sequence selected from any one of SEQ ID NOS: 1-40; and (b) a second polypeptide (denoted in the figure as "therapeutic (protein)"), the second polypeptide being a therapeutic polypeptide heterologous to the first polypeptide, the second polypeptide being delivered by passing through the gastrointestinal lining and into the bloodstream after oral administration to a subject. Through an active endocytosis process, the polypeptide construct is absorbed into the apical cell wall, translocates and exits through the basal wall, where the first polypeptide is naturally cleaved by thrombin in the blood, thereby delivering the therapeutic polypeptide into the bloodstream. [Figure 1B]1 shows an overview of a drug delivery system comprising a polypeptide construct, the drug delivery system comprising: (a) a first polypeptide, the first polypeptide comprising an amino acid sequence at least 80% identical to an amino acid sequence selected from any one of SEQ ID NOs. 1-40; and (b) a second polypeptide, the second polypeptide being heterologous to the first polypeptide. In one embodiment, the peptide according to SEQ ID NOs. 1-40 is attached at the N-terminus or C-terminus of the therapeutic polypeptide. The therapeutic polypeptide may be a biological agent, a peptide-based agent, or a macromolecular agent not suitable for oral administration. The disclosed drug delivery method provides a means to convert drug delivery limited to IV / SQ to delivery by PO. Shown in FIG. 1B are representative examples of polypeptide constructs containing therapeutic polypeptides, such as erythropoietin (PT-EPO), GLP-1, GLP-1 agonists (PT-GA-1, PT-GA2), and octreotide (PT-OCT), with other therapeutic proteins listed in Table 2. [Figure 1C] 1A-1C show schematics of targeted delivery polypeptide constructs and targeted delivery systems, in which the polypeptide is conjugated to a therapeutic polypeptide, such as a biologic agent, and the conjugation is via ligation of the polypeptide to a protein. (Note: the constructs shown in FIGS. 1B and 1C are not drawn to scale.) [Diagram 2]1 shows the in vivo uptake of polypeptide constructs comprising a polypeptide having an amino acid sequence having sequence identity to SEQ ID NO: 1-40, and a heterologous polypeptide, as determined by a fluorescence assay in a Caco-2 cell model (see Example 1). Caco-2 cells are cultured in the presence of a polypeptide and a polypeptide construct comprising a heterologous polypeptide, and analyzed using a fluorescence assay to determine the percentage of cellular uptake of the polypeptide construct. The polypeptide constructs are, from left to right, BSA (bovine serum albumin), elosulfase alpha, factor VIII, g-CSF, belatacept, glucarpidase, erythropoietin (EPO), and a polypeptide construct comprising a polypeptide according to SEQ ID NO: 1 bound to factor IX. Several peptide constructs were tested for uptake by Caco-2 cells, including various peptide constructs comprising truncated polypeptides. Although not shown in the graph, truncated polypeptides (as short as 20 amino acids in length, as represented by the polypeptides according to SEQ ID NO: 1-20) were confirmed to enhance uptake of heterologous polypeptides by Caco-2 cells compared to controls. [Diagram 3] Figure 1 shows an outline of an in vivo animal study using Sprague-Dawley rats to test a method of targeted delivery of an oral dosage form of human erythropoietin. Sprague-Dawley rats (n=8) were administered a composition comprising a polypeptide construct of SEQ ID NO: 41 (designated "PT-EPO" in the figure). The polypeptide construct comprised a polypeptide according to SEQ ID NO: 1 and a heterologous polypeptide according to SEQ ID NO: 43. Compositions comprising PT-EPO at concentrations of 2.5 mg / kg, 1 mg / kg, and 0.25 mg / kg in PBS were administered orally (per os) (PO). Compositions comprising PT-EPO at concentrations of 0.5 mg / kg in PBS were administered intravenously (IV) as a separate control / reference for bioavailability. Blood was collected from treated animals (PO and IV) at time intervals following administration including 0, 5, 15, 30, and 60 minutes, as well as 2, 4, 8, and 24 hours. The samples were tested to assess the presence or absence of human erythropoietin in the bloodstream of the subject animals. [Figure 4] Figures 4A and 4B show the uptake and bioavailability of targeted delivery methods for oral administration of erythropoietin (see Examples 2-5). Figure 4A. A composition comprising a polypeptide construct of sequence ID 41 (PT-EPO) was administered PO and IV in three doses. Serum was collected at various time points over a 24-hour period, including 0, 5, 15, 30, and 60 minutes, as well as 2, 4, 8, and 24 hours after administration. At all doses administered, a polypeptide according to sequence ID 56 (human erythropoietin with glycine and alanine at the N-terminus) was detected in the serum of treated rats. Figure 4B. Western blot was used to separate human erythropoietin with glycine and alanine at the N-terminus from other serum proteins. The nucleotide sequence of this band was determined and confirmed to be the amino acid sequence corresponding to the full-length sequence of human erythropoietin, with two additional amino acid residues ("GA") remaining. This data confirms that the polypeptide construct of sequence ID 41 crossed the gastrointestinal (GI) barrier when administered orally, and that the polypeptide of sequence ID 56 was introduced into the bloodstream. [Diagram 5] 1 shows the presence of a polypeptide of sequence ID 57 in the serum of rats following oral and intravenous administration of a composition comprising a polypeptide construct of sequence ID 41. The data indicates that following administration of the composition, the polypeptide construct is cleaved to produce two polypeptide fragments: a polypeptide according to sequence ID number 56 and sequence ID number 57. [Figure 6] An outline of an animal model study to test the efficacy of a composition comprising a polypeptide according to sequence ID41 for targeted delivery of erythropoietin across the gastrointestinal barrier is shown. Sprague-Dawley rats (n=8) were administered 600 μg (daily PO) of a composition comprising a polypeptide construct with sequence identity corresponding to sequence ID41 (see Example 3). A corresponding control group (n=8) was administered a solvent control (PBS solution containing 10 mM maltose) PO. Blood samples were taken on days 0, 14, and 28 and tested for the presence of human erythropoietin. Hemoglobin levels were also measured. [Figure 7]Human erythropoietin was detected in Sprague-Dawley rats (n=8) administered (daily PO) a composition containing a polypeptide construct having sequence identity corresponding to sequence ID41, indicating that the composition can cross the intestinal barrier and deliver erythropoietin to the bloodstream of the animal. [Figure 8] Figure 2 shows the therapeutic effect of a composition comprising a polypeptide construct according to sequence ID 41 after oral administration (600 μg PO daily) to Sprague-Dawley rats. The mean hemoglobin levels of treated animals increased over time (measured in weeks) after oral administration. [Figure 9] The therapeutic effect in a second animal (dog) model after oral administration of a composition comprising a polypeptide construct having sequence identity corresponding to sequence ID 41 is shown (see Example 5). After oral administration of a composition comprising a polypeptide having sequence identity corresponding to sequence ID 41, hemoglobin and hematocrit levels were increased in a subject dog (beagle). The doses were 1 mg / kg, 5 mg / kg, 50 mg / kg, and 125 mg / kg, each dissolved in PBS and administered PO as a single dose. [Figure 10] 1 shows summary data for red blood cell counts, hemoglobin levels, and hematocrit levels following a single PO administration of a composition comprising a polypeptide construct according to sequence ID41, compared to a control (see Example 5). [Figure 11]1 shows the uptake in a Caco-2 cell model of a polypeptide construct comprising a polypeptide having sequence identity to SEQ ID NO:1 and linked to one or more heterologous polypeptides having sequence identity to SEQ ID NO:55-56, wherein the heterologous polypeptide comprises a GLP-1 agonist (see Example 6). In the figure, exemplary polypeptide constructs include a polypeptide construct comprising a polypeptide according to sequence ID 1 bound to a heterologous polypeptide comprising a polypeptide according to sequence ID 55 (exenatide analogue) (referred to as "PT-GA1" and corresponding to the polypeptide construct having sequence identity to sequence ID 42), a polypeptide construct comprising a polypeptide according to sequence ID 1 bound to a heterologous polypeptide comprising a polypeptide according to sequence ID 56 (semaglutide / liraglutide analogue) (referred to as "PT-GA2" and corresponding to the polypeptide construct having sequence identity to sequence ID 44), and a polypeptide construct comprising a polypeptide according to sequence ID 1 bound to a semaglutide / liraglutide analogue referred to as PT-GA2 is referred to as PT-GA2 (and corresponds to the polypeptide construct according to sequence ID 46). [Figure 12] The in vivo therapeutic effect of a composition comprising a polypeptide construct according to sequence ID 42 or 44 is demonstrated by the reduction in blood glucose levels after oral administration of the composition (dosage of 600 μg in PBS). [Figure 13] 1 shows an outline of a polypeptide having sequence identity to sequence ID21, with a modified moiety at the C-terminus comprising a modified lysine residue (Lys(N3), a ligand for ligating and conjugating the polypeptide to a second, heterologous polypeptide). [Figure 14] FIG. 1 outlines a chemical formula representing a heterologous polypeptide comprising an octreotide analog (pentynoyl-octreotide as a modification) for ligation (binding) to a polypeptide having sequence identity to SEQ ID NOs: 21-40 (see Example 7). [Figure 15]Figures 15A-15D outline a formula comprising a polypeptide construct (designated "PT-OCT") comprising a polypeptide according to sequence ID 21 ligated via click chemistry to a heterologous polypeptide, where the heterologous polypeptide is an octapeptide (an octreotide analogue) according to sequence ID 53 (and modified according to Figure 14). [Figure 16] An outline of click chemistry reactions using alkyne- and azide-modified peptides is shown (see Example 7 and FIG. 15). [Figure 17] FIG. 1 shows the in vitro uptake of polypeptide constructs having sequence identity corresponding to sequence ID numbers 41, 49, and 50. Caco-2 cells were treated with a composition comprising a polypeptide construct having sequence identity corresponding to sequence ID 41 (denoted in the figure as PT-EPO), a polypeptide construct having sequence identity corresponding to sequence ID 49 (denoted in the figure as PT-OCT "fusion"), and a polypeptide construct having sequence identity corresponding to sequence ID 50 (denoted as PT-OCT "linked") at a concentration of 5 μg / mL for 2 hours. Based on a fluorescence assay, it was confirmed that more than 30% of the constructs were taken up by Caco-2 cells, indicating the ability of the composition to pass through the gastrointestinal tract. The side-by-side comparison also validates the use of click chemistry ligation as a method for generating polypeptide constructs as disclosed herein, along with traditional expression vectors and other recombinant methods for generating polypeptide constructs. [Figure 18] The therapeutic effect of a peptide construct comprising a polypeptide having sequence identity to sequence ID50 (denoted in the figure as "PT-OCT") and the same construct following cleavage with thrombin (denoted in the figure as "PT-OCT (slice)"). Each polypeptide construct (full length and "slice") causes a decrease in relative glucose secretion by glucose stimulated islet cells (compared to control). The ability of the polypeptide constructs to inhibit insulin secretion from glucose stimulated islet cells was thus confirmed. [Figure 19]We show that a polypeptide construct (designated as PT-OCT) comprising a polypeptide with sequence identity corresponding to sequence ID50 was taken up by Caco-2 cells in vitro and that a composition comprising a polypeptide construct comprising a polypeptide with sequence identity corresponding to sequence ID50 results in targeted delivery of a therapeutic polypeptide when administered (PO) to rats (untreated cells and cells treated with a polypeptide construct with sequence identity corresponding to sequence ID41 were used as controls). For the in vivo study, Wistar rats (n=2) were administered (PO) a composition comprising a polypeptide construct with sequence identity corresponding to sequence ID50 at a concentration of 600 μg / animal (in PBS). Blood samples were taken from the animals at 0, 3, and 5 hours after administration. The samples were analyzed and showed elevated levels of the polypeptide fragment according to sequence ID53. Thus, oral administration of a composition comprising a peptide construct was confirmed to result in targeted delivery of a heterologous polypeptide across the gastrointestinal barrier. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Oral administration of certain therapeutic agents, including proteins, peptides, and other macromolecules, is extremely challenging for a variety of reasons. Thus, injection or hydrophilic routes of administration are practiced as the only routes of administration for certain therapeutic agents. The digestive system is inherently designed to break down molecules prior to absorption. The poor bioavailability of biologics and peptide-based drugs remains an active area of ​​research. The present disclosure provides a promising avenue for site-specific drug delivery and improves the oral bioavailability of biologics from less than 1% to over 50%, enabling the specific delivery of therapeutic agents that were previously not suitable or formulated for oral administration.

[0017] The present disclosure provides one or more of the following major advantages for achieving targeted delivery of polypeptide-based therapeutics by the oral route: a) preventing proteolytic activity that degrades the therapeutic in the stomach and intestine, b) providing protease-resistant therapeutic polypeptide analogs that retain biological activity, c) stabilizing the therapeutic or polypeptide by conjugation with a polypeptide that acts as a "shielding molecule", and / or d) improving passive therapeutic or polypeptide transport (diffusion) across the intestinal epithelial membrane.

[0018] The present disclosure provides compositions and methods for formulating polypeptide-based therapeutics for oral delivery. Although polypeptide-based therapeutics are difficult to administer by the oral route, they have several advantages over small molecule drugs. First, proteins often perform a highly specific and complex set of functions that cannot be mimicked by simple chemical compounds. Second, because the actions of proteins are highly specific, protein therapeutics are often less likely to interfere with normal biological processes and cause side effects. Third, because many of the proteins used as therapeutics are produced naturally in the body, these drugs are often well tolerated and less likely to cause an immune response. Fourth, for diseases where genes have mutations or deletions, protein therapeutics can provide an effective alternative treatment without the need for gene therapy, which is currently unavailable for most genetic diseases. Fifth, the time required for clinical development and FDA approval of protein therapeutics is faster than that of small molecule drugs.

[0019] Relatively few protein therapeutics are purified from native sources, such as pancreatic enzymes from pig and piglet pancreases and alpha-1-proteinase inhibitor from pooled human plasma, but most are now produced by recombinant DNA technology and purified from a wide range of organisms. Recombinant protein production systems include bacteria, yeast, insect cells, mammalian cells, and genetically modified animals and plants. The choice of system is determined by the cost of production and the protein modifications (e.g., glycosylation, phosphorylation, proteolytic cleavage) required for biological activity. For example, bacteria do not perform glycosylation reactions, and each of the other biological systems listed above produces a different type or pattern of glycosylation. The glycosylation pattern of a protein can dramatically affect the activity, half-life, and immunogenicity of the recombinant protein in the body. For example, the half-life of native erythropoietin (see below), a growth factor important for red blood cell production, can be increased by increasing the glycosylation of the protein. Darbepoetin-a is an erythropoietin analogue that has been engineered to contain two additional amino acids that are substrates for N-linked glycosylation. When expressed in Chinese hamster ovary cells, the analogue is synthesized with five N-linked glycans instead of three. This modification increases the half-life of darbepoetin by three-fold compared to erythropoietin.

[0020] Recombinantly produced proteins have several further advantages compared to non-recombinant proteins. First, transcribing and translating precise human genes results in higher specific activity of the protein and less chance of immunological rejection. Second, recombinant proteins are often produced more efficiently and cheaply, and in potentially unlimited quantities. One prominent example is the protein-based treatment of Gaucher disease. Gaucher disease is a chronic congenital disorder of lipid metabolism caused by a deficiency of the enzyme β-glucocerebrosidase (also known as glucosylceramidase) and is characterized by enlarged liver and spleen, increased skin pigmentation, and painful bone lesions. Initially, β-glucocerebrosidase purified from human placenta was used to treat the disease, but this required purifying the protein from 50,000 placentas per patient per year, and there were obvious practical limitations to the availability of purified protein. Recombinant forms of β-glucocerebrosidase were subsequently developed and introduced. Not only would this recombinant form be available in sufficient quantities to treat many more patients with the disease, but it would also eliminate the risk of transmissible diseases (e.g., viral or prion) associated with purifying the protein from human placenta, demonstrating a third advantage of recombinant proteins over non-recombinant proteins: reduced exposure to animal and human diseases.

[0021] A fourth advantage is that recombinant techniques allow the modification of proteins or the selection of specific genetic variants to improve function or specificity. Here again, recombinant β-glucocerebrosidase provides an interesting example. When this protein is made recombinantly, a mannose residue can be added to the protein by changing the amino acid arginine to histidine. This mannose is recognized by the endocytic glycan receptors of macrophages and many other cell types, allowing the enzyme to enter these cells more efficiently and cleave intracellular lipids that have accumulated to pathological amounts, resulting in improved therapeutic outcomes. Finally, recombinant techniques allow the production of proteins that provide novel functions or activities, as discussed below.

[0022] Delivery of certain therapeutic proteins, including heterologous polypeptides, proteins, and peptides, and other macromolecular therapeutics, has been limited to injection or parenteral administration.Therefore, provided herein are compositions and methods for targeted delivery of therapeutics through gastrointestinal lining.In embodiments, the disclosed compositions and methods improve the oral bioavailability of polypeptides and proteins from less than 1% to more than 50%, even for therapeutics that were previously considered not suitable for oral administration or not formulated for oral administration.

[0023] definition The following terms are used in this disclosure to describe different embodiments. These terms are used for descriptive purposes only and are not intended to limit the scope of any aspect of the subject matter claimed herein.

[0024] As used herein, "active agent" refers to a biological, chemical, or molecular moiety that has an activity that produces a therapeutic effect.

[0025] As used herein, a "composition" or "formulation" refers (interchangeably) to an active agent in a particular form, such as an aqueous solution, solid, semisolid, or aerosol, for administration by oral or parenteral routes. Optionally, the formulation includes a pharma- ceutically acceptable carrier, excipient, and / or one or more additives. The formulations disclosed herein may contain other known active agents in combination with the active agents described herein.

[0026] The term "fusion protein" as used herein refers to a synthetic, semi-synthetic, or recombinant protein molecule that contains all or part of two or more different proteins, and / or peptides, and / or polypeptides. For example, provided herein are fusion proteins that contain a polypeptide and a heterologous polypeptide linked to each other. In some embodiments, the fusion protein is synthesized in vitro. In some embodiments, the two or more different polypeptides and / or peptides that the fusion protein comprises are produced separately and then covalently linked. In some embodiments, the fusion protein is expressed as a recombinant protein.

[0027] As used herein, an amino acid sequence or nucleotide sequence is "heterologous" to another sequence to which it is operably linked when the two sequences are not naturally related. Such linkage is not necessarily covalent. For example, provided herein is a fusion protein or recombinant protein comprising a polypeptide and a heterologous polypeptide or protein, where the polypeptide and the heterologous polypeptide / protein are not naturally related. Also provided herein is a polypeptide construct comprising a polypeptide and a heterologous polypeptide.

[0028] The term "linker" as used herein refers to a cleavable or non-cleavable bond between a polypeptide and a heterologous polypeptide. Linkers take many forms, as will be appreciated by those of skill in the art. A linker can be a bond between two polypeptides, specifically resulting from a bond between the atoms of two amino acids, or a bond formed between the atoms of a modification or functional group on one or more amino acids.

[0029] As used herein, "peptide transporter" or "PT" is a term used to refer to a polypeptide having sequence identity to SEQ ID NOs: 1-40.

[0030] As used herein, a "polypeptide" refers to a polymer of three or more amino acids in a tandem sequence linked via peptide bonds. The term "polypeptide" includes proteins, protein fragments, protein analogs, oligopeptides, and the like. The term "polypeptide" contemplates a polypeptide encoded by a nucleic acid, produced by recombinant techniques, isolated from a suitable source, or synthesized. The term "polypeptide" further contemplates a polypeptide as defined above, including chemically modified amino acids, or amino acids covalently or non-covalently linked to other molecules, functional groups, ligation ligands, or labeling ligands.

[0031] The term "polypeptide construct" as used herein refers to a synthetic, semi-synthetic, or recombinant single molecule that comprises all or part of two or more different proteins and / or polypeptides. For example, provided herein is a polypeptide construct that comprises a first polypeptide and a second polypeptide, where the second polypeptide is heterologous to the first polypeptide. The second polypeptide that is heterologous to the first polypeptide is also referred to herein as a "heterologous polypeptide." In some embodiments, the polypeptide construct is synthesized in vitro. In some embodiments, the two or more different proteins and / or polypeptides that the polypeptide construct comprises are produced separately and then combined.

[0032] As used herein, "SEQ ID" or "SEQ ID NO" refers (interchangeably) to a protein, polypeptide, peptide fragment, or analog thereof, and includes modifications thereof, and refers to an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence designated by the number according to the numbers listed in Table 1.

[0033] The term "sequence identity" as used herein refers to the identity between two nucleic acid molecules, polypeptides, or amino acids, expressed as the identity or similarity between the sequences. Sequence identity can be measured as a percentage of identity, the higher the percentage, the more identical the sequences are. The percentage of identity is calculated over the entire length of the sequence. Homologs or orthologs of amino acid sequences have relatively high sequence identity when aligned by standard methods. This homology is more pronounced when comparing orthologous proteins from more closely related species (e.g., human and mouse sequences) with orthologous proteins from more distantly related species (e.g., human and nematode sequences). Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Nat. Acad Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:23744, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Carpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed discussion of sequence alignment methods and homology calculations. The level of sequence identity can be determined using the GCG program package (Devereux et al., Nucleic Acids Research 12:387, 1984), BLASTP, BLASTN, FASTA (Altschul et al., J. Mol. Biol. 215:403 (1990), and the ALIGN program (version 2.0).The well-known Smith Waterman algorithm can also be used to determine similarity. BLAST programs are publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST2.0 at http: / / www.ncbi.nlm.nih.gov / blast / ). Amino acid residues may be post-translationally modified or attached or modified with other functional or non-functional molecular groups. Of course, such modified amino acid residues are included in the amino acid sequence and within the scope of the compositions described herein. For example, polypeptides having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the specific polypeptides described herein and preferably exhibiting substantially the same function, and polynucleotides encoding such polypeptides, are contemplated. In comparing sequences, the above method considers various substitutions, deletions, and other modifications. In some embodiments, the polypeptide comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 conservative amino acid substitutions compared to any one of SEQ ID NOS: 1-50. As used herein, the terms "conservative amino acid substitutions" and "conservative modifications" refer to amino acid modifications that do not significantly affect or alter the function and / or activity of the proteins disclosed herein that contain the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the proteins of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to physicochemical properties, such as charge and polarity. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid from the same group.For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine, negatively charged amino acids include aspartic acid and glutamic acid, and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Additionally, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine. Nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine.

[0034] As used herein, "subject" or "individual" or "animal" or "patient" or "mammal" refers to a subject for whom treatment is sought or for whom diagnosis, prognosis, or therapy is desired, particularly a mammalian subject, e.g., a human.

[0035] As used herein, a "therapeutic polypeptide" refers to an ordered set of amino acids, proteins, and / or polypeptide-based pharmaceutical agents that can be administered to a subject to elicit a biological or medical response in a tissue, system, animal, or human that is desired, for example, by a researcher or clinician. A therapeutic polypeptide can elicit two or more biological or medical responses. A therapeutic polypeptide can be used for therapeutic purposes, i.e., for the treatment of a disorder in a subject. It should be noted that although a therapeutic polypeptide can be used for therapeutic purposes, the present disclosure is not limited to such uses, as the polypeptide can also be used for in vitro studies. Illustrative, but non-exhaustive, examples of therapeutic polypeptides are provided in Table 2, which is not intended to limit the scope of the present disclosure or interpretation of the claims.

[0036] The terms "treat", "treating" or "treatment" as used herein, and other grammatical equivalents as used herein, include alleviating, reducing or ameliorating the symptoms of a disease or condition, preventing further symptoms, improving or preventing the underlying metabolic cause of the symptoms, inhibiting a disease or condition, e.g., preventing the onset of a disease or condition, alleviating a disease or condition, regressing a disease or condition, alleviating a condition caused by a disease or condition, arresting the symptoms of a disease or condition, and preventing. These terms further include achieving therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to eradication or amelioration of the underlying disease being treated. Therapeutic benefit is also achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disease, such that an improvement is observed in the patient, even though the patient may still be suffering from the underlying disease. For prophylactic benefit, the compositions can be administered to patients at risk of developing a particular disorder or to patients who report one or more physiological symptoms even though no diagnosis has been made.

[0037] A "therapeutically effective amount" or "effective amount" as used herein is an amount of a biologically active agent / therapeutic polypeptide that can achieve a clinically relevant endpoint in a subject when administered once or repeatedly to the subject. Such an effect need not be absolute to be beneficial. The appropriate dose of the composition depends on the route of administration, such as oral, injection, or infusion, and depends on the subject being treated as well as the severity of the condition being treated. Scaling methods, such as allometric scaling, can be used to predict appropriate and exemplary dose ranges for administration of the compositions to adults, as disclosed herein. Dose scaling is an empirical approach, well characterized and understood in the art. This approach assumes that there are some unique characteristics regarding anatomical, physiological, and biochemical processes between species, and that possible differences in pharmacokinetics / physiological times are, as such, accounted for by the scaling. Determination of a therapeutically effective amount is within the capabilities of one of ordinary skill in the art, especially in light of the detailed disclosure provided herein.

[0038] As used herein, a "vector" is a nucleic acid molecule that is preferably self-replicating in a suitable host and transfers an inserted nucleic acid molecule into and / or between host cells. The term includes vectors that function primarily for the insertion of DNA or RNA into a cell, replicating vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. It also includes vectors that provide more than one of the above functions. An "expression vector" is a polynucleotide that is transcribed and translated into a polypeptide(s) upon introduction into a suitable host cell. An "expression system" generally refers to a suitable host cell comprised of an expression vector that functions to obtain a desired expression product.

[0039] Disclosed herein are polypeptides, polypeptide fragments, heterologous polypeptides, and polypeptide constructs formed therefrom having sequence identity corresponding to SEQ ID NOS: 1-59 identified and set forth in Table 1. [Table 1] TIFF2024545981000002.tif181141TIFF2024545981000003.tif185141TIFF2024545981000004.tif18614 1TIFF2024545981000005.tif191141TIFF2024545981000006.tif194141TIFF2024545981000007.tif11141

[0040] Compositions are disclosed that include a polypeptide according to the formula: Xaa01Xaa02Xaa03Xaa04Xaa05 Xaa06Xaa07Xaa08Xaa09Xaa10 Xaa11Xaa12Xaa13Xaa14Xaa15 Xaa16Xaa17Xaa18Xaa19Xaa20 Xaa21Xaa22Xaa23Xaa24Xaa25 Xaa26Xaa27Xaa28Xaa29Xaa30 Xaa31Xaa32Xaa33Xaa34Xaa35 Xaa36Xaa37Xaa38Xaa39Xaa40 Xaa41Xaa42Xaa43Xaa44Xaa45 Xaa46Xaa47Xaa48Xaa49Xaa50 Where: Xaa01=M,A,V,I,L Xaa02=A,G,S Xaa03=D,E Xaa04=D,E Xaa05=A,G,S Xaa06=G,A,S Xaa07=A,G,S Xaa08=A,G,S Xaa09=G,A,S Xaa10=G,A,S Xaa11=P Xaa12=G,A,S Xaa13=G,A,S Xaa14=P Xaa15=G,A,S Xaa16=G,A,S Xaa17=P Xaa18=G,A,S Xaa19=M,A,V,I,L Xaa20=M,T,I,G,A,S Xaa21=N,G,A,Q Xaa22=N,Q,R,K Xaa23=R,K,G,A,S Xaa24=G,A,S Xaa25=G,A,F,Y,W,H Xaa26=F,Y,W,R,K Xaa27=R,K,G,A,S Xaa28=G,A,S Xaa29=G,A,F,Y,W,H Xaa30=F,Y,W,G,A,S Xaa31=G,A,S Xaa32=S,T,G,A Xaa33=G,A,I,V,L,M Xaa34=R,K Xaa35=G,A,S Xaa36=R,K Xaa37=G,A,S Xaa38=R,K Xaa39=G,A,S Xaa40=R,K Xaa41=G,A,S Xaa42=R,K Xaa43=G,A,S Xaa44=R,K Xaa45=G,A,S Xaa46=R,K Xaa47=G,A,S Xaa48=R,K Xaa49=G,A,S Xaa50=A,G,S,K It is.

[0041] Disclosed are compounds comprising a polypeptide of the following formula: H-Met-Ala-Asp-Asp-Ala 5 -Gly-Ala-Ala-Gly-Gly 10 -Pro-Gly-Gly-Pro-Gly 15 -Gly-Pro-Gly-Met-Gly 20 -Asn-Arg-Gly-Gly-Phe 25 -Arg-Gly-Gly-Phe-Gly 30 -Ser-Gly-Ile-Arg-Gly 35 -Arg-Gly-Arg-Gly-Arg 40 -Gly-Arg-Gly-Arg-Gly 45 -Arg-Gly-Arg-Gly-Lys(N3) 50 -OH wherein the polypeptide is capable of being linked to a heterologous polypeptide via a terminal lysine residue, and the compound, when administered to a subject, provides targeted delivery of the heterologous polypeptide.

[0042] Disclosed herein is a heterologous polypeptide according to sequence ID 52 modified for ligation to a polypeptide, comprising: propionoic acid-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, said modified heterologous polypeptide being designed for conjugation to a polypeptide, said polypeptide comprising Lys(N3) 50 It is a heterologous polypeptide that is modified at its terminus to contain a modified lysine residue that contains an --OH.

[0043] Disclosed herein is a compound comprising a formula comprising H-Met-Ala-Asp-Ala-Gly-Ala-Ala-Gly-Gly-Pro-Gly-Gly-Pro-Gly-Gly-Pro-Gly-Met-Gly-Asn-Arg-Gly-Gly-Phe-Arg-Gly-Gly-Phe-Gly-Ser-Gly-Ile-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Nle(triazole-propionyl-D-Phe-Cys-Phe-D-Tru-Lys-Thr-Cys-Thr-ol)-OH, wherein the compound provides targeted delivery of the polypeptide when administered to a subject by oral route.

[0044] Disclosed herein is a polypeptide construct suitable for delivering a heterologous polypeptide across the gastrointestinal lining when administered via oral route to a subject, said peptide construct comprising a polypeptide linked to said heterologous polypeptide, said polypeptide having at least 90% sequence identity to a peptide according to SEQ ID NO:1-40, said heterologous polypeptide being a therapeutic polypeptide, said polypeptide being linked to said heterologous polypeptide by a linker.

[0045] Said polypeptide and said heterologous polypeptide are directly or indirectly linked via covalent and / or ionic bonds. In one embodiment, said polypeptide construct comprises a polypeptide and a heterologous polypeptide. In an embodiment, said polypeptide and said heterologous polypeptide are linked by ionic bonds. Ionic bonds refer to bonds resulting from electrostatic attraction between oppositely charged ions. In an embodiment, said polypeptide and said heterologous polypeptide are linked by covalent bonds. Covalent bonds refer to the mutual sharing of one or more pairs of electrons between two atoms. In one embodiment, said polypeptide and said heterologous polypeptide are linked by amide bonds or peptide bonds.

[0046] In some embodiments, the polypeptide is linked to the N-terminus of the heterologous polypeptide. In some embodiments, the polypeptide is linked to the C-terminus of the heterologous polypeptide. In some embodiments, the C-terminus of the polypeptide is linked to the N-terminus of the heterologous polypeptide. In some embodiments, the N-terminus of the polypeptide is linked to the C-terminus of the heterologous polypeptide. In some embodiments, the N-terminus of the heterologous polypeptide is linked to the N-terminus of the polypeptide. In some embodiments, the C-terminus of the heterologous polypeptide is linked to the C-terminus of the polypeptide. The term "linked" does not necessarily require that the polypeptide and the heterologous polypeptide are directly linked to each other. In embodiments, the polypeptide and the heterologous polypeptide are linked via a linker, such as a cleavable or non-cleavable additional moiety.

[0047] The polypeptide construct comprises two or more polypeptides and one heterologous polypeptide. In some embodiments, the polypeptide construct comprises at least the following components in the indicated orientation: polypeptide-heterologous polypeptide-polypeptide. In some embodiments, the polypeptide construct comprises at least the following components in the indicated orientation: polypeptide-polypeptide-heterologous polypeptide. In some embodiments, the polypeptide construct comprises at least the following components in the indicated orientation: heterologous polypeptide-polypeptide-polypeptide.

[0048] In some embodiments, the polypeptide is linked to the heterologous polypeptide via a linker. In embodiments, the linker is a polypeptide linker at least 1, at least 2, at least 3, at least 5, at least 7, at least 10 amino acids in length. In embodiments, the polypeptide linker is 1-20 amino acids in length. The linker may include natural and non-naturally occurring amino acids. The linker linking the polypeptide to the heterologous polypeptide may include a flexible portion and / or a rigid portion. In embodiments, the linker is a flexible linker. In embodiments, the linker is a rigid linker. In embodiments, the linker is a polypeptide linker comprising one or more, or a plurality of, glycines and serines. In one embodiment, the linker is a cleavable linker. In one embodiment, the linker is a lysine or a plurality of lysine residues. In one embodiment, the linker is a non-cleavable linker. In one embodiment, the linker is a helical linker. In one embodiment, the linker is a non-helical linker. In one embodiment, the linker is a strong non-covalent interaction such as biotin-streptavidin, hi one embodiment, the linker is an amide bond formed between an alkyl-modified peptide on a polypeptide and an azide-modified peptide on a heterologous polypeptide.

[0049] In one embodiment, some or all of the components that make up the polypeptide construct are produced separately, for example by recombinant expression or chemical synthesis, and then combined. In an embodiment, some or all of the components that make up the polypeptide construct, or the entire polypeptide construct, are produced in a recombinant host cell or synthesized from recombinant nucleic acid. A "recombinant host cell" is a host cell that contains a recombinant nucleic acid. As used herein, the term "recombinant nucleic acid" refers to a nucleic acid that has been removed from its naturally occurring environment, or that is not associated with all or part of the nucleic acid that is adjacent or proximal to the nucleic acid when present in nature, or that is operably linked to a nucleic acid with which it is not naturally associated, or that is not naturally occurring, or that contains modifications that are not present in the nucleic acid in nature (e.g., artificially introduced insertions, deletions, point mutations, etc.), or that has been integrated into a chromosome at a heterologous moiety. The term includes cloned DNA isolates, and nucleic acids that contain chemically synthesized nucleotide analogs.

[0050] A variety of expression vectors have been developed for efficient synthesis of polypeptide constructs in prokaryotic cells such as bacteria and eukaryotic cell systems, including but not limited to yeast and mammalian cell culture systems. Vectors can include segments of chromosomal, non-chromosomal, and synthetic DNA sequences. Also provided are cells containing expression vectors for expressing the polypeptide constructs disclosed herein. Expression vectors are usually replicable in the host organism, either as episomes or as an integral part of the host chromosomal DNA. In general, expression vectors include selectable markers (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, or neomycin resistance) to allow detection of cells transformed with the desired DNA sequence (see, e.g., U.S. Patent No. 4,704,362 to Itakura et al.).

[0051] Expression of the polypeptide constructs disclosed herein may occur in prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insect, fungal, avian, and mammalian cells in vivo or in situ, or host cells of mammalian, insect, avian, or yeast origin. Mammalian cells or tissues may be from humans, primates, hamsters, rabbits, rodents, cows, pigs, sheep, horses, goats, dogs, or cats, although any other mammalian cells may be used.

[0052] E. coli is one prokaryotic host particularly useful for cloning the polynucleotides of the present invention. Other suitable microbial hosts include bacilli such as Bacillus subtilis, and Enterobacteriaceae such as Salmonella, Serratia, and Pseudomonas.

[0053] Other microbes such as yeast are also useful for expression. Saccharomyces and Pichia are exemplary yeast hosts, with appropriate vectors optionally having expression control sequences (e.g., promoters), origins of replication, termination sequences, and the like. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for methanol, maltose, and galactose utilization, and the like.

[0054] Furthermore, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved, for example, by using the yeast ubiquitin hydrolase system. The fusion proteins thus produced can be processed in vivo or purified and processed in vitro, allowing the synthesis of polypeptide constructs with specified amino-terminal sequences. Furthermore, problems associated with retention of the methionine residue from the start codon in direct yeast (or bacterial) expression may be avoided. Sabin et al., 7 Bio / Technol. 705 (1989); Miller et al., 7 Bio / Technol. 698 (1989). When yeast is cultured in glucose-rich medium, glycolytic enzymes are produced in large quantities. Any of a range of yeast gene expression systems incorporating promoter and termination elements from active expression genes encoding these glycolytic enzymes can be utilized for the polypeptide constructs. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and termination signals of the phosphoglycerate kinase gene can be utilized.

[0055] Production of a polypeptide construct in an insect can be accomplished, for example, by infecting an insect host with a baculovirus engineered to express a transmembrane polypeptide, by methods known to those of skill in the art.

[0056] In addition to microorganisms, mammalian tissue cultures are also used for the expression and production of polypeptide constructs. Expression vectors for these cells can include expression control sequences such as replication origins, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary processing information such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

[0057] Vectors containing sequences encoding a polypeptide construct of interest can be introduced into host cells by well-known methods that vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics, or viral-based transfection are utilized for other cellular hosts. (See generally, Sambrook et al., Molecular Cloning: A Laboratory Manual [Cold Spring Harbor Press, 2nd ed., 1989]). Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally, Sambrook et al., supra). To produce transgenic animals, the transgene is microinjected into fertilized eggs or incorporated into the genome of an embryonic stem cell, the nucleus of which is transferred into a nucleated egg.

[0058] Provided herein is a method of producing a polypeptide construct disclosed herein, the method comprising providing a cell that expresses a polypeptide construct herein, and isolating the polypeptide construct.

[0059] Provided herein is a method for producing a polypeptide construct disclosed herein, the method comprising the steps of providing a polypeptide according to SEQ ID NO:21-40 and ligating the polypeptide to a heterologous polypeptide, wherein the ligation is performed by a chemical reaction (such as click chemistry) utilizing modified amino acid residues at the terminus of the polypeptide.

[0060] In one embodiment, some or all of the components that make up the polypeptide construct are joined by chemical ligation. Click chemistry is one exemplary ligation method, and various methods for joining molecules by click chemistry are well known in the art. "Click chemistry" is a term introduced by researchers at the Scripps Research Institute to refer to chemistry tailored to rapidly and reliably generate substances by joining small units. The term "click chemistry" applies to reactions that are highly efficient, broad in scope, and stereospecific. The products are easily isolated, the reactions are simple to perform with inexpensive reagents, and can be carried out in mild solvents such as water. The Huisgen 1,3-dipolar cycloaddition is perhaps the most widely studied click reaction. A variant of this reaction, the copper-catalyzed azide-alkyne cycloaddition (CuAAC), also fits well with the concept of click chemistry and is one of the most popular prototype click reactions to date.

[0061] Classical click chemistry methods usually rely on heterobifunctional crosslinkers such as N-hydroxysuccinimide (NHS)-linker-maleimide or similar two-step processes. Proteins are attached to solid supports using amino-reactive NHS and thiol-reactive maleimide. Other methods combine strain-promoted azide-alkyne cycloaddition (SPAAC) click reactions and OaAEP1(C247A)-based enzymatic ligation. These methods are well known in the art and the present disclosure can be applied by those skilled in the art.

[0062] In some embodiments, the polypeptide, one or both of the heterologous polypeptides, and / or the polypeptide constructs comprise chemical modifications to one or more amino acids and / or the addition or attachment of functional moieties. Such amino acid modifications include, but are not limited to, phosphorylation, methylation (e.g., lysine methylation [mono-, di-, or tri-methylation], and arginine methylation [mono-, asymmetric, or symmetric dimethylation]), acetylation, ubiquitination, myristoylation, palmitoylation, isoprenylation, prenylation, acylation, glycosylation, hydroxylation, iodination, oxidation, sulfation, selenoylation, sumoylation, citrullination, deamidation, carbamylation, ADP-ribosylation, ubiquitination, nitrosylation, lysine crotonylation, formylation, propionyl lysylation, butyryl lysylation, or any combination thereof. In some embodiments, the polypeptide construct is covalently modified with one or more lipids, including but not limited to fatty acids, cholesterol, isoprenoids, phospholipids, and diacylglyceryl lipids. In some embodiments, the polypeptide construct is linked to a functional moiety, including but not limited to a diagnostic moiety, a detectable moiety, a moiety useful for ligation or purification, or a targeting moiety. The linkage to the functional moiety may or may not be at one of the termini of the polypeptide construct. A moiety may have more than one function. In one embodiment, the modifications include an alkyl-modified peptide on the terminus of a first polypeptide and an azide-modified peptide on a second (heterologous) polypeptide, which promote the formation of an amide bond between the modified peptide on the first polypeptide and the modified peptide on the second polypeptide to generate the polypeptide construct.

[0063] Examples of moieties useful for purification include, but are not limited to, albumin binding protein (ABP), alkaline phosphatase (AP), AU1 epitope, AU5 epitope, bacteriophage T7 epitope (T7-tag), bacteriophage V5 epitope (V5-tag), biotin carboxy carrier protein (BCCP), bluetongue virus tag (B-tag), calmodulin binding peptide (CBP), chloramphenicol acetyltransferase (CAT), cellulose binding domain (CBP), chitin binding domain (CBD), choline binding domain (CBD), dihydrofolate reductase (DHFR), E2 epitope, FLAG epitope, galactose binding protein (GBP), and the like. ), Green Fluorescent Protein (GFP), Glu-Glu (EE-Tag), Glutathione S-Transferase (GST), Human Influenza Hemagglutinin (HA), HaloTag®, Histidine Affinity Tag (HAT), Horseradish Peroxidase (HRP), HSV Epitope, Ketosteroid Isomerase (KSI), KT3 Epitope, LacZ, Luciferase, Maltose Binding Protein (MBP), Myc Epitope, NusA, PDZ Domain, PDZ Ligand, Polyarginine (Arg-Tag), Polyaspartic Acid (Asp-Tag), Polycysteine ​​(Cys-Tag), Polyhistidine (His-Tag), Polyphenylalanine (Phe-Tag), Profinity These include eXact, Protein C, S1-tag, S-tag, streptavidin-binding peptide (SBP), Staphylococcal Protein A (Protein A), Staphylococcal Protein G (Protein G), Strepttag, Streptavidin, Small Ubiquitin-Like Modifier (SUMO), Tandem Affinity Purification (TAP), T7 epitope, Thioredoxin (Trx), TrpE, Ubiquitin, Universal, and VSV-G.

[0064] Examples of detectable moieties include, but are not limited to, fluorescent moieties or labels, imaging agents, radioisotope moieties, radio-opaque moieties, etc., such as detectable labels, such as biotin, fluorophores, chromophores, spin resonance probes, or radioactive labels. Non-limiting examples of fluorophores include fluorescent dyes (e.g., fluorescein, rhodamine, etc.) and other luminescent molecules (e.g., luminal). Fluorophores may be environmentally sensitive such that their fluorescence changes when located near one or more residues in a modified protein that undergo a conformational change upon binding to a substrate (e.g., dansyl probe). Non-limiting examples of radioactive labels include small molecules that contain atoms with one or more low sensitivity nuclei (13C, 15N, 2H, 125I, 123I, 99Tc, 43K, 52Fe, 67Ga, 68Ga, 111In, etc.). Other useful moieties are known in the art.

[0065] Provided herein is a polypeptide construct comprising: (a) a polypeptide comprising an amino acid sequence at least 80% identical to an amino acid sequence selected from any one of SEQ ID NOS: 1-40; and (b) a heterologous polypeptide. In some embodiments, the polypeptide comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOS: 1-40. In some embodiments, the polypeptide comprises any one of SEQ ID NOS: 1-40. In some embodiments, the polypeptide comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOS: 1. In some embodiments, the polypeptide comprises SEQ ID NOS: 1. In embodiments, the polypeptide comprises an N-terminal or C-terminal lysine. Also contemplated are polypeptides that contain one or more truncations, internal deletions, internal insertions, substitutions, or modifications compared to any of the polypeptide sequences disclosed herein. For example, one skilled in the art may wish to truncate a polypeptide sequence disclosed herein to alter the stability of the polypeptide or to increase the ease or cost of manufacturing the polypeptide. Truncated polypeptides (50-30 amino acids, and 30-20 amino acids) have been tested and shown to exhibit the properties of the polypeptide according to SEQ ID NO:1 (full length) for targeted delivery of heterologous polypeptides.

[0066] Provided herein is a polypeptide construct comprising: (a) a polypeptide comprising a modified terminal lysine; and (b) a heterologous polypeptide, wherein the heterologous polypeptide is attached to the polypeptide via an amide bond formed between the heterologous polypeptide and the modified terminal lysine of the polypeptide.

[0067] Provided herein is a polypeptide construct comprising: (a) a polypeptide; and (b) a heterologous polypeptide, wherein the heterologous polypeptide is a therapeutic polypeptide. It should be noted that although a therapeutic polypeptide may be used for therapeutic purposes, the present disclosure is not limited to therapeutic uses, as the polypeptide may also be used for in vitro studies.

[0068] In some embodiments, the therapeutic polypeptide is a hormone, an interferon, an interleukin, a growth factor, a tumor necrosis factor, a thrombolytic agent, an enzyme, an antibody, an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, or an artificial protein scaffold.

[0069] In some embodiments, the hormone is erythropoietin. In some embodiments, the hormone is human erythropoietin. In one embodiment, the hormone is epoetin. Non-limiting examples of erythropoietin include Epogen® (epoetin-alpha), Procit® (epoetin alfa-epbx), and Retacrit® (epoetin alfa-epbx), and pegylated epoetin. In some embodiments, the hormone is glucagon-like peptide 1 (GLP-1) or a GLP-1 agonist. Non-limiting examples of GLP-1 agonists include Exendin4, semaglutide (including but not limited to Wegovy® and Ozempic®), liraglutide (including but not limited to Victoza®), exenatide (including but not limited to Byetta® and Bydureon®), and the like. In some embodiments, the hormone is insulin. In some embodiments, the insulin is insulin aspart, insulin lispro, insulin glulisine, insulin detemir, degludec insulin, and glargine insulin.

[0070] In some embodiments, the therapeutic polypeptide is somatostatin, a somatostatin analog, glucagon, galsulfase, nesiritide, or taliglucerase alpha. Non-limiting examples of somatostatins include, but are not limited to, Sandostatin® LAR Depot (octreotide acetate), MYCAPSSA® (octreotide), which uses a transient permeation enhancer (TPE®) to move octreotide from the stomach into the bloodstream. TPE® is an oil suspension of octreotide, which contains a number of excipients and can transiently alter the integrity of the epithelial barrier by opening intestinal epithelial tight junctions resulting from transcellular perturbation. It is questionable whether permeation enhancers (PEs) cause irreversible epithelial damage and tight junction opening sufficient to allow co-absorption of bystander pathogens, lipopolysaccharides and their fragments, or payloads with exotoxins and endotoxins that may be associated with sepsis, inflammation, and autoimmune diseases. Most PEs appear to cause membrane damage to varying degrees, but are rapidly reversible, and overall evidence of pathogen co-absorption is generally lacking. However, it is unclear whether the intestinal epithelial damage-repair cycle will persist during repeated dosing regimens for chronic treatment. The peptides according to SEQ ID NOs. 1-40 do not act as PEs, but rather allow proteins to be taken up into intestinal cells and exported back through the other side to the blood, thereby avoiding all of the problems associated with PE.

[0071] In some embodiments, the therapeutic polypeptide is a polypeptide as disclosed in Table 1 or Table 2, or a variant thereof. "Variant" refers to a polypeptide that contains one or more changes compared to a parent polypeptide, including, but not limited to, amino acid additions, substitutions, insertions, deletions, or post-translational modifications, wherein the variant retains at least 10% of the therapeutic activity of the parent polypeptide. Also provided are heterologous polypeptides that are biosimilar versions of any of the heterologous polypeptides disclosed herein.

[0072] In some embodiments, the heterologous polypeptide is a mammalian polypeptide. In some embodiments, the heterologous polypeptide is a human polypeptide.

[0073] In some embodiments, provided is a polypeptide construct comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 41-50. In some embodiments, provided is a polypeptide construct comprising an amino acid sequence selected from any one of SEQ ID NOs: 41-50.

[0074] In some embodiments, provided is a polypeptide construct comprising: (a) a first polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 1-40, 58, 59; (b) a heterologous polypeptide; and, optionally, (c) a second polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 1-40. In some embodiments, provided is a polypeptide construct comprising: (a) a first polypeptide comprising any one of SEQ ID NOs: 1-40, 58, or 59; (b) a heterologous polypeptide; and, optionally, (c) a second polypeptide comprising any one of SEQ ID NOs: 1-40, 58, or 59.

[0075] In some embodiments, provided is a polypeptide construct comprising: (a) a first polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from any one of SEQ ID NOS: 1-40, 58, and 59; (b) a heterologous polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOS: 41-50; and, optionally, (c) a second polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from any one of SEQ ID NOS: 1-40. In some embodiments, provided is a polypeptide construct comprising: (a) a first polypeptide comprising any one of SEQ ID NOs: 1-40, 58, 59; and (b) a heterologous polypeptide. [Table 2] TIFF2024545981000009.tif59141

[0076] Also provided herein are nucleic acids, genomes and vectors that contain the nucleic acids that code for the polypeptide constructs disclosed herein.The term "nucleic acid" as used herein refers to any length of polymer of nucleotides, either ribonucleotides or deoxyribonucleotides.Thus, this term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrid, or polymers that contain purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0077] Provided herein is a nucleic acid comprising (i) a promoter and (ii) a transgene encoding a polypeptide construct disclosed herein, the transgene being operably linked to the promoter. As used herein, "operably linked" refers to both expression control sequences contiguous with the transgene and expression control sequences acting in trans or at a distance to control the expression of the transgene. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (e.g., Kozak consensus sequences), sequences that enhance protein stability, and sequences that enhance protein processing and / or secretion, as desired.

[0078] In one aspect, provided is a cell comprising a transgene encoding a polypeptide construct disclosed herein. Provided is a method of making a polypeptide construct disclosed herein, comprising (i) providing a cell comprising a transgene encoding a IL-polypeptide construct disclosed herein, and (ii) expressing the polypeptide construct in the cell. In some embodiments, the polypeptide construct is substantially purified from the cell. In some embodiments, provided is a cell comprising a transgene encoding a polypeptide construct disclosed herein, the cell secreting the polypeptide construct. In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. Provided herein is an isolated cell.

[0079] Provided herein is a pharmaceutical composition comprising the polypeptide construct disclosed herein, formulated with one or more pharma- ceutically acceptable excipients.The active agent and excipient(s) are formulated into compositions and dosage forms according to methods known in the art.The pharmaceutical compositions disclosed herein are specially formulated in solid or liquid form, including those adapted for oral administration.

[0080] Therapeutic compositions comprising the polypeptide constructs disclosed herein may be formulated with one or more pharma- ceutically acceptable excipients, which may be pharma- ceutically acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, carriers, manufacturing aids (e.g., lubricants, magnesium talc, calcium or zinc stearate, or stearate), solvents or encapsulating materials, bulking agents, salts, surfactants, and / or preservatives, that are involved in carrying or transporting the therapeutic compound for administration to a subject. Examples of materials that can function as pharma- ceutically acceptable excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; celluloses and their derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; gelatin, talc, waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as ethylene glycol and propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar, buffers, water, isotonic saline, pH buffer solutions, and other non-toxic compatible substances used in pharmaceutical formulations.

[0081] Bulking agents are compounds that add mass to a pharmaceutical formulation and contribute to the physical structure of the formulation in lyophilized form. Suitable bulking agents according to the present disclosure include mannitol, glycine, polyethylene glycol, and sorbitol.

[0082] The use of a surfactant can reduce aggregation of the reconstituted protein and / or reduce the formation of particulates in the reconstituted formulation. The amount of surfactant added is such that it reduces aggregation of the reconstituted protein and minimizes the formation of particulates after reconstitution. Suitable surfactants according to the present disclosure include polysorbates (e.g., polysorbate 20 or 80), poloxamers (e.g., poloxamer 188), triton, sodium dodecyl sulfate (SDS), sodium laurel sulfate, sodium octyl glycoside, lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine, lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine, linoleyl-, myristyl-, or cetyl-betaine, lauroamidopropyl-, cocamide propyl-, glyceryl ... Examples of suitable oleyl esters include oleyl-, linoleyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl), myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine, sodium methyl cocoyl-, or disodium methyl oleyl-taurate, and polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronic, PF68, etc.).

[0083] Preservatives are used in the formulations disclosed herein. Suitable preservatives for use in the formulations disclosed herein include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl, and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Other suitable excipients are described in standard pharmaceutical textbooks, such as "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19th Ed. Mack Publishing Company, Easton, Pa., (1995).

[0084] In an embodiment, a pharmaceutical composition comprises a polypeptide construct for oral administration, said composition may be in the form of a solid, semi-solid, gel, or liquid, including in the form of a tablet, capsule, lozenge, or aqueous solution.

[0085] Provided herein is a method for transporting a polypeptide construct comprising a heterologous polypeptide from the gastrointestinal tract of a subject in need thereof to the circulatory system of the subject, the method comprising orally administering to the subject a polypeptide construct disclosed herein or a pharmaceutical composition comprising a polypeptide construct disclosed herein. Provided herein is a polypeptide construct comprising a heterologous polypeptide for use in a method for transporting a polypeptide construct from the gastrointestinal tract of a subject in need thereof to the circulatory system of the subject, the method comprising orally administering to the subject a polypeptide construct disclosed herein or a pharmaceutical composition comprising a polypeptide construct disclosed herein. In an embodiment, the subject is a mammal. In an embodiment, the subject is a human.

[0086] In an embodiment, the polypeptide construct comprising a polypeptide and a heterologous polypeptide is absorbed into the apical cell wall of the intestine, travels through the basal wall into the circulatory system, and is excreted. The circulatory system includes the heart, blood vessels (including arteries, veins, and capillaries), and blood. In an embodiment, the polypeptide construct is absorbed by the stomach wall. In an embodiment, the heterologous polypeptide is separated from the remainder of the polypeptide construct once the heterologous polypeptide is located in the circulatory system. In an embodiment, after separation from the remainder of the polypeptide construct, the heterologous polypeptide comprises an N-terminal or C-terminal appendage derived from the remainder of the polypeptide construct. In an embodiment, the N-terminal or C-terminal appendage is selected from A, GA, RGA, GRGA, or a combination thereof.

[0087] Provided herein is a method of administering to a subject a therapeutically effective amount of a polypeptide construct disclosed herein.

[0088] Disclosed is a method of translocating a therapeutic polypeptide across the gastrointestinal lining of a subject and into the circulatory system of the subject, comprising ingesting a pharmaceutical composition comprising a polypeptide construct comprising a first polypeptide comprising a polypeptide having sequence identity selected from SEQ ID NO: 1-40 bound to a heterologous polypeptide, wherein the heterologous polypeptide is a therapeutic polypeptide, wherein the subject is a human, and wherein the therapeutic polypeptide is cleaved in whole (or in part) from the first polypeptide prior to (or at) entering the circulatory system, and further wherein the therapeutic polypeptide present in the circulatory system optionally comprises an N-terminal or C-terminal appendage from the first polypeptide selected from A, GA, RGA, GRGA, or a combination thereof.

[0089] Provided herein is a method of treating anemia in a subject in need thereof, comprising administering to the subject a polypeptide construct comprising erythropoietin. Provided herein is a polypeptide construct comprising erythropoietin. Provided herein is the use of a polypeptide construct comprising erythropoietin in the manufacture of a medicament for treating anemia. In an embodiment, the erythropoietin is epoetin alpha or pegylated epoetin. In an embodiment, the polypeptide construct comprises SEQ ID NO: 41.

[0090] Provided herein is a method of treating diabetes in a subject in need thereof, the method comprising administering to the subject a composition comprising a polypeptide construct comprising a polypeptide having sequence identity to SEQ ID NOs: 1-40 and a heterologous polypeptide, the heterologous polypeptide being selected from the group consisting of liraglutide, semaglutide, octreotide, GLP-1, insulin, or variants or analogs thereof. Provided herein is the use of the polypeptide construct comprising one of liraglutide, semaglutide, octreotide, GLP-1, insulin in the manufacture of a medicament for treating diabetes. In an embodiment, the polypeptide construct comprises SEQ ID NOs: 42-50.

[0091] Said polypeptide construct provided herein can be administered orally.In an embodiment, said polypeptide construct is administered once, twice, three times, four times, five times, six times a day.Said polypeptide construct is administered every other day, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once a month, once every eight weeks (or once every two months), once every twelve weeks (or once every three months), or once every twenty-four weeks (once every six months). The polypeptide construct is administered for a period of about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks, or about 52 weeks, or longer.

[0092] The effective amount, or possibly therapeutically effective amount, of the polypeptide construct disclosed herein can be determined by methods known in the art. For example, the appropriate dose of the polypeptide disclosed herein depends on the route of administration and on the subject to be treated as well as the severity of the condition to be treated. Scaling methods such as allometric scaling can be used to predict appropriate and exemplary dose ranges for administration of the compositions as disclosed herein to adults. Dose scaling is an empirical approach, well characterized and understood in the art. This approach assumes that there are some unique characteristics regarding anatomical, physiological, and biochemical processes between species, and as such, possible differences in pharmacokinetic / physiological times are accounted for by scaling.

[0093] In one embodiment, provided herein is a composition comprising a polypeptide construct, further comprising an additional therapeutic agent. Such additional agents include, but are not limited to, antibacterial agents, cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, and anti-infective agents. The agents used in such combination therapy fall into one or more of the above categories. The administration of the polypeptide construct and the additional therapeutic agent may be simultaneous or sequential. The administration of the polypeptide construct and the additional therapeutic agent may be separate or in mixture.

[0094] In order to facilitate a better understanding of the present disclosure, the following specific examples are given: The following examples should not be read to limit or define the entire scope of the present disclosure, and the examples are provided for illustrative purposes, and not for limitation with respect to the subject matter claimed herein. EXAMPLES

[0095] To evaluate the efficacy of polypeptides comprising amino acid sequences having sequences identified according to SEQ ID NO: 1-40 to facilitate targeted delivery of therapeutic polypeptides, a series of peptide conjugates were constructed comprising a polypeptide according to SEQ ID NO: 1-40 conjugated to a heterologous polypeptide comprising a therapeutic polypeptide. The polypeptide conjugates can be studied in vitro using Caco-2 cells to evaluate the uptake and bioactivity of the therapeutic polypeptide.

[0096] The human epithelial cell line Caco-2 (available from Sigma Aldrich) is widely used as a model of the intestinal epithelial barrier. One of the most advantageous properties of this cell line, originally derived from a colon carcinoma, is its ability to spontaneously differentiate into a monolayer of cells with many properties typical of absorptive enterocytes with a brush border layer as found in the small intestine. To mimic the stereological conditions of the in vivo intestine, Caco-2 cells can be cultured on permeable filter inserts (available from Becton Dickenson, Corning, Costar, etc.). Cultivation of Caco-2 cells on filter supports improves the morphological and functional differentiation of the cells. It is well known that polarized Caco-2 monolayers are a reliable correlate for studies of the absorption of drugs and other compounds after oral ingestion in humans. Several studies have compared Caco-2 permeability coefficients with absorption data in humans and found a high correlation, especially when compounds are transported by passive small cell transport mechanisms (Lea T. Caco-2 Cell Line. In: Verhoeckx K, Cotter P, Lopez-Exposito I, et al., editors. The Impact of Food Bioactives on Health: in vitro and ex vivo models [Internet]. Cham (CH): Springer; 2015. Chapter 10. Available at: https: / / www.ncbi.nlm.nih.gov / books / NBK500149 / doi:10.1007 / 978-3-319-16104-4_10).

[0097] Various polypeptide constructs were labeled using Alexa Fluor™ Labeling Kit (ThermoFisher) to generate fluorescently labeled polypeptide constructs. Caco-2 cells were seeded in 12-well plates and incubated with labeled polypeptides at concentrations of 2-10ug / mL for 4 hours. After incubation, the plates were read on a Tecan Infinite M Nano+ and after several gentle washes with PBS, fresh medium was added and the plates were read again. The % of fluorescence remaining in the dish represents the relative uptake of the polypeptide construct compared to the amount initially added to the cells (see Figure 2). Uptake of the polypeptide constructs by Caco-2 cells suggests that the constructs are suitable delivery agents for therapeutic polypeptides across the gastrointestinal lining, with the polypeptide according to SEQ ID NOs. 1-40 acting as a "shuttle" to transport the therapeutic peptide to which it is attached from the intestine to the bloodstream. Thus, the Caco-2 cell model assay is a screening tool for various polypeptide therapeutics to confirm their suitability as an orally administered formulation. EXAMPLES

[0098] Human erythropoietin (Epo) is a 30.4 kDa glycoprotein hormone containing a single chain of 165 amino acid residues to which four carbohydrate chains are attached. Epo is a key element in the feedback control of red blood cell production in the bone marrow. Epoetin is a recombinant form of human erythropoietin (containing a chain of 166 amino acid residues) that is used to promote differentiation of progenitor cells into red blood cells in the treatment of anemia (among other therapies). Epoetin alpha, sold under the brand name Epogen®, is a synthetic protein that aids in the body's production of red blood cells and is primarily used to treat anemia. Epogen® (like many macromolecular therapeutics) is not absorbed in the intestine and must be administered intravenously. These properties made human erythropoietin useful as a test molecule for oral formulation compositions using the methods disclosed herein (see FIG. 3). A polypeptide construct was constructed that includes a polypeptide having sequence identity according to sequence ID1 attached to the amino terminus of the human erythropoietin ("Epo") sequence. This ligation resulted in a chimeric protein containing a polypeptide having amino acid sequence identity corresponding to sequence ID41.

[0099] The polypeptide according to sequence ID41 was cloned by inserting the (human) erythropoietin sequence upstream of sequence ID1 into the pBluescript bacterial expression vector (Sigma Aldrich). This bacterial construct culture was grown under standard culture conditions to express the polypeptide according to sequence ID41. The bacterial slurry containing the polypeptide according to sequence ID41 was centrifuged and resuspended in 25 ml column buffer (CB) per liter of culture. The bacterial cells were lysed by freeze-thawing and then passed through a 20-gauge needle. The lysed cells were centrifuged and the supernatant was diluted by adding 125 ml cold CB to 25 ml of crude extract. The diluted crude extract was applied to a Protein A microbead column containing antibodies against human erythropoietin, washed with 12 column volumes of CB, and eluted with elution buffer after washing. The amount of isolated polypeptide according to sequence ID41 was determined by bicinchoninic acid assay (BSA) assay, the amount of protein was measured by ELISA, and purity was assessed by the ratio of protein to sequence ID41 protein in the eluate.

[0100] Eight 12-week-old male Wistar rats were administered a composition comprising a polypeptide according to sequence ID41 (400 μg in 200 μL PBS) PO or a control (200 μL PBS only) PO once daily for 4 weeks. All treated rats were bled via the tail vein on days 0, 14, and 28 (see FIG. 7).

[0101] Two weeks after dosing, a fragment of the polypeptide according to sequence ID 56 was detected in the blood of seven of eight animals (range 0.8-23 ng / mL) and in all animals by week 4 (1.2-22 ng / mL), indicating that the polypeptide construct is able to cross the gastrointestinal lining and that the therapeutic agent can pass from the gastrointestinal (GI) tract into the bloodstream after oral administration.

[0102] To assess whether compositions comprising a polypeptide construct according to sequence ID41 were biologically active following oral administration, the animals' hemoglobulin levels were measured following administration. After two weeks of administration, the mean hemoglobulin levels increased from 8.79 gm / dl to 9.211 gm / dl, while the controls remained relatively constant at 8.87 gm / dl and 8.84 gm / dl, respectively. At the end of administration (four weeks), the control group's hemoglobulin levels remained below 9 with a mean value of 8.96 gm / dl, while the control group's levels further increased to 9.64 gm / dl. Thus, compositions comprising a polypeptide construct comprising an amino acid sequence according to sequence ID41, when administered orally, not only crossed the gastrointestinal lining of the GI tract and entered the animal's bloodstream, but also remained biologically and therapeutically active following uptake into the bloodstream. EXAMPLES

[0103] Four Sprague-Dawley rats were administered a 600 μg dose of a composition comprising a polypeptide according to sequence ID41, and then exsanguination occurred 4 hours (n=2) and 6 hours (n=2) after administration (see FIG. 6). Blood collected from the animals was then processed to remove most of the blood proteins, size-selected by Western blot, and then peptides were removed by PAGE. Bands between 15 and 25 kDA were excised from the electrophoretic gel and sequenced. The sequence of the serum-derived peptide obtained was found to be a polypeptide according to sequence ID56 (corresponding to the erythropoietin sequence [sequence ID51]) with two amino acids (GA) added to the amino terminus. This indicates that after administration, 48 of the 50 amino acids of sequence ID1 were cleaved from sequence ID41 (see FIG. 4B).

[0104] To assess bioavailability, ten Sprague-Dawley rats were administered a composition comprising a polypeptide according to sequence ID41 by oral gavage at a concentration of 2.5 mg / kg (n=5) or 1 mg / kg (n=5). Blood was collected from each animal at 0, 5, 15, 30, 60 minutes, and 2, 4, 8, and 24 hours after administration. The amount of a composition comprising a polypeptide according to sequence ID41 or a fragment thereof in the blood was measured and compared to the value of animals administered the same composition by intravenous injection at a dose of 0.5 mg / kg (n=5). Bioavailability (F) is the percentage of an administered drug that reaches the systemic circulation. Mathematically, bioavailability is equal to the ratio of the area under the plasma drug concentration curve versus time (AUC) of an extravascular formulation compared to the AUC of an intravascular formulation. The AUC is utilized because it is proportional to the dose that entered the systemic circulation.

[0105] To determine the absolute bioavailability of a drug, plasma drug concentration versus time plots of the drug after intravenous (iv) and extravascular (non-iv, i.e., oral) administration were determined. Absolute bioavailability is the dose-corrected area under the curve (AUC) for non-iv administration divided by the AUC for iv administration. Thus, the absolute bioavailability of a drug administered intravenously is 100% (f=1), while the absolute bioavailability of a drug administered by other routes is usually less than 1.

[0106] According to this concentration vs. time plot / AUC model, the average F value of the polypeptide constructs containing amino acid sequence identity to sequence ID 41 was found to be 0.18, ranging from 0.062 to 0.32. max ) is the average C max was 202.6mIUnits / ml, and the average t max The time to reach maximum concentration was 180 minutes. All animals receiving oral administration of a composition comprising a polypeptide of sequence ID 41 achieved therapeutic levels of serum Epogen®. EXAMPLES

[0107] Sprague-Dawley rats were administered a composition comprising sequence ID41 PO (doses of 2.5 mg / kg, n=5, 1 mg / kg, n=5, 0.25 mg / kg, n=5) and also IV (doses of 0.5 mg / kg, n=5), blood was collected at 0, 5, 15, 30, 60 minutes, and 2, 4, 8, and 24 hours post-dosing, and systemic levels of the peptide were compared between PO and IV treated animals. In PO treated animals, the peptide according to sequence ID42 was detected in the blood as early as 30 minutes post-dosing, with peak levels between 4 and 6 hours, an F value of 0.3, and circulating levels between 150 and 240 mIU / mL sustained for 24 hours post-dosing (see FIG. 4A).

[0108] To test the hypothesis that the polypeptide comprising SEQ ID NO:1 or a fragment thereof is cleaved from the peptide construct comprising SEQ ID NO:41 upon uptake into the bloodstream, an ELISA assay was performed to detect the 50-aaPT sequence in the blood following administration. As seen with the detection of Epo, the polypeptide comprising SEQ ID NO:57 was detected as early as 30 minutes after administration, with levels peaking at 4 hours. However, unlike Epo levels, the concentration of the polypeptide comprising SEQ ID NO:57 decreased to less than 40% within 8 hours after administration and was undetectable within 24 hours, confirming cleavage and degradation of the polypeptide (see FIG. 5).

[0109] To determine the efficacy of a composition comprising a polypeptide construct comprising an amino acid sequence identity to SEQ ID NO: 41 (designated "PT-EPO"), Sprague-Dawley rats (n=8) were administered PT-EPO at 2 mg / kg daily (PO) for 28 days. Blood was collected on days 0, 14, and 28 to measure Epo and hemoglobin concentrations. Two weeks after PT-EPO administration, an average of 10.1125 pg / mL of Epo was detected in the animals' serum, and this level increased to 11.425 pg / mL by day 28 (see FIG. 7). PT-EPO administration was also associated with a significant increase in hemoglobin levels, from 16.35 ng / mL at the start of administration to 17.1375 ng / mL on day 14 and 17.9375 ng / mL on day 28 (p<0.05), while control animals showed no significant change (see FIG. 8). EXAMPLES

[0110] The bioavailability of an orally administered composition comprising a polypeptide construct (PT-EPO) comprising an amino acid sequence having sequence identity to sequence ID 41 was evaluated in a 14-day safety study in a canine animal model (beagle). The animals were administered (PO) a composition comprising PT-EPO at daily doses of 0, 5, 50, and 125 mg / kg for 14 consecutive days. After completion of the study, pathological analysis of the animals revealed that PT-EPO had no adverse effects on major organ function. The safety study also showed that there were no adverse effects when a high dose (125 mg / kg) of PT-EPO was administered daily for 14 days to dogs. Furthermore, after administration (PO) of a composition comprising PT-EPO for 14 days, treated animals had a significantly higher red blood cell count (6.74 x 10 6 Cells / ul vs 7.26 x 10 6 The levels of hemoglobin (15.85 g / dl vs. 17.125 g / dl) and hematocrit (46.8% vs. 49.65%) increased, confirming the bioavailability of the orally administered composition containing the polypeptide construct (see Figures 9 and 10). EXAMPLES

[0111] GLP-1 agonists such as exenatide and liraglutide are desirable candidates for formulation as orally administered therapeutics. We wished to test whether the polypeptides according to SEQ ID NO: 1-40, when conjugated to a GLP-1 agonist as a polypeptide construct, would enhance the transport of the GLP-1 agonist from the stomach to the blood after oral administration. The amino acid sequences of exenatide and liraglutide were used to clone the downstream sequence of the polypeptide containing the amino acid sequence according to SEQ ID NO: 1, and an expression vector system was used to generate peptide constructs containing the amino acid sequences according to SEQ ID NO: 42 and 44 (referred to as "PT-GA1" and "PT-GA2" in the figures, respectively). We also generated a polypeptide construct according to SEQ ID NO: 46 ("PT-GA2B"), which contains the liraglutide sequence flanked by the polypeptides according to SEQ ID NO: 1 at both the carboxy and amino termini of the liraglutide sequence. The polypeptide constructs were tested in the Caco-2 uptake assay (Example 1), with uptake of 28.7% for PT-GA1, 31.4% for PT-GA2, and 29.4% for PT-GA2B (see FIG. 11). To test the efficacy and bioavailability in vivo, Sprague-Dawley rats were administered (PO) 600 ug of a composition comprising a polypeptide construct selected from PT-GA1, PT-GA2, or PT-GA2B, and blood was drawn at 0, 4, and 6 hours after administration to measure blood glucose levels. After administration, the blood glucose levels of PT-GA1-treated animals decreased from 107 mg / dL at 0 hours to 118 mg / dL at 4 hours and to 87.5 mg / dL at 6 hours after administration, while those of PT-GA2 and PT-GA2B were 98 mg / dL, 126,111 mg / dL, and 102 mg / dL, 105 mg / dL, and 104 mg / dL, respectively (see FIG. 12). EXAMPLES

[0112] Alternative strategies for generating the peptide constructs disclosed herein include chemical synthesis and ligation. In one example, a polypeptide according to SEQ ID NO:21-40 having a modified terminal residue is chemically ligated to a heterologous polypeptide having a modified terminal residue. For example, the terminal lysine of the polypeptide can be an alkyl-modified peptide, and the terminal residue of the heterologous polypeptide can be an azide-modified peptide, where the alkyl-modified peptide reacts with the azide-modified peptide to generate an amide bond between the polypeptide and the heterologous polypeptide (see FIG. 16).

[0113] In one embodiment shown in Figure 13, the terminal amino acid of the peptide according to sequence ID 1-20 comprises a modified lysine residue, Lys(N3) = Fmoc-L-Lys(N3)-OH. In one exemplary formula, the modified peptide according to sequence ID 21 is H-Met-Ala-Asp-Asp-Ala 5 -Gly-Ala-Ala-Gly-Gly 10 -Pro-Gly-Gly-Pro-Gly 15 -Gly-Pro-Gly-Met-Gly 20 -Asn-Arg-Gly-Gly-Phe 25 -Arg-Gly-Gly-Phe-Gly 30 -Ser-Gly-Ile-Arg-Gly 35 -Arg-Gly-Arg-Gly-Arg 40 -Gly-Arg-Gly-Arg-Gly 45 -Arg-Gly-Arg-Gly-Lys(N3) 50 -OH, wherein the polypeptide is conjugated via the lysine terminal residue to a heterologous polypeptide, including a therapeutic polypeptide, and the heterologous polypeptide is a modified polypeptide according to the formula: Propynoic acid-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol.

[0114] In one embodiment, copper(I)-catalyzed alkyne azide 1,3-dipolar cycloaddition (CuAAC) or "click" reaction is utilized to conjugate peptides according to SEQ ID NO:21-40 to heterologous polypeptides. The copper-catalyzed click reaction is a versatile reaction that can be performed under a variety of reaction conditions, including various solvents, a wide pH and temperature range, and different copper sources, with or without additional ligands or reducing agents. The reaction is highly selective and can be performed in the presence of other functional groups. The 1,4-disubstituted triazole products of the CuAAC reaction are suitable for isosteres for amide conjugation.

[0115] In one example, the heterologous polypeptide is an octapeptide shown in Figure 14, which mimics the pharmacological effect of natural somatostatin (similar to Octreotide sold under the brand name Sondostatin®), and is ligated to a polypeptide according to SEQ ID NOs: 21-40 (modified) to generate a polypeptide construct according to SEQ ID NO: 50 (referred to herein as "PT-OCT" or "PT-OCT click") shown in Figures 15A-15D.

[0116] The compound produced by ligation comprises the formula: H-Met-Ala-Asp-Asp-Ala-Gly-Ala-Ala-Gly-Gly-Pro-Gly-Gly-Pro-Gly-Gly-Pro-Gly-Met-Gly-Asn-Arg-Gly-Gly-Phe-Arg-Gly-Gly-Phe-Gly-Ser-Gly-Ile-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Nle(triazole-propionyl-D-Phe-Cys-Phe-D-Tru-Lys-Thr-Cys-Thr-ol)-OH.

[0117] A compound having the following characteristics: Appearance: White to off-white powder. Identity: Mass spectrometry MW(average)=5822.5±lamu, (M+4H)4+ / 4=1456.9 / (M+4H)5+ / 5=1165.7. After deconvolution: MW=5823.6amu. Purity: RP-HPLC 90%. Net peptide content (NPC) (nitrogen analysis): 83.5%. Moisture Content (Karl Fischer USP <921> 5.2%, and Total mass balance (NPC + counterion (acetate)): 90-105% / 97%.

[0118] Polypeptide constructs generated by ligation methods such as click chemistry can also be tested using in vitro methods described herein, including a Caco-2 uptake assay to assess uptake of the polypeptide construct into Caco-2 cells, which mimics uptake of the polypeptide construct through the gastrointestinal barrier (see Example 1).

[0119] Additionally, a polypeptide construct comprising a polypeptide having an amino acid sequence identity to SEQ ID NO: 49 (utilizing the expression vector method disclosed herein, referred to as "PT-OCT fusion") and a polypeptide construct comprising a polypeptide having an amino acid sequence identity to SEQ ID NO: 50 (utilizing a chemical "click" ligation method, referred to as "PT-OCT click") were generated and compared side-by-side in vivo and in vitro. The PT-OCT fusion and PT-OCT click constructs were shown to be effective in eliciting uptake in vitro (Caco-2 cells). In one example, approximately 38% of the polypeptide constructs (PT-OCT fusion and PT-OCT click) administered to Caco-2 cells were taken up by the Caco-2 cells (see FIG. 17, where uptake by PT-EPO was also performed for reference).

[0120] To determine whether a polypeptide construct comprising a polypeptide having amino acid sequence identity to SEQ ID NO: 49 or 50 is biologically active after administration (PO), the ability of PCT-OCT to inhibit insulin secretion from glucose-stimulated islets was tested. Twenty-five human islets (from donor islets procured from an IIDP-approved islet center) were seeded in triplicate in a 96-well plate containing 100 μl of CMRL islet medium (available from MediaTech, et al.). The islets were preincubated with either 100 μl (50%) serum (serum obtained from pancreatic donor), 100 μl (50%) serum + PT-OCT, or 100 μl (50%) serum + PT-OCT exposed to thrombin for 45 minutes to cleave the polypeptide sequence from the therapeutic octapeptide of the polypeptide construct. Following preincubation, the medium and serum were removed and replaced with KREB buffer containing 16.7 mM glucose and incubated for an additional 30 minutes, at which point the KREB buffer was withdrawn. Supernatant insulin concentrations were measured using a commercially available ELISA kit (available from Abcam, etc.). Both polypeptide constructs (PT-OCT and PCT-OCT "slices") demonstrated the ability to inhibit insulin secretion in glucose-stimulated islets, with the polypeptide construct containing full-length PT-OCT reducing insulin secretion by 46%, while the polypeptide construct containing the thrombin-cleaved PCT-OCT "slices" inhibiting insulin expression in glucose-stimulated islets by over 61% (see Figures 18 and 19).

[0121] The foregoing examples are supplemented by the accompanying figures and Tables 1 and 2. Some of the examples and figures refer to compositions and constructs of the disclosure other than SEQ ID NOs, the nomenclature of which is set forth in Table 1.

[0122] Polypeptides according to sequence IDs 1-40 have been shown to be safe and effective for targeted drug delivery. The use of peptides in pharmaceutical formulations is generally believed to be rapidly cleaved by proteolytic enzymes and rapidly cleared from the blood circulation by the liver and kidneys. These pharmacodynamic properties can be modulated by various modification and stabilization approaches (Vlieghe et al., 2010). One of the best-known concepts for stabilizing peptides is lipidation, which incorporates fatty acids into the peptide (Zhang and Bulaj, 2012). Lipidation of the polypeptide constructs disclosed herein is also contemplated by the present disclosure. Fatty acids bind to serum albumin and prevent proteolytic cleavage by proteases in the blood, thereby extending circulation time (Frokjaer and Otzen, 2005). Liraglutide (Victoza®) and semaglutide (Ozempic®) (Marso et al., 2016), long-acting glucagon-like peptide-1 (GLP-1) receptor agonists used to treat type 2 diabetes and obesity, are examples of this approach. Peptides are generally considered safe because they have low immunogenicity and produce non-toxic metabolites (Ahrens et al., 2012).

[0123] While the foregoing information emphasizes the aspects disclosed herein as illustrations and examples for purposes of clarity of understanding, it will be apparent that certain changes and modifications can be made within the scope of the subject matter claimed herein. It will be apparent to one skilled in the art that features described in connection with any of the above aspects and various embodiments can be applied interchangeably between the different embodiments.

[0124] The above aspects and embodiments are examples intended to illustrate various features of the subject matter claimed herein. All publications and patent applications disclosed in this specification are indicative of the level of ordinary skill in the art to which the subject matter of the disclosure and claims pertains.

[0125] It will be understood that numerical values ​​will contain some degree of experimental error. Thus, the use of the modifier "about" (or "approximately") preceding a numerical error indicates the incorporation of the possible experimental error associated with the numerical value. The absence of the modifier "about" (or "approximately") preceding an experimentally obtained numerical value does not imply that the numerical value is free from some degree of experimental error.

[0126] Throughout this description and the claims, the terms "comprise" and "contain" and variations thereof mean "including, but not limited to," and are not intended to (and do not) exclude other moieties, additives, ingredients, or steps. Throughout this description and the claims, the singular encompasses the plural, unless the context dictates otherwise. Where indefinite articles are used, the specification is understood to contemplate the plural as well as the singular, unless the context requires otherwise.

[0127] It is understood that features, properties, compounds, chemical moieties, or groups described in connection with a particular aspect, embodiment, or example are applicable to other aspects, embodiments, or examples described herein, unless inconsistent therewith. All features disclosed herein (including the accompanying claims, abstract, and drawings), and / or all steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The subject matter claimed herein is not limited to the details of the foregoing embodiments. The subject matter claimed herein extends to any novel one or any novel combination of features disclosed herein (including the accompanying claims, abstract, and drawings), or any novel one or any novel combination of steps of any method or process so disclosed.

[0128] All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A polypeptide construct comprising: (a) a first polypeptide comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from any one of SEQ ID NOS: 1-40; and (b) a second polypeptide, wherein the second polypeptide is heterologous to the first polypeptide; and A polypeptide construct comprising:

2. The polypeptide construct of claim 1, wherein the first polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 1-40.

3. The polypeptide construct of claim 2, wherein the first polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOS: 1 to 40.

4. The polypeptide construct of claim 1, wherein the first polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or 21.

5. The polypeptide construct of claim 4, wherein the first polypeptide comprises sequence ID number 1 or 21.

6. The polypeptide construct of claim 1, wherein the first polypeptide and the second polypeptide are linked via a linker.

7. The polypeptide construct of claim 1, wherein the first polypeptide and the second polypeptide are linked via a covalent bond, an ionic bond, or a non-covalent bond.

8. 8. The polypeptide construct of claim 7, (a) the N-terminus of the second polypeptide is linked to the C-terminus of the first polypeptide; or (b) the N-terminus of the first polypeptide is linked to the C-terminus of the second polypeptide; Polypeptide constructs.

9. The polypeptide construct of claim 7, wherein the covalent bond is an amide bond.

10. The polypeptide construct of claim 6, wherein the first polypeptide and the second polypeptide are linked via a polypeptide linker.

11. The polypeptide construct of claim 10, wherein the polypeptide linker is a flexible linker.

12. 12. The polypeptide construct of claim 11, wherein the flexible linker comprises a plurality of amino acids selected from glycine and / or serine.

13. The polypeptide construct of claim 10, wherein the polypeptide linker is a rigid linker.

14. The polypeptide construct of claim 1, further comprising a third polypeptide comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from any one of SEQ ID NOs: 1 to 40.

15. The polypeptide construct of claim 14, wherein the third polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from any one of SEQ ID NOs: 1 to 40.

16. The polypeptide construct of claim 15, wherein the third polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOS: 1 to 40.

17. 15. The polypeptide construct of claim 14, wherein the third polypeptide is linked to the first or second polypeptide via an ionic bond.

18. 15. The polypeptide construct of claim 14, wherein the third polypeptide is linked to the first or second polypeptide via a covalent bond.

19. 19. The polypeptide construct of claim 18, (a) the N-terminus of the second polypeptide is linked to the C-terminus of the first polypeptide; and (b) the C-terminus of the second polypeptide is linked to the N-terminus of the third polypeptide; Polypeptide constructs.

20. 19. The polypeptide construct of claim 18, wherein the covalent bond is an amide bond.

21. 20. The polypeptide construct of claim 18, wherein the third polypeptide is linked to the first or second polypeptide via a polypeptide linker.

22. 22. The polypeptide construct of claim 21, wherein the polypeptide linker is a flexible linker.

23. 23. The polypeptide construct of claim 22, wherein the flexible linker comprises a plurality of glycines and serines.

24. 22. The polypeptide construct of claim 21, wherein the polypeptide linker is a rigid linker.

25. 20. The polypeptide construct of claim 18, wherein a third polypeptide is linked to the first or second polypeptide via click chemistry.

26. The polypeptide construct of claim 1, wherein the second polypeptide is or comprises a therapeutic protein.

27. 27. The polypeptide construct of claim 26, wherein the therapeutic protein is a hormone, interferon, interleukin, growth factor, tumor necrosis factor, thrombolytic agent, enzyme, antibody, Fc fusion protein, anticoagulant, blood factor, bone morphogenetic protein, or artificial protein scaffold.

28. 28. The polypeptide construct of claim 27, wherein the hormone is erythropoietin.

29. 29. The polypeptide construct of claim 28, wherein the erythropoietin is epoetin alfa or pegylated epoetin.

30. 28. The polypeptide construct of claim 27, wherein the hormone is a glucagon-like peptide 1 (GLP-1) agonist.

31. The polypeptide construct according to claim 30, wherein the GLP-1 agonist is semaglutide, exenatide, or liraglutide.

32. 28. The polypeptide construct of claim 27, wherein the hormone is insulin.

33. 33. The polypeptide construct of claim 32, wherein the insulin is insulin aspart, insulin lispro, insulin glulisine, insulin detemir, degludec insulin, or glargine insulin.

34. 27. The polypeptide construct of claim 26, wherein the therapeutic protein is somatostatin, a somatostatin analog, glucagon, galsulfase, nesiritide, or taliglucerase alfa.

35. The polypeptide construct of claim 1, wherein the polypeptide construct comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 41, 44-52.

36. The polypeptide construct of claim 35, wherein the polypeptide construct comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 41, 44-52.

37. The polypeptide construct of claim 35, wherein the polypeptide construct comprises an amino acid sequence selected from any one of SEQ ID NOs: 41, 44-52.

38. 10. The polypeptide construct of claim 1, wherein the polypeptide construct is conjugated to one or more of a cytotoxin, a fluorescent label, and an imaging agent.

39. 10. The polypeptide construct of claim 1, wherein the polypeptide construct comprises one or more amino acid modifications.

40. A pharmaceutical composition comprising the polypeptide construct of claim 1 and optionally a pharmaceutically acceptable excipient.

41. 41. The pharmaceutical composition of claim 40, wherein the pharmaceutical composition further comprises an excipient, a stabilizer, a permeation enhancer, a protease inhibitor, or any combination thereof.

42. 41. The pharmaceutical composition of claim 40, wherein the pharmaceutical composition is formulated for oral administration.

43. A polypeptide construct according to claim 1 or a pharmaceutical composition according to claim 40 for use as a medicament.

44. A polypeptide construct according to claim 28, or a pharmaceutical composition comprising said polypeptide construct, for use in the treatment of anemia.

45. A polypeptide construct or pharmaceutical composition for use as described in claim 44, wherein the polypeptide construct comprises the amino acid sequence of sequence ID number 41.

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