Polypeptides and compositions for oral administration and methods of use thereof

Abstract

Disclosed herein are compositions and constructs related to a polypeptide, PT-X'n-PTA-Hm (1 ), or pharmaceutically acceptable salts thereof, wherein PT is a peptide transport or fragment thereof; X'n is linker comprising a triazole moiety, n = 0 or 1; PTA is a peptide therapeutic agent selected from the group consisting of a modified semaglutide, a modified tirzepatide, caplacizumab, ozoralizumab; and Hm is a histidine tag where m is 0 to 6; as well as uses thereof.

Description

POLYPEPTIDES AND COMPOSITIONS FOR ORAL ADMINISTRATION AND METHODS OF USE THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U. S. Provisional Patent Application Nos. 63 / 724239, filed on November 22, 2024; 63 / 839,371, filed on July 7, 2025; and 63 / 839,404, filed on July 7, 2025, each of which are hereby incorporated by reference.BACKGROUND

[0002] Tirzepatide is a synthetic peptide that functions as a dual agonist of the glucosedependent insulinotropic polypeptide (GIP) receptor and the glucagon-like peptide-1 (GLP-1) receptor. See Bokvist and Mounjaro Label. Tirzepatide is useful for the treatment of type 2 diabetes mellitus, obesity, and obstructive sleep apnea. An issue with tirzepatide is that it is administered subcutaneously (SQ). The SQ administration can cause significant patient compliance challenges due to injection site discomfort and fear of needles.

[0003] Semaglutide is a synthetic peptide that functions as an agonist of the GLP-1 receptor. See Ozempic Label. Semaglutide is useful for the treatment of type 2 diabetes mellitus, obesity, among other things. An issue with certain marketed forms of semaglutide (e.g., Ozempic and Wegovy) is that it is administered subcutaneously.

[0004] Nanobodies are single domain antigen recognizing proteins. Compared to other antigen recognizing proteins (e.g. antibodies) Nanobodies are typically characterized by a relatively low molecular weight (less than 50k Daltons) and lack of glycosylation leading to the ability to produce in E. coli. systems while maintaining a high affinity for the target antigen. The route of nanobody administration requires an injection (i.e. intravenous, intramuscular, or subcutaneous). Therapeutic administration of therapeutic nanobodies is complicated by their relatively short circulatory half-life leading to the need for frequent invasive dosing to maintain therapeutic levels of drug.REFERENCE TO SEQUENCE LISTING

[0005] The application contains a Sequence Listing which is submitted electronically in accordance with WIPO Standard ST.26. The Sequence Listing is entitled “IMG029.xml”, created on NOVEMBER 22, 2025, and is incorporated herein by reference in its entirety. The information recorded in the electronically submitted Sequence Listing file is part of the description of the present application.SUMMARY

[0006] Compositions comprising peptide-linked therapeutics for targeted delivery of the therapeutic across the gastrointestinal lining following oral administration to a subject are disclosed.

[0007] The present disclosure provides one or more of the following main advantages to achieve targeted delivery of a peptide-linked therapeutic by the oral route: a) prevents proteolytic activity that degrades the therapeutic in the stomach and gut, b) provides proteaseresistant therapeutic analogs that retain biological activity, c) stabilize the therapeutic by conjugation to a peptide that acts as a “shielding molecule”, and / or d) improve passive therapeutic or peptide transport (diffusion) through the epithelial membrane of the intestine.

[0008] Provided herein are compositions and methods for targeted delivery of a therapeutic across the gastrointestinal lining. In various embodiments, the disclosed compositions include but are not limited to a composition comprising a modified GLP-1 agonist and have been shown to improve the oral bioavailability from less than 1% to 50% or more. These compositions and methods are useful as therapeutics which were previously not considered suitable or formulated for oral administration.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 Represents an example of the peptide-linked therapeutics contemplated herein. Shown is a modified semaglutide conjugated to a peptide transporter, in one example a 50-mer peptide is linked to a modified semaglutide (shown in top row; see also SEQ ID NO: 10) and in another example a 31-mer is linked to a modified semaglutide (shown in second row; see also SEQ ID NO: 9). Linker “R2” is also shown.

[0010] FIG. 2 Shows one embodiment of a modified semaglutide-PT conjugate, wherein a 50mer peptide transporter is linked to a modified semaglutide (referred to herein as PT-2). See also SEQ ID NO: 10.

[0011] FIG 3. Shows one embodiment of a modified semaglutide-PT conjugate, wherein a 31mer peptide transporter is linked to a modified semaglutide (referred to herein as PT-3). See also SEQ ID NO: 9.

[0012] FIG. 4A Shows one embodiment of a modified semaglutide-PT conjugate, wherein a 31mer peptide transporter is linked, via a triazole linkage, to a modified semaglutide. See also SEQ ID NO: 9.

[0013] FIG. 4B Shows a modified semaglutide wherein the modification includes an azide moiety for ligation to the peptide transporter. See also SEQ ID NO: 8.

[0014] FIG. 4C Shows a 31 -mer (n = 0) or a 32-mer peptide (n = 1 ) transporter with an alkyne moiety available for ligation to the modified semaglutide.

[0015] FIG. 5 Shows that oral administration of a composition comprising PT-2 (SEQ ID NO: 10) results in a reduction in body weight in ZDF rats compared to untreated controls. The graph measures relative weight change overtime for rats treated vs untreated.

[0016] FIG. 6 Shows that a composition comprising PT-2 (SEQ ID NO: 10) has a bioavailability of approximately 25%.

[0017] FIG. 7 Shows that oral administration of a composition comprising PT-2 (SEQ ID NO: 10) results in a reduction in blood glucose in ZDF rats compared to untreated controls. Data was calculated as normalized change relative to day 1 blood glucose levels.

[0018] FIG. 8A - 8C: FIG. 8A shows one embodiment of a modified tirzepatide-PT conjugate (referred to herein as PT-4; see also SEQ ID NO: 12) whose conjugate is formed by a triazole between the PT and modified tirzepatide; FIG. 8B shows modified tirzepatide (see SEQ ID NO: 11 ); FIG. 8C shows 31-mer (n = 0) a 32-mer PT (n = 1) with azide group is linked to a modified tirzepatide with an alkyne (FIG 8B).

[0019] FIG. 9 Shows the results of an insulin sensitivity assay using islet cells of a composition comprising PT-4 (SEQ ID NO: 12) versus saline; results show the increase in insulin response observed in islet cells treated with PT-4 versus control.

[0020] FIG. 10 Shows the bioavailability results in rats treated with a composition comprising PT-4 (SEQ ID NO: 12) at 2.5 mg / kg PO (oral administration) vs. unmodified tirzepatide at 0.5 mg / kg SC (sub-cutaneous administration); results show that the orally administered composition comprising PT-4 has a similar bioavailability profile as IV-administered unmodified tirzepatide (control).

[0021] FIG. 11 Shows results of ZDF rats dosed daily (P. O.) with either a composition comprising PT-4 (SEQ ID NO: 12) or water (control). Weights were measured and percent change in weight was ascertained. Rats dosed PO with PT-4 were found to have a decrease in weight after 7 days of dosing.

[0022] FIG. 12 A and B Shows that a 50-mer peptide transporter is cleaved from a caplacizumab-PT conjugate (PT-6) (SEQ ID NO: 14) following treatment with thrombin. FIG 12A shows the caplacizumab-PT conjugate (PT-6) and 12B shows the Caplacizumab free of the 50-mer PT following treatment with thrombin, confirming that treatment with thrombin cleaves the 50-mer PT from the caplacizumab.

[0023] FIG. 13 Shows that a composition comprising a caplacizumab-PT conjugate (SEQ ID NO: 13) when administered orally (0.1 mg / mL) in mice is taken up into thebloodstream, as evidenced by plasma concentration at 1, 3 and 7 hours post-dose (measured as ng / mL) using an ELISA assay against a His-tag on the caplacizumab-PT conjugate.

[0024] FIG. 14 Shows the results of a RIPA test with human plasma incubated with ristocetin (which induces platelet aggregation by activating vWF). The ristocetin-induced platelet aggregation (RIPA) test is used to assess vWF function by observing platelet aggregation in response to ristocetin. Precent aggregation was measured (with and without inhibitor) after 10 minutes. The data shows that each of caplacizumab-PT conjugate (PT-6) (SEQ ID NO: 14), PT-6 (cleaved), and unmodified caplacizumab (control) reduce aggregation in human plasma at concentrations less than 10 nM.

[0025] FIG. 15 Shows the pK profile of a Caplacizumab-PT conjugate (SEQ ID NO: 13). DETAILED DESCRIPTION

[0026] The following terms are used in this disclosure to describe different embodiments. These terms are used for explanation purposes only and are not intended to limit the scope for any aspect of the subject matter claimed herein.

[0027] As used herein “composition” or “formulation” refer (interchangeably) to an active agent (i.e., a therapeutic) in a specific presentation, such as an aqueous solution, solid, semi solid or aerosol for administration by oral or parenteral route. If needed, the formulation may comprise a carrier, e.g., one or more pharmaceutically acceptable carriers, excipients and / or one or more additives. The formulations disclosed herein may contain other known active agents, in combination with the active agents described herein.

[0028] As used herein the term “peptide transport leader” or “peptide transporter”, or simply “PT” refers to a peptide sequence that is operably linked to an active agent, or therapeutic, (e.g., semaglutide, tirzepatide, caplacizumab, or ozoralizumab), and facilitates epithelial transport and systemic bioavailability. Conservative amino acid substitutions, truncations, or extensions are encompassed in other embodiments.

[0029] As used herein “peptide therapeutic agent” or “PTA” refers to an active agent or therapeutic that may be linked to a peptide transporter, wherein the PT facilitates epithelial transport of the PTA.

[0030] As used herein “polypeptide” is a polymer of amino acids of three or more amino acids in a serial array, linked through peptide bonds. The term “polypeptide” includes proteins, protein fragments, protein analogues, oligopeptides and the like. The term “polypeptide” contemplates polypeptides that are encoded by nucleic acids, produced through recombinant technology, isolated from an appropriate source, or are synthesized. The term “polypeptide” further contemplates polypeptides as defined above that include chemically modified amino acids or amino acids covalently or noncovalently linked to othermolecules, functional groups, ligation ligands, or labeling ligands. Polypeptide and peptide are used interchangeably herein.

[0031] As used herein " SEQ ID”, or “SEQ ID NO” refer (interchangeably) to a protein, polypeptide, peptide fragment, or analogue thereof, and including any modification thereto, having an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence specified by number, according to the number listed in Table 1.

[0032] As used herein, the term "sequence identity" refers to the identity between two nucleic acid molecules, polypeptides, or amino acids, expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. The percentage identity is calculated over the entire length of the sequence. Homologs or orthologs of amino acid sequences possess a relatively high degree of sequence identity when aligned using standard methods. This homology is more significant when the orthologous proteins are derived from species which are more closely related (e.g., human and mouse sequences), compared to species more distantly related (e.g., human and C. elegans sequences).Methods of alignment of 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 consideration of sequence alignment methods and homology calculations. The level of sequence identity may 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 may also be used to determine similarity. The BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0 at http: / / www.ncbi.nlm.nih.gov / blast / ). Amino acid residues may be post-translationally modified or conjugated or modified with other functional or non-functional molecular groups; naturally, such modified amino acid residues are included in the amino acid sequences 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 to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotides encoding such polypeptides, are contemplated. In comparing sequences, the above methods account for various substitutions, deletions, and other modifications. 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 presently disclosed proteins comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the proteins of this disclosure by standard techniques known in theart, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties such as charge and polarity. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, histidine, negatively charged amino acids include aspartic acid, glutamic acid, neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, 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; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine.

[0033] As used herein, “subject” or “individual” or “animal” or “patient” or “mammal” refers to a subject, in particular a mammalian subject, for which treatment is sought, or a diagnosis, prognosis or therapy is desired, for example, to a human.

[0034] As used herein, the terms “treat,” “treating” or “treatment,” and other grammatical equivalents as used herein, include alleviating, abating or ameliorating a disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and prophylaxis. The terms further include achieving a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a patient at risk of developing a particular disorder, or to a patient reporting one or more of the physiological symptoms, even though a diagnosis may not have been made.

[0035] As used herein, a “therapeutically effective amount” or “effective amount”, is an amount of biologically active agent / therapeutic polypeptide capable of achieving a clinically relevant endpoint in a subject when administered in one or repeated doses to the subject. Such effect need not be absolute to be beneficial. The appropriate dose of the composition may depend on the route of administration, such as oral, injection or infusion, and may depend on the subject being treated as well as the severity of the condition to be treated. Using scaling methods, such as allometric scaling, it is possible to predict suitable and exemplary dosage ranges for the administration of compositions, as disclosed herein, to adult humans. Dose scaling is an empirical approach, is well characterized and understood in the art. This approach assumes that there are some unique characteristics on anatomical, physiological, andbiochemical process among species, and the possible difference in pharmacokinetics / physiological time is, as such, accounted for by scaling. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0036] Disclosed peptides relate to PT-X'n-PTA-Hm(1 ) or pharmaceutically acceptable salts thereof, where PT is a peptide transport, X’nis linker comprising a triazole moiety that may be present / absent (i.e., n = 0 or 1), PTA is a peptide therapeutic agent, and Hmis a histidine tag where m is 0 to N (e.g., N= 0, 1, 2, 3, 4, 5, or 6). The transporter can be either on N or C terminal of the PTA provided the peptide transporter has its C-terminal facing away from the his-tag labeled protein.

[0037] For illustration, PT may be comprised of 50 amino acids and may be represented as follows:

[0038] Xaa01 Xaa02 Xaa03 Xaa04 Xaa05 Xaa06 Xaa07 Xaa08 Xaa09 Xaa10

[0039] Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20

[0040] Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Xaa30

[0041] Xaa31 Xaa32 Xaa33 Xaa34 Xaa35 Xaa36 Xaa37 Xaa38 Xaa39 Xaa40

[0042] Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50

[0043] where

[0044] Xaa01 = M, A, V, I, L;

[0045] Xaa02 = A, G, S;

[0046] Xaa03 = D, E;

[0047] Xaa04 = D, E;

[0048] Xaa05 = A, G, S;

[0049] Xaa06 = G, A, S;

[0050] Xaa07 = A, G, S;

[0051] Xaa08 = A, G, S;

[0052] Xaa09 = G, A, S;

[0053] Xaa10 = G, A, S;

[0054] Xaa11 = P;

[0055] Xaa12 = G, A, S;

[0056] Xaa13 = G, A, S;

[0057] Xaa14 = P;

[0058] Xaa15 = G, A, S;

[0059] Xaa16 = G, A, S;

[0060] Xaa17 = P;

[0061] Xaa18 = G, A, S;

[0062] Xaa19 = M, A, V, I, L;

[0063] Xaa20 = G, A, S, N;

[0064] Xaa21 = N, G, A, Q;

[0065] Xaa22 = R, K, Q;

[0066] Xaa23 = G, A, S;

[0067] Xaa24 = G, A, S;

[0068] Xaa25 = F, Y, W, L, H;

[0069] Xaa26 = R, K, Q;

[0070] Xaa27 = G, A, S;

[0071] Xaa28 = G, A, S;

[0072] Xaa29 = F, Y, W, L, M;

[0073] Xaa30 = G, A, S;

[0074] Xaa31 = S, A, T, N;

[0075] Xaa32 = G, S, N;

[0076] Xaa33 = I, V, M;

[0077] Xaa34 = R, K, Q;

[0078] Xaa35 = G, A, S, N;

[0079] Xaa36 = R, K, Q;

[0080] Xaa37 = G, A, S, N;

[0081] Xaa38 = R, K, Q;

[0082] Xaa39 = G, A, S, N;

[0083] Xaa40 = R, K, Q;

[0084] Xaa41 = G, A, S, N;

[0085] Xaa42 = R, K, Q;

[0086] Xaa43 = G, A, S, N;

[0087] Xaa44 = R, K, Q;

[0088] Xaa45 = G, A, S, N;

[0089] Xaa46 = R, K, Q;

[0090] Xaa47 = G, A, S, N;

[0091] Xaa48 = R, K, Q;

[0092] Xaa49 = G, A, S, N; and

[0093] Xaa50 = A, G, V, L.

[0094] In view of the foregoing, it will be appreciated that PT or a pharmaceutically acceptable salt thereof may be a peptide comprising 50 amino acids having the following primary sequence.MADDA5GAAGG10PGGPG15GPGMG20NRGGF25RGGFG30SGIRG35RGRGR40GRGRG45RGRGA50, which is referred to herein as PT(1-50). Using the PT(1-50) sequence a reference, contemplated herein are additional peptide transporters designated as PT(1-49), PT(20-49), PT(19-49), PT(20-50), where the parenthetical numbers refer to the numbered amino acids of PT(1-50), and contemplated variants thereof (see above Xaa01Xaa02Xaa03... Xaa50). For instance, an exemplary peptide fragment of PT(1-50) is PT(19-49), which refers to MG20NRGGF25RGGFG30SGIRG35RGRGR40GRGRG45RGRG. Anotherexemplary peptide fragment of PT(1-50) is PT(20-50), which refers to G20NRGGF25RGGFG30SGIRG35RGRGR40GRGRG45RGRGA50.

[0095] Additional PT fragments include, for example PT(Xaa,a-Xaa,co), where a is any number from 2-37 and co is 49 or 50. For instance, using the PT(1-50) example, when a is 37 and co is 50, the PT fragment comprises GRGR40GRGRG45RGRGA50, and when a is 37 and co is 49, the PT fragment comprises GRGR40GRGRG45RGRG. These PT fragments are disclosed in U. S. Provisional Patent Application No. 63 / 839,371.

[0096] As related to PT-X’n-PTA-Hm (1), it will be appreciated that X' may be absent (i.e., n = 0), and thus, PT is covalently tethered to PTA via a peptide bond. In certain instances, n is 1, and X' comprises a triazole moiety derived from, for example from the reaction product of an azido-derivative and an alkynyl derivative. For instance, the azido-derivative may be based on lysine, such as 6-azido-L-lysinyl (or KN3), or any suitable azide derivative of an amino-substituted organic radical, such as L-ornithine, L-2,4-diaminobutyric acid, 5-aminovaleric acid, y-aminobutyric acid, and the like. One will appreciate that click chemistry may be employed to couple a PT-fragment comprising a modified terminal amino acyl derivative (AAD) comprising an azido moiety (AAD-N3, such as KN3) with a PTA comprising a modified terminal AAD comprising an alkynyl (i.e., -C=CH) moiety (e.g., AAD-C=CH), such as, N8-(pent-4-ynoyl-L-lysine, (S)-N-(L-seryl)-2-aminopent-4-ynamide, and the like, thereby forming a linker comprising a triazolyl moiety shown in, for example, FIG. 1.

[0097] In certain embodiments, the linker (X) comprising a triazole moiety may be derived from a PT-azido derivative (see FIG. 8C) and a PTA-alkynyl derivative (see FIG. 8B). Accordingly, modified PTs include, e.g., PT(20-49)X (e.g., X = AADN3, such as KN3), and PT(20-50)X (e.g., X = AADN3, such as KN3), and PT(19-49)X (e.g., X = AADN3, such as KN3), and PT(19-50)X (e.g., X = AADN3, such as KN3), and PT(20-48)X (e.g., X = AADN3, such as KN3).

[0098] In other embodiments, the linker (X) comprising a triazole moiety may be derived from a PT-alkynyl derivative (see FIG. 4C) and a PTA-azido derivative (see FIG. 4B). Accordingly, modified PTs include, e.g., PT(20-49)X (e.g., X = AAD-C=CH), and PT(20-50)X (e.g., X = AAD-C=CH), and PT(19-49)X (e.g., X = AAD-C=CH), and PT(19-50)X (e.g., X = AAD-C=CH), and PT(20-48)X (e.g., X = AAD-C=CH).

[0099] As related to PTA, of PT-X’n-PTA-Hm(1), where n = 1 and m = 0, specific PTAs include but are not limited to modified versions of semaglutide (see SEQ ID No. 8) and tirzepatide (see SEQ ID No. 11).

[0100] As related to PTA, of PT-X’n-PTA-Hm(1), where n = 0 and m = 6, specific PTAs include but are not limited to caplacizumab and ozoralizumab; and where m=0, specific PTAs include but are not limited to caplacizumab and ozoralizumab.

[0101] Disclosed herein are peptides, polypeptides, polypeptide fragments, heterologous polypeptides, therapeutic polypeptides, and polypeptide constructs formed therefrom, and pharmaceutically acceptable salts thereof, with sequence identities as identified and set forth in Table 1.Table 1: List of SequencesSEQ ID Amino Acid Sequence DescriptionNO:MADDA5GAAGG10PGGPG15GPGMG20NRGGF25RGGFG30SGIRG35R PT(1-49)X (X = KN3(6-azido-L-lysinyl)), 1 GRGR40GRGRG45RGRGX50also referred to herein as a 50-mer peptide transporter. (C - N direction) MADDA5GAAGG10PGGPG15GPGMG20NRGGF25RGGFG30SGIRG35R PT(1-50) (No linker; for expression with 2 GRGR40GRGRG45RGRGA50nanobody)PT(19-49)X (X = KN3(or N6-diazo-L- 3 MG20NRGGF25RGGFG30SGIRG35RGRGR40GRGRG45RGRG49X50lysinyl), also referred to herein as a 31-mer peptide transporter, ora 31-mer.PT(19-50)X (X = KN3 (or N6-diazo-l_- 4 MG20NRGGF25RGGFG30SGIRG35RGRGR40GRGRG45RGRGA50X51lysinyl)), also referred to herein as a 32-mer peptide transporter, or a 32-mer.PT(20-50)X (X = KN3(or N6-diazo-L- 5 G20NRGGF25RGGFG30SGIRG35RGRGR40GRGRG45RGRGA50X lysinyl), also referred to herein as a 31-mer peptide transporter, ora 31-mer.(PT(20-49)X (X = KN3(or N6-diazo-L- 6 G20NRGGF25RGGFG30SGIRG35RGRGR40GRGRG45RGRG49X lysinyl), also referred to herein as a 30-mer peptide transporter, or a 30-mer.(PT(20-48)X (X = KN3(or N6-diazo-L- 7 G20NRGGF25RGGFG30SGIRG35RGRGR40GRGRG45RGR48X lysinyl), also referred to herein as a 29-mer peptide transporter, or a 29-mer.H-His-Aib-Glu-Gly-Thr5-Phe-Thr-Ser-Asp-Val-Ser10-Ser-Tyr-Leu- Glu-Gly15-Gln-Ala-Ala-Lys(R1) -Glu20-Phe-lle-Ala-Trp-Leu-Val25- Modified semaglutide8 R1= C18diacid-y-Glu-AEEA-AEEA; see FIG Arg-Gly-Arg-Gly-Lys(Na) 4B (azido (N3) on modified semaglutide)Modified semaglutide linked to 32-mer PT (SEQ ID NO. 3), aka PT(19-49)-X’-Sema H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- (where X' = K'-Rj-K'), also referred to herein Gly-Gln-Ala-Ala-Lys-(R1) -Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg- as “PT-3”9 Gly-Lys-(R2)-Lys-Gly-Arg-Gly-Arg-Gly-Arg- Gly-Arg-Gly-Arg-Gly-Arg- Gly-Arg-Gly-Arg-lle-Gly-Ser-Gly-Phe-Gly-Gly-Arg-Phe-Gly-Gly-Arg- R1= C18diacid-y-Glu-AEEA-AEEA Asn-Gly-Met-H R2= triazolylModified semaglutide linked to 50-mer PT H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu- Gly-Gln-Ala-Ala-Lys-(RI) -Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg- (SEQ ID NO. 1), aka PT(1-49)-X'-Sema Gly-Lys-(R2)-Lys-Gly-Arg-Gly-Arg-Gly-Arg- Gly-Arg-Gly-Arg-Gly-Arg- (where X' = K'-R2-K’), also referred to herein 10 as “PT-2”.Gly-Arg-Gly-Arg-lle-Gly-Ser-Gly-Phe-Gly-Gly-Arg-Phe-Gly-Gly-Arg- Asn-Gly-Met-Gly-Pro-Gly-Gly-Pro-Gly-Gly-Pro-Gly-Gly-Ala-Ala-Gly- Ala-Asp-Asp-Ala-Met-H R1= C18diacid-y-Glu-AEEA-AEEA R2= triazolyl [Y{Aib}EGTFTSDYSI{Aib}LDKIAQ{Lys(PEG2-PEG2-y-Glu- Exemplary tirzepatide analog, where S' = 11C20DIACID)}AFVQWLIAGGPSSGAPPPS' (S)-N-(L-seryl)-2-aminopent-4-ynamide.Modified tirzepatide linked to 32-mer (SEQ ID NO. 4), aka PT(20-49)-X’-TZP, where X’ is K’-R2-S', also referred to herein as “PT- 4."2-amino-isobutyric acid;Y{Aib)EGTFTSDYSI{Aib}LDKIAQ{Lys(PEG2-PEG2-gamma-glu- {Lys(PEG2-PEG2-Y-Glu-C20DIACID)} is 12 C20DIACID)}AFVQWLIAGGPSSGAPPPS{Pra}{Amidation}-tri azole identically found in tirzepatide (see Mountjaro Label, Sec. 11; see also Bokvist Lys{AGRGRGRGRGRGRGRGRIGSGFGGRFGGRNGM(US9474780B2), 8:55-10:55); where S{Pra}{Amidation}-triazole Lys refers to the conjugate linker obtained from click chemistry coupling of KN3(6-azido-L-lysinyl) from SEQ ID NO: 4 and the alkynyl moiety of (S)-N-(L-seryl)-2-aminopent-4-ynamide ofSEQ ID NO: 11.MADDAGAAGGPGGPGGPGMGNRGGFRGGFGSGIRGRGRGRGRGRGRGRGA- EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGK GRELVAAISRT GGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAA GVRAEDGRV 50mer-Caplacizumab with His-tag RTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLS CAASGRTFSY (“PT-6-His" or PT(1-50)-Caplacizumab-H6) NPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAK RMVYLQMNS LRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSH HHHHH MADDAGAAGGPGGPGGPGMGNRGGFRGGFGSGIRGRGRGRG RGRGRGRGA- EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGK GRELVAAISRT GGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAA 50mer-Caplacizumab GVRAEDGRV RTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLS (“PT-6” or PT(1-50)-Caplacizumab CAASGRTFSY NPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAK RMVYLQMNS LRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS MG20NRGGF25RGGFG30SGIRG35RGRGR40GRGRG45RGRGA5“- EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGK GRELVAAISRT GGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAA GVRAEDGRV RTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLS 32mer-Caplacizumab with His-tag CAASGRTFSY or PT(19-50)-Caplacizumab-H6 NPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAK RMVYLQMNS LRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSH HHHHH MG20NRGGF25RGGFG30SGIRG35RGRGR40GRGRG45RGRGA50- QPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRT GGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAA GVRAEDGRV RTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLS 32mer-Caplacizumab CAASGRTFSY or PT(19-50)-Caplacizumab-H6 NPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAK RMVYLQMNS LRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS MADDA5GAAGG10PGGPG15GPGMG20NRGGF25RGGFG30SGIRG35RGRGR40GRGRG45RGRGA50- EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGK GLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPE DTAVYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESG 50mer-Qzoralizumab with His Tag GGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI (“PT-5-His” or PT(1-50)-Qzoralizumab-H6) GGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGS LRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPD SVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQ GTLVTVSSHHHHHHMADDA5GAAGG10PGGPG15GPGMG2° NRGGF25RGGFG3°SGIRG35RGRGR40GRGRG45RGRGA50- EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGK GLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPE DTAVYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESG 50mer-Ozoralizumab18 GGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI (“PT-5” or PT(1-50)-ozoralizumab) GGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGS LRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPD SVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQ GTLVTVSS MG20NRGGF25RGGFG30SGIRG35RGRGR40GRGRG45RGRGA5“- EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGK GLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPE DTAVYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESG 32mer-Ozoralizumab with His-tag 19 GGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI GGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGS (PT-5 or PT(19-50)-ozoralizumab-H6) LRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPD SVKGRFTISRDNAKNTLYLQMNSLRPEDTAVY YCARSPSGFNRGQGTLVTVSSHHHHHH MG20NRGGF25RGGFG30SGIRG35RGRGR40GRGRG45RGRGA50- EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGK GLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPE DTAVYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESG20 GGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS 32mer-Ozoralizumab GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI (or PT(19-50)-ozoralizumab) GGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGS LRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPD SVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQ GTLVTVSS

[0102] Also disclosed herein is a composition comprising polypeptide PT-X’n-PTA-Hm (1) or pharmaceutically acceptable salts thereof.

[0103] Also disclosed herein is composition comprising a peptide leader sequence comprising the formula(s):H-Met-Gly-Asn-Arg-Gly-Gly-Phe-Arg-Gly-Gly-Phe-Gly-Ser-Gly-lle-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Ala-Lys(N3)-OH (PT(19-50)KN3; or a peptide leader sequence comprising the formula: H-Met-Gly-Asn-Arg-Gly-Gly-Phe-Arg-Gly-Gly-Phe-Gly-Ser-Gly-lle-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly--Lys(N3)-OH (PT(19-49)KN3); or a peptide leader sequence comprising H-Gly-Asn-Arg-Gly-Gly-Phe-Arg-Gly-Gly-Phe-Gly-Ser-Gly-lle-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Ala-Lys(N3)-OH (PT(20-50)X; or a peptide leader sequence comprising H-Gly-Asn-Arg-Gly-Gly-Phe-Arg-Gly-Gly-Phe-Gly-Ser-Gly-lle-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly--Lys(N3)-OH(PT(20-49)X.

[0104] In one example, peptide fragments consisting of the amino acid sequences MGNRGGFRGGFGSGIRGRGRGRGRGRGRGRG, GNRGGFRGGFGSGIRGRGRGRGRGRGRGRG, and MGNRGGFRGGFGSGIRGRGRGRGRGRGRGRGA,GNRGGFRGGFGSGIRGRGRGRGRGRGRGRGA, and GNRGGFRGGFGSGIRGRGRGRGRGRGRGR (SEQ ID Nos: 3-7) were synthesized, N terminally conjugated to fluorescein isothiocyanate (FITC) via an isothiocyanate-amine thiourea linkage, purified (e.g., >95% by RP HPLC), and applied to human Caco-2 intestinal epithelial cells, where confocal microscopy and flow cytometry demonstrated concentration and time dependent intracellular accumulation of the PT-FITC conjugates that exceeded signals from equimolar free FITC and a scrambled sequence control; trypan blue quenching reduced membrane associated fluorescence while retaining intracellular signal, uptake at 37°C exceeded uptake at 4°C, and competition with unlabeled PT reduced internalization, collectively indicating active internalization rather than surface adherence. The data supports that the PTs function as a Gl epithelial uptake (cell penetrating) motifs suitable for delivering linked “cargos” — including small molecules, peptides, proteins, therapeutics i.e., PTAs), and nanoparticles — to intestinal epithelial cells and supporting claims to compositions and methods employing the PTs to enhance cellular uptake and, optionally, transepithelial transport in vitro (e.g., Caco-2 Transwell assays) and in vivo. The data also support use of SEQ ID NO(s): 3-7 as a modular uptake element for oral delivery as described further herein, with the modified GLP-1 agonists, and can be used with other cargo, including but not limited to peptide drug conjugates, protein therapeutics, and other carriers formulated for oral delivery.

[0105] In one embodiment, a peptide leader sequence is linked to a modified GLP-1 agonist, such as a modified semaglutide or tirzepatide by a method comprising a click chemistry reaction between the Lys(Ns) of the polypeptide and the Lys((N£-4-pentynoyl) residue of the modified GLP-1 agonist; or the peptide leader sequence (SEQ ID NO. 2) may be linked to a modified nanobody.

[0106] Disclosed herein is a composition comprising a modified semaglutide according to the following formula(s):

[0107] H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-GIn-Ala-Ala-Lys(R1) -Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-Lys-R2-Lys-Gly-Arg-Gly-Arg-Gly-Arg- Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-lle-Gly-Ser-Gly-Phe-Gly-Gly-Arg-Phe-Gly-Gly-Arg-Asn-Gly-Met-Gly-Pro-Gly-Gly-Pro-Gly-Gly-Pro-Gly-Gly-Ala-Ala-Gly-Ala-Asp-Asp-Ala-Met-H (or PT(19-49)-K'-R2-K'-Sema;

[0108] and

[0109] H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-GIn-Ala-Ala-Lys(R1) -Glu-Phe-lle-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-Lys-R2-Lys-Gly-Arg-Gly-Arg-Gly-Arg- Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg -lle-Gly-Ser-Gly-Phe-Gly-Gly-Arg-Phe-Gly-Gly-Arg-Asn-Gly-Met-H (or PT(19-49)-K’-R2-K’-Sema;

[0110] or pharmaceutically acceptable salts thereof.

[0111] Disclosed herein is a composition comprising a modified tirzepatide according to the following formula(s):

[0112] Y{Aib}EGTFTSDYSI{Aib}LDKIAQ{Lys(PEG2-PEG2-gamma-glu-C20DIACID)}AFVQWLIAGGPSSGAPPPS{Pra}{Amidation}-tri azole Lys{AGRGRGRGRGRGRGRGRIGSGFGGRFGGRNGM (PT(20-49)-X’-TZP, where X’ is K’-R2-S’).

[0113] Disclosed herein is a composition comprising PT-PTA-Hm; or PT-X'n-PTA-Hm(1), or pharmaceutically acceptable salts thereof.

[0114] In some embodiments, the composition comprises a chemical modification to one or more amino acids, and / or the addition or conjugation of a functional moiety. Such amino acid modifications include, but are not limited to, phosphorylation, methylation (e.g., lysine methylation (mono-, di-, or trimethylation) and arginine methylation (mono, asymmetric dimethylation, 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, propionyllysine, butyryllysine, a polyhistadine tag, 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, or a detectable moiety, a moiety useful for ligation or purification, or a targeting moiety. The conjugation 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, modification includes an alkyl modified peptide on the terminus of a first polypeptide and an azide modified peptide on the second (heterologous) polypeptide, which facilitate the formation of an amide bond between the modified peptides on the first polypeptide and the second polypeptide, in order to generate a polypeptide construct (i.e., two polypeptides linked together).

[0115] The compositions disclosed herein may formulated with one or more pharmaceutically-acceptable excipients, which can be a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), solvent or encapsulating material, involved in carrying or transporting the therapeutic compound for administration to the subject, bulking agent, salt, surfactant and / or a preservative. Some examples of materials which can serve as pharmaceutically-acceptable excipients include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose 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; buffering agents; water; isotonic saline; pH buffered solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0116] In embodiments, a pharmaceutical composition comprises a therapeutic for oral administration, wherein the composition may be in the form of a solid, a semi-solid, a gel or a liquid, including the form of a tablet, a capsule, a lozenge, or an aqueous solution. The compounds disclosedherein are modifications of existing therapeutics that cause many side effects and exhibit low bioavailability due to their administration being intravenous.

[0117] An effective amount, or therapeutically effective amount, as the case may be, of a composition disclosed herein can be determined by methods known in the art. For example, the appropriate dose of a composition disclosed herein may depend on the route of administration and may depend on the subject being treated as well as the severity of the condition to be treated. Using scaling methods, such as allometric scaling, it is possible to predict suitable and exemplary dosage ranges for the administration of compositions, as disclosed herein, to adult humans. Dose scaling is an empirical approach, is well characterized and understood in the art. This approach assumes that there are some unique characteristics on anatomical, physiological, and biochemical process among species, and the possible difference in pharmacokinetics / physiological time is, as such, accounted for by scaling.

[0118] To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given. The following examples should not be read to limit or define the entire scope of the disclosure; the examples are offered by way of illustration and not by way of limitation with respect to subject matter claimed herein.EXAMPLES

[0119] The following examples illustrate, without limitation, representative materials, methods, and certain embodiments of the invention and is not intended to limit the scope of the claims. Unless indicated otherwise, all numerical values are approximate. Variations in species, doses, schedules, formulations, and analytical parameters may be employed to achieve comparable outcomes. Definitions used in the examples include: RT: room temperature; EIA / ELISA: enzyme immunoassay / enzyme-linked immunosorbent assay; SA-HRP: streptavidin-horseradish peroxidase; TMB: 3, 3', 5,5'-tetramethylbenzidine substrate; Cmax: maximum observed plasma concentration; AUC(O-t): area under the plasma concentration-time curve from time zero to last measurable concentration; ng h / mL and ng / mL are reported as measured; additional Pharmacokinetics (PK) parameters [e.g., Tmax (time to maximum concentration), f! (elimination half-life), CL / F (apparent clearance of the compound following extravascular administration)] may be determined in other embodiments; IV: intravenous; PBS: phosphate-buffered saline; ZDF: Zucker Diabetic Fatty.Example 1 - Systemic Exposure of Modified Semaglutide Linked to a Peptide Transport Leader Sequence (PT-3) Following Oral Administration Compared to Intravenous Semaglutide

[0120] In one embodiment, the systemic exposure of semaglutide following an intravenous (IV) dose was compared with the exposure achieved after oral administration of a composition comprising a modified semaglutide construct according to SEQ ID NO. 9 (PT-3).

[0121] Test Articles and Dosing

[0122] In one embodiment, the reference compound, unmodified semaglutide, was administered as a single intravenous dose at 17 pg / kg. The composition comprising PT-3 was administered orally (PO)as a single dose at 10 pg / kg or 30 pg / kg. The compositions were prepared and delivered under controlled laboratory conditions to ensure accurate dosing and reproducibility of exposure data.

[0123] In one embodiment, the study was conducted using domestic swine (Sus scrota domesticus) maintained under standard husbandry conditions suitable for pharmacokinetic evaluation. Animals were housed individually and provided a standardized diet and water ad libitum, with an acclimation period prior to dosing consistent with accepted practices for swine pharmacokinetic studies.

[0124] In one embodiment, systemic exposure was evaluated using standard pharmacokinetic parameters, including maximum plasma concentration (Cmax, ng / mL) and area under the plasma concentration-time curve (AUC(O-t), ng-h / mL). Plasma concentrations of semaglutide and PT-3 were determined using a validated enzyme-linked immunosorbent assay (ELISA).

[0125] Administration of semaglutide by the intravenous route at a dose of 17 pg / kg yielded a mean Cmax of 24.3 ng / mL and an AUC(O-t) of 844.7 ng-h / mL. Oral administration of a composition comprising PT-3 (SEQ ID No. 9) at 10 pg / kg resulted in a mean Cmax of 10.3 ng / mL and an AUC(O-t) of 8.95 ng-h / mL. At a higher oral dose of 30 pg / kg, a composition comprising PT-3 achieved a mean Cmax of 73.4 ng / mL and an AUC(0-t ) of 14.7 ng-h / mL.

[0126] These results demonstrate that oral administration of a composition comprising PT-3 (SEQ ID NO. 9) produced detectable systemic exposure, with plasma concentrations that scaled with dose. The findings further establish that a composition comprising PT-3 is capable of achieving measurable bioavailability following oral administration, providing a benchmark for comparison to intravenous semaglutide and supporting the potential for oral delivery of peptide-linked semaglutide formulations.

[0127] The results show that the enhanced oral absorption observed for a composition comprising PT-3 results from the presence of the peptide transport leader (SEQ ID No. 3), which facilitates epithelial transport and / or confers increased stability within the gastrointestinal tract. The observed systemic exposure confirms that a composition comprising PT-3 retains sufficient integrity for systemic delivery and may serve as a prototype or benchmark for oral formulations of semaglutide and related GLP-1 receptor agonists.Table 2: Individual and Mean AUC and Cmax for Semaglutide and a semaglutide-PT construct according to SEQ ID O.9 (PT-3).Semaglutide 17 ug / kg PT-3 Low 10 ug / kg x 7 days PT-3 High 30 mg / kg x 7 days AUCo-t AUCo-t AUC0.t Cmax(ng / mL) (ng*h / mL) Cmax(ng / mL) (ng*h / mL) (ng*h / mL) 1002 8.62 1075 2001 12.24 1006 3003 123.02 4648.303 1101 26.9 747.5 2002 8.53 867 3101 75.43 2866.908 1103 37.43 711.6 2103 10.2 7.21 3102 21.8 1730Mean 24.3 844.7 Mean 10.3 626.7 Mean 73.4 3082

[0128] Semaglutide Cmax and AUG (0-t) were 24.3 ng / mL and 844.7 ng*hr / mL. PT-3 was detected in plasma after dosing and exposures (Cmax and AUG) increased with dose. Cmax was 10.3 and 73.4 ng / mL at 10 ug / kg and 30 ug / kg. respectively. AUG (0-t) was 626.7 and 3082 ng*hr / mL, respectively.

[0129] The AUG values for semaglutide (17 ug / kg x 1), PT-3 (10 ug / kg x 7) and a composition comprising PT-3 (30 ug / kg x 7) adjusted for cumulative dose were 49.7, 8.95, and 14.7 ng*hr / mL, respectively. The molecular weights of semaglutide and a composition comprising a semaglutide construct according to SEQ ID NO. 9 are 4113 and 7283 g / mol, respectively. As a result, estimated dose and molecular weight adjusted relative bioavailability of a composition comprising PT-3 vs (IV) semaglutide was 10.9-18.2%.

[0130] To enable accurate quantification of circulating semaglutide, serum samples were subjected to a modified extraction procedure followed by ELISA using a commercially available semaglutide ELISA kit (e.g., BMA, S-1530). Reagents were equilibrated to ambient room temperature (RT) prior to use. Unless stated otherwise, volumes are per well for a 96 well microplate.

[0131] In one embodiment, a 1X EIA buffer was prepared by diluting 50 mL of a 20X EIA buffer concentrate with 950 mL of distilled or deionized water to achieve a 1:20 dilution. Five milliliters of the resulting 1X EIA buffer were then added to the antiserum and mixed gently by vortexing at low speed. In one embodiment, a semaglutide standard stock solution at 0.001 mg / mL (1,000 ng / mL) was prepared by serial dilution from an initial 5 mg / mL material. The material was first diluted 1:5 in EIA buffer to obtain a 1 mg / mL solution, followed by three successive 1:10 dilutions to yield the final concentration of 0.001 mg / mL.

[0132] In one embodiment, a calibration series of standards was prepared from this stock. A standard SI solution at 100 ng / mL was obtained by combining 0.1 mL of the 1,000 ng / mL stock with 0.9 mL of EIA buffer. Standards S2 through S6 were then generated by five sequential 1:4 dilutions of S1, in which 0.2 mL of the preceding standard was mixed with 0.6 mL of EIA buffer at each step. A biotinylated tracer was prepared by adding 5 mL of EIA buffer and mixing gently. A streptavidin-HRP (SA-HRP) reagent was prepared by diluting 0.06 mL of SA-HRP into 12 mL of EIA buffer. A TMB substrate working solution was prepared by adding 0.425 mL of TMB substrate stock to 8.5 mL of TMB substrate buffer and mixing thoroughly.

[0133] In one embodiment, all ELISA kit reagents were equilibrated to room temperature prior to use. Serum samples, following extraction, were dispensed into duplicate wells of the assay plate at 0.05 mL (50 pL) per well. Calibration standards SO (EIA buffer blank) through S6 were loaded into duplicate weils at 0.05 mL per well, and blank wells received 0.075 mL of EIA buffer. The plate was then sealed and incubated at room temperature on a plate shaker operating at approximately 60 rpm for one hour.Foliowing incubation, 0.025 mL of biotinylated tracer was added to each well, the plate was resealed, and incubation was continued at room temperature with shaking (approximately 60 rpm) for two hours.

[0134] After incubation, the wells were decanted by inversion (“flick” method) to remove liquid. The plate was washed by adding 0.3 mL of EIA buffer per well, shaking gently for one to two minutes, and decanting. This washing procedure was repeated five times in total, after which the top surface of the plate was blotted gently on an absorbent pad. Streptavidin-HRP was added at 0.1 mL per well, and the plate was incubated at room temperature with shaking for one hour, followed by another five washes as described above.

[0135] In one embodiment, 0.1 mL of TMB substrate solution was added to each well, and the plate was incubated at room temperature with gentle shaking for up to 15 minutes. Absorbance at 650 nm could be recorded at 5 and 10 minutes to monitor color development, if desired. The reaction was stopped by adding 0.1 mL of 2 N HCI to each well, and absorbance was measured at 450 nm. A standard curve was generated by plotting absorbance versus concentration for standards SO through S6, and sample concentrations were determined by interpolation from this standard curve.

[0136] Note: An extraction step was performed prior to ELISA to minimize matrix effects and enhance recovery; the extraction employed a modified protocol suited to Semaglutide detection in serum. The same extraction was applied to standards as appropriate to maintain matrix parity with samples. Example 2 - Intestinal Uptake, Oral Bioavailability, and In Vivo Efficacy of Modified Semaglutide Linked to a Peptide Transport Leader Sequence

[0137] In one embodiment, fluorescent labeling of the peptide-semaglutide constructs was performed using Alexa Fluor labeling kits (Thermo Fisher Scientific). Caco-2 human epithelial colorectal adenocarcinoma cells (Sigma-Aldrich) were used as an in vitro model of intestinal epithelial transport. Additional reagents included phosphate-buffered saline (PBS), 12-well tissue culture plates, and a Tecan Infinite M Nano+ plate reader.

[0138] In one embodiment, animal studies utilized Zucker Diabetic Fatty (Charles River Laboratories) rats for single-dose oral efficacy evaluations and male wild-type Sprague Dawley rats (7-9 weeks of age; Charles River Laboratories) for pharmacokinetic and bioavailability studies. Quantitation of drug in plasma was performed using an enzyme-linked immunosorbent assay (ELISA; Creative Diagnostics).

[0139] In one embodiment, in vitro intestinal uptake was evaluated using differentiated Caco-2 cell monolayers. Cells were seeded into 12-well plates and cultured until confluent. Fluorescently labeled semaglutide-PT constructs were applied at a concentration of 10 pg / mL and incubated for three hours at 37 °C. Following incubation, the apical supernatant was collected, and monolayers were washed gently three times with PBS and replenished with fresh medium. Fluorescence in both the cell-associated fractions and the supernatants was quantified using a Tecan Infinite M Nano+ plate reader. Total fluorescence, percentage uptake (cell-associated), and percentage secreted (supernatant) were calculated relative to total input fluorescence.

[0140] In one embodiment, oral efficacy was evaluated in ZDF rats following administration of a single oral dose of a composition comprising SEQ ID NO. 10 (PT-2). Untreated animals served as controls. Blood glucose levels were measured serially for six days post-dose, and body-weight gain was recorded over the same period. See Fig. 7.

[0141] In one embodiment, pharmacokinetic and oral bioavailability studies were performed in Sprague Dawley rats. Test compositions were administered by oral gavage at various concentrations, and a separate cohort received intravenous (IV) administration of the same constructs. Blood was collected pre-dose and at multiple post-dose time points. Plasma concentrations of PT-2 were quantified by ELISA, and systemic exposure (AUC) and absolute oral bioavailability (F%) were calculated using noncompartmental analysis based on AUC(po), AUC(iv), and dose normalization.

[0142] Results from the in vitro Caco-2 assay demonstrated that the control construct (semaglutide without the leader peptide) exhibited a relative bio-absorbance of approximately 30%, SEQ ID NO. 9 and 10 exhibited relative bio-absorbances of approximately 27% and 34%, respectively. These findings indicate that both semaglutide constructs linked to a peptide leader facilitate measurable transcellular association and uptake across intestinal epithelial monolayers.

[0143] Oral efficacy studies in diabetic ZDF rats demonstrated that untreated controls exhibited an average 40% increase in blood glucose over six days, whereas animals receiving oral doses of a composition comprising PT-2 exhibited an average 13% decrease in blood glucose by day six post-dose. See Fig. 5. Treated animals also exhibited reduced body weight gain relative to untreated controls. These data demonstrate that orally administered peptide transporter-linked semaglutide retains biological activity sufficient to lower blood glucose and mitigate weight gain following a single oral administration.

[0144] In one embodiment, pharmacokinetic analyses in Sprague Dawley rats demonstrated that the absolute oral bioavailability (F%) of peptide-linked semaglutide was approximately 25%, based on ELISA-derived plasma exposure relative to IV dosing of unmodified semaglutide. For comparison, the reported oral bioavailability of unmodified semaglutide is approximately 0.4% to 1%, with clinical values near 0.8% under optimized dosing conditions (administration with <120 mL water followed by a 30-minute fast). These results indicate that covalent modification of semaglutide with a peptide leader markedly enhances systemic exposure following oral administration compared to unmodified semaglutide.

[0145] The results demonstrate that conjugation of semaglutide to a peptide transport leader sequence significantly enhances its oral uptake, systemic exposure, and pharmacologic efficacy. In vitro, both compositions comprising PT-2 or PT-3 exhibited measurable transcellular association consistent with energy-dependent uptake. In vivo, orally administered PT-2 and PT-3 reduced hyperglycemia and bodyweight gain in diabetic rats following a single oral dose. Pharmacokinetic data further confirmed that chemical modification of semaglutide to generate peptide transporter-linked constructs increased absolute oral bioavailability approximately 20- to 25-fold compared with literature reports for unmodified semaglutide. See Fig. 6.

[0146] The data confirm that semaglutide conjugated to a peptide transport leader sequence achieves therapeutically relevant plasma concentrations and pharmacologic efficacy following oral administration. Equivalent results are expected for peptide conjugates comprising leader sequences of comparable length, charge distribution, and hydrophobicity, provided that overall structural integrity and receptor affinity are maintained.

[0147] Equivalent fluorescent labels, plate readers, or ELISA kits may be substituted without departing from the scope of the example. Dosing levels, sampling schedules, and wash steps can be varied as needed; comparable results are expected provided that total exposure and assay sensitivity are maintained.Example 3 - Oral Administration of modified Tirzepatide-PT conjugate (PT-4)

[0148] In one embodiment, a composition comprising a modified tirzepatide-peptide conjugate (PT-4) was prepared by covalently linking a tirzepatide analog (SEQ ID NO. 11 ) to a peptide transport leader (SEQ ID NO. 4). The conjugation was achieved using solid-phase peptide synthesis of the two individual peptide fragments followed by azide-alkyne cycloaddition to ligate the fragments into a single linear construct. The final product comprised a peptide conjugate in which tirzepatide was linked at its N-terminus to the transport leader sequence through a stable triazole linkage.

[0149] In one embodiment, the biological activity of a composition comprising a tirzepatide-peptide construct according to SEQ ID NO. 12 (PT-4) was evaluated for glucose-dependent insulinotropic activity and oral bioavailability. Glucose sensitivity was first assessed in vitro using isolated human pancreatic islet cells. The cells were incubated under two glucose conditions: a basal concentration (5 mM, 90 mg / dL) and a stimulatory concentration (20 mM, 360 mg / dL). KREBS buffer served as the vehicle for all incubations. Cells were exposed to each glucose condition for one hour in the presence of a PT-4 at an effective concentration equivalent to 2.5 mg / kg. See Fig. 9.

[0150] Following incubation, insulin secretion was quantified using a commercially available ELISA kit (Crystal Chem Insulin ELISA #90095). Islet cells treated with PT-4 demonstrated a marked increase in insulin release under high-glucose conditions relative to saline-treated controls, indicating that the conjugated peptide retained glucose-dependent biological activity following conjugation. See Fig. 9.

[0151] In one embodiment, oral bioavailability of the PT-4 peptide conjugate was evaluated in comparison to unmodified tirzepatide using a rodent pharmacokinetic model. Male rats were randomized into two cohorts (n = 6 per group). Cohort I received a single oral dose of PT-4 at 2.5 mg / kg, while Cohort II received unmodified tirzepatide subcutaneously at 0.5 mg / kg. Blood samples (0.2 mL) were collected from each animal at 1, 3, 7, 24, and 72 hours post-administration. Plasma was isolated from whole blood and extracted using a C18 column (BMA, S5000). The resulting samples were analyzed by enzyme-linked immunosorbent assay (ELISA; Fisher Scientific, NC2747160) to determine concentrations of both unmodified tirzepatide and the PT-4 conjugate. See Fig. 10.

[0152] Pharmacokinetic parameters were calculated using noncompartmental analysis. PT-4 exhibited a Cmax of 6,678.7 ng / mL, a Tmax of 7 hours, an AUC°° of 539,311.9 ng h / mL, and a volume of distribution of 393.5 ml_. By comparison, unmodified tirzepatide administered subcutaneously demonstrated a Cmax of 5,141.1 ng / mL, a Tmax of 24 hours, an AUC°° of 284,734.6 ng h / mL, and a volume of distribution of 27.9 mL.

[0153] These data demonstrate that PT-4 achieved measurable systemic exposure following oral administration in rodents. The observed pharmacokinetic parameters indicate that oral bioavailability of a composition comprising a PT-4 conjugate was significant and comparable to, or greater than, the systemic exposure achieved with subcutaneous administration of unmodified tirzepatide.

[0154] In one embodiment, PT-4 was further evaluated for effects on body weight in male Zucker Diabetic Fatty (ZDF) rats. Animals were housed under standard conditions with food (PicoLabs 5P76) and water provided ad libitum. Rats were randomized into two treatment groups: a control group receiving water and a treatment group receiving oral PT-4 once daily for 7-8 consecutive days. Body weight was recorded daily, and percent change from baseline was calculated for each animal relative to its initial weight on Day 0. At Day 7, animals receiving PT-4 exhibited an average body weight reduction of approximately 2% relative to baseline, whereas control animals exhibited an average 1% weight gain, resulting in a net difference of approximately 3 percentage points. By Day 8, the PT-4-treated group demonstrated an average 3% weight loss relative to baseline, while the control group exhibited an average 1.5% weight gain, for a net difference of approximately 4.5 percentage points. See Fig. 11.

[0155] The results demonstrate that conjugation of tirzepatide to a transport leader sequence and the resulting tirzepatide-PT conjugate according to SEQ ID NO. 12 (PT-4) confers oral bioavailability while retaining glucose-dependent insulinotropic activity.

[0156] In vitro, PT-4 stimulated insulin secretion in isolated human pancreatic islets under high-glucose conditions, confirming preservation of glucose responsiveness following conjugation. In vivo, oral administration of PT-4 in rodents resulted in measurable plasma exposure and pharmacokinetic parameters consistent with systemic bioavailability. The observed AUC and Cmax values indicated that the PT-4 conjugate achieved oral absorption sufficient to produce pharmacodynamic effects comparable to injectable tirzepatide formulations. Moreover, short-term administration of PT-4 in diabetic rats produced a time-dependent reduction in body weight relative to water-treated controls, demonstrating that orally delivered PT-4 retains therapeutic activity consistent with GLP-1 receptor agonism. The data suggest that the enhanced oral absorption of PT-4 results from the transport leader sequence facilitating epithelial uptake and providing protection against gastrointestinal degradation.Example 4 - Modified Caplacizumab

[0157] In one embodiment, modified forms of caplacizumab were generated and evaluated for their ability to retain biological activity and achieve oral bioavailability. Caplacizumab is a nanobody that targets von Willebrand factor (vWF) and is clinically used for the treatment of acquired thromboticthrombocytopenic purpura (aTTP), a rare blood clotting disorder characterized by microvascular thrombosis and thrombocytopenia. Caplacizumab acts by inhibiting the interaction between vWF and platelets, thereby preventing pathological platelet aggregation and thrombus formation in small blood vessels.

[0158] In one embodiment, modified caplacizumab constructs were produced by genetic expression in a bacterial vector. The constructs caplacizumab-50mer (PT-6 and PT-6-His) were expressed in E. coli using a recombinant expression vector. Following cell lysis, the recombinant proteins were purified via affinity chromatography to greater than 95% purity. Preparative yields were approximately 10 mg per batch.

[0159] In one embodiment, thrombin cleavage analysis was used to confirm the presence and correct processing of the peptide leader extension (50mer, SEQ ID NO. 2). Mass spectrometric analysis demonstrated a molecular weight of approximately 33 kDa for the uncleaved 50mer-caplacizumab construct and approximately 29 kDa for the cleaved product, corresponding to the expected molecular weights before and after removal of the 50mer sequence. See Fig. 12A and 12B.

[0160] In one embodiment, the binding affinity of caplacizumab-50mer (PT-6) to vWF was evaluated using surface plasmon resonance (SPR). vWF was immobilized on a sensor chip using a two-step process. The chip surface was first activated with N-hydroxysuccinimide and 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) followed by immobilization of vWF. Unreacted coupling sites were subsequently blocked with ethanolamine.

[0161] The SPR assay was performed at 25 °C using HBS-EP+buffer as the running buffer. Caplacizumab-50mer (30 pg / mL) was injected over the vWF-coated surface during the association phase, followed by running buffer during the dissociation phase. Sensorgram data were processed using a standard 1:1 binding model to determine kinetic and equilibrium constants.

[0162] The observed binding parameters for caplacizumab-50mer were: ka = 2.96 x 106M1s1, kd = 2.64 x 10-2s'1, and KD = 8.9 x 10“9M (8.9 nM). These values demonstrate that incorporation of the 50mer peptide transport leader did not significantly impair vWF binding affinity. Of note, when the 50mer sequence was reversed, the resulting reversed-50mer construct exhibited a lower binding affinity to vWF, indicating a directional dependence of the leader sequence on interaction strength.

[0163] In one embodiment, the oral uptake of caplacizumab-50mer (also referred to as ImPh-CALP1, see Fig. 15) was evaluated in vivo following oral administration in rodents. Male Sprague-Dawley rats (6-8 weeks old, n = 6) received a single oral dose of 2.5 mg / kg via gavage. Blood samples were collected from the tail vein at 1, 3, 7, 24, and 72 hours post-dose. Plasma was separated by cold centrifugation and stored at -80 °C until analysis. See Fig. 15.

[0164] Plasma concentrations of caplacizumab-50mer (PT-6-His) were determined using an anti-Hex-His ELISA (His-tag detection; manufacturer’s protocol). The results demonstrated detectable plasma levels of 50mer-caplacizumab at 1, 3, and 7 hours post-dose, confirming systemic absorption following oral administration. See Fig. 13.

[0165] In one embodiment, the biological activity of a composition comprising PT-6 was further evaluated using the ristocetin-induced platelet aggregation (RIPA) assay in human plasma. This assay measures platelet aggregation in response to ristocetin, which activates vWF and induces platelet-vWF binding.

[0166] Human plasma samples were incubated with a composition comprising PT-6, unmodified caplacizumab, or control vehicle prior to addition of ristocetin. Percent platelet aggregation was measured after 10 minutes in the presence and absence of inhibitor. Both caplacizumab and PT-6 inhibited ristocetin-induced aggregation at concentrations below 10 nM, demonstrating that the composition comprising PT-6 retained functional inhibitory activity against vWF-mediated platelet aggregation. See Fig. 14.

[0167] Of further interest, when the 50mer sequence was reversed in orientation, the resulting reversed-50mer construct exhibited reduced binding affinity and weaker inhibition in the RIPA assay, consistent with the directional dependence observed in the SPR binding studies.

[0168] The results confirm that incorporation of a peptide transport leader sequence into caplacizumab yields a modified nanobody that maintains high-affinity binding to vWF and retains functional inhibitory activity. Thrombin cleavage and mass spectrometry verified the molecular integrity and expected processing of the fusion constructs. SPR and RIPA assays demonstrated that the 50mer-caplacizumab construct preserved binding kinetics and biological function equivalent to native caplacizumab.

[0169] In vivo oral administration of caplacizumab-50mer appears to facilitate epithelial uptake and / or protects the nanobody from proteolytic degradation in the gastrointestinal tract. These results collectively demonstrate that peptide transport leader-modified caplacizumab constructs are bioactive, orally absorbed, and retain vWF binding properties comparable to unmodified caplacizumab, supporting their potential use as orally administered biologic therapeutics.Example 5 - Modified Ozoralizumab

[0170] In one embodiment, modified forms of ozoralizumab were generated and evaluated for biological activity and binding affinity. Ozoralizumab is a monoclonal antibody that binds and neutralizes tumor necrosis factor-alpha (TNF-a), a pro-inflammatory cytokine involved in the pathogenesis of autoimmune and inflammatory diseases such as rheumatoid arthritis (RA). TNF-a plays a central role in driving chronic inflammation and tissue damage in RA and related disorders. By inhibiting TNF-a signaling, ozoralizumab modulates the inflammatory response, reducing joint inflammation, tissue destruction, and disease progression.

[0171] In one embodiment, a peptide transport leader sequence comprising 50 amino acids was attached to ozoralizumab, using a bacterial expression vector, to enhance stability and oral bioavailability. The resulting construct, designated 50mer-ozoralizumab (PT-5), was expressed in an E. coli-basedexpression system using a recombinant vector encoding the full-length fusion sequence. The expressed protein was recovered by cell lysis and purified via affinity chromatography.

[0172] The purified 50mer-ozoralizumab was obtained at a 10 mg preparative scale with a purity exceeding 95%, as confirmed by SDS-PAGE and analytical HPLC. The final construct contained a C-terminal His-tag (SEQ ID NO:5) to facilitate purification and subsequent bio-layer interferometry analysis.

[0173] In one embodiment, the binding affinity of PT-5 for TNF-a was evaluated using bio-layer interferometry (BLI). The 50mer-ozoralizumab fusion protein was immobilized on a nickel-nitrilotriacetic acid (Ni-NTA) biosensor via its His-tag. Recombinant human TNF-a was used as the analyte in the association phase, followed by dissociation in running buffer. Data were processed using a 1:1 binding model to determine kinetic rate constants and equilibrium affinity.

[0174] The observed binding parameters for PT-5 were: ka = 3.46 x 10sM'1s“1; kd = 7.52 x 10'5s'1; KD = 2.17 x io-10M (0.217 nM). For comparison, the unmodified ozoralizumab exhibited the following parameters: ka = 1.43 x 105M '1s '1; kd = 6.43 x 10'5s'1; KD = 4.48 x 10'10M (0.448 nM). These results indicate that the 50mer-ozoralizumab construct exhibited approximately twofold higher binding affinity for TNF-a compared to unmodified ozoralizumab. The enhanced binding is consistent with improved stability and conformational preservation of the antigen-binding domain following incorporation of the peptide transport leader.

[0175] In one embodiment, the results confirm that the incorporation of a 50-amino acid transport leader sequence into ozoralizumab yields a biologically active construct that maintains, and in some cases enhances, binding affinity to TNF-a. The improved affinity may result from stabilization of the antibody's variable region or reduced steric hindrance at the TNF-a binding site. The results support that the peptide transport leader sequence may confer additional structural stability and facilitate absorption across biological membranes, supporting its potential role in improving oral or mucosal delivery of antibody fragments or nanobody-based therapeutics.

[0176] Unless otherwise indicated, volumes in the Examples are approximate and may vary ±10% without materially affecting results.

[0177] In one embodiment, dosing regimens of the various PT-conjugated PT As and constructs described herein may include once-daily, twice-daily, or alternate-day, or weekly schedules. Exemplary dose ranges include about 1 pg / kg -10 mg / kg, about 5-50 pg / kg, or about 10-30 pg / kg, depending on the frequency of dosing, concentration, and condition treated.

[0178] The methods described herein may be applied to other mammalian species, including mice, rats, dogs, and non-human primates, with routine adjustments to dose and sampling intervals. Plasma may be substituted with serum matrices provided analytical validation supports comparability.

[0179] Alternative analytical methods such as immunoassay, LC-MS / MS, or hybrid ligandbinding assays may be used, with or without extraction, provided they achieve acceptable accuracy, precision, and sensitivity for the analyte of interest.

[0180] In one embodiment, peptide-conjugates (e.g., PT-2, PT-3, PT-4, PT-5, PT-6) may be formulated with pharmaceutically acceptable excipients suitable for oral, parenteral, or mucosal delivery. Excipients may include buffers, stabilizers, permeation enhancers, or solubilizing agents in either solid or liquid dosage forms.

[0181] Although the foregoing information highlights aspects disclosed herein by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the subject matter claimed herein. It will be clear to a person skilled in the art that features described in relation to any of the aspects and various embodiments described above can be applicable interchangeably between the different embodiments.

[0182] The aspects and embodiments described above are examples to illustrate various features of the subject matter claimed herein. All publications and patent applications disclosed herein are indicative of the level of those skilled in the art to which this disclosure and the subject matter of the claims pertains.

[0183] It will be understood that a numerical value may be associated with a certain amount of experimental error. Thus, recitation of the qualifier "about" (or “approximately”) prior to a numerical error is meant to embody the experimental error that may be associated with the recited numerical value. To the extent that a numerical value obtained experimentally is not preceded by the expression "about" (or “approximately”) does not mean that the numerical value is not associated with a certain amount of experimental error.

[0184] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components, or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. Where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0185] Features, characteristics, compounds, chemical moieties, or groups described in conjunction with a particular aspect, embodiment, or example are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The subject matter claimed herein is not restricted to the details of any foregoing embodiments. The subject matter claimed herein extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0186] 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 to be incorporated by reference.ADDITIONAL ASPECTS

[0187] Aspect 1. A polypeptide, PT-X’n-PTA-Hm(1), or pharmaceutically acceptable salts thereof, wherein PT is a peptide transport or fragment thereof; X’nis linker comprising a triazole moiety, n = 0 or 1; PTA is a peptide therapeutic agent selected from the group consisting of a modified semaglutide, a modified tirzepatide, caplacizumab, ozoralizumab; and Hmis a histidine tag where m is 0 to 6.

[0188] Aspect 2. The polypeptide of Aspect 1, wherein PT comprises:

[0189] Xaa01 Xaa02 Xaa03 Xaa04 Xaa05 Xaa06 Xaa07 Xaa08 Xaa09 Xaa10

[0190] Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20

[0191] Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Xaa30

[0192] Xaa31 Xaa32 Xaa33 Xaa34 Xaa35 Xaa36 Xaa37 Xaa38 Xaa39 Xaa40

[0193] Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50

[0194] wherein

[0195] Xaa01 = M, A, V, I, L;

[0196] Xaa02 = A, G, S;

[0197] Xaa03 = D, E;

[0198] Xaa04 = D, E;

[0199] Xaa05 = A, G, S;

[0200] Xaa06 = G, A, S;

[0201] Xaa07 = A, G, S;

[0202] Xaa08 = A, G, S;

[0203] Xaa09 = G, A, S;

[0204] Xaa10 = G, A, S;

[0205] Xaa11 = P;

[0206] Xaa12 = G, A, S;

[0207] Xaa13 = G, A, S;

[0208] Xaa14 = P;

[0209] Xaa15 = G, A, S;

[0210] Xaa16 = G, A, S;

[0211] Xaa17 = P;

[0212] Xaa18 = G, A, S;

[0213] Xaa19 = M, A, V, I, L;

[0214] Xaa20 = G, A, S, N;

[0215] Xaa21 = N, G, A, Q;

[0216] Xaa22 = R, K, Q;

[0217] Xaa23 = G, A, S;

[0218] Xaa24 = G, A, S;

[0219] Xaa25 = F, Y, W, L, H;

[0220] Xaa26 = R, K, Q;

[0221] Xaa27 = G, A, S;

[0222] Xaa28 = G, A, S;

[0223] Xaa29 = F, Y, W, L, M;

[0224] Xaa30 = G, A, S;

[0225] Xaa31 = S, A, T, N;

[0226] Xaa32 = G, S, N;

[0227] Xaa33 = I, V, M;

[0228] Xaa34 = R, K, Q;

[0229] Xaa35 = G, A, S, N;

[0230] Xaa36 = R, K, Q;

[0231] Xaa37 = G, A, S, N;

[0232] Xaa38 = R, K, Q;

[0233] Xaa39 = G, A, S, N;

[0234] Xaa40 = R, K, Q;

[0235] Xaa41 = G, A, S, N;

[0236] Xaa42 = R, K, Q;

[0237] Xaa43 = G, A, S, N;

[0238] Xaa44 = R, K, Q;

[0239] Xaa45 = G, A, S, N;

[0240] Xaa46 = R, K, Q;

[0241] Xaa47 = G, A, S, N;

[0242] Xaa48 = R, K, Q;

[0243] Xaa49 = G, A, S, N; and

[0244] Xaa50 = A, G, V, L.

[0245] Aspect 3. The polypeptide of any one of Aspects 1-2, wherein PT comprises PT(a-co), where a = 1-37 and co =49-50.

[0246] Aspect 4. The polypeptide of any one of Aspects 1-3, wherein PT comprises PT(1-50) and / or a fragment thereof selected from the group consisting of PT(1-49), PT(19-49), PT(20-49), PT(19-50), and PT(20-50); and wherein the PTA is a modified semaglutide; or wherein PT-X’n-PTA-Hm(1) comprises any one of SEQ ID NOs: 9-10.

[0247] Aspect 5. A composition for oral delivery comprising the polypeptide of Aspect 4 and an acceptable carrier.

[0248] Aspect 6. A method of treating a condition in a subject in need thereof the method comprising administering orally therapeutically effective amount of the composition of Aspect 5, wherein the condition is type 2 diabetes, obesity, obstructive sleep apnea, or a combination thereof.

[0249] Aspect 7. The polypeptide of any one of Aspects 1-3, wherein PT comprises PT(1-50) and / or a fragment thereof selected from the group consisting of PT(1-49), PT(19-49), PT(20-49), PT(19-50), and PT(20-50); and wherein the PTA is a modified tirzepatide; or wherein PT-X’n-PTA-Hm (1) comprises SEQ ID NO: 12.

[0250] Aspect 8. A composition for oral delivery comprising the polypeptide of Aspect 7 and an acceptable carrier.

[0251] Aspect 9. A method of treating a condition in a subject in need thereof comprising administering orally a therapeutically effective amount of the composition of Aspect 8, wherein the condition is type 2 diabetes, obesity, obstructive sleep apnea, or a combination thereof.

[0252] Aspect 10. The polypeptide of any one of Aspects 1-3, wherein PT comprises PT(1-50) and / or a fragment thereof selected from the group consisting of PT(1-50), PT(1-49), PT(19-49), PT(20-49), PT(19-50), and PT(20-50); wherein n = 0; and wherein the PTA is caplacizumab; or wherein PT-X’n-PTA-Hm(1) comprises any one of SEQ ID NOs: 13-16.

[0253] Aspect 11. A composition for oral delivery comprising the polypeptide of Aspect 10 and an acceptable carrier.

[0254] Aspect 12. A method of treating acquired Thrombotic Thrombocytopenic Purpura (aTTP) in a subject in need thereof the method comprising administering orally to the subject a therapeutically effective amount of the composition of Aspect 11.

[0255] Aspect 13. A method of inhibiting von Willibrand factor in a subject in need thereof comprising administering orally to the subject a therapeutically effective amount of the composition of Aspect 11.

[0256] Aspect 14. The polypeptide of any one of Aspects 1-3, wherein PT comprises PT(1-50) and / or a fragment thereof selected from the group consisting of PT(1-50), PT(1-49), PT(19-49), PT(20-49), PT(19-50), and PT(20-50); wherein n = 0; and wherein the PTA is ozoralizumab; or wherein PT-X’n-PTA-Hm(1) comprises any one of SEQ ID NOs: 17-20.

[0257] Aspect 15. A composition for oral delivery comprising the polypeptide of Aspect 14 and an acceptable carrier.

[0258] Aspect 16. A method of treating inflammation by targeting TNF-alpha in a subject in need thereof the method comprising administering orally a composition comprising a therapeutically effective amount of the composition of Aspect 15.

[0259] Aspect 17. A method of treating an autoimmune disorder characterized by inflammation, such as rheumatoid arthritis, in a subject in need thereof, the method comprising administering orally a composition comprising a therapeutically effective amount of the composition of Aspect 15.CITED INFORMATION

[0260] Mounjaro® (tirzepatide) injection, for subcutaneous use, prescribing information, as of May 28, 2025 (" Mounjaro Label").

[0261] Ozempic® (semaglutide) injection, for subcutaneous use, prescribing information, as of January 28, 2025 (“Ozempic Label”).

[0262] U. S. Patent No. 9,474,780 B2, GIP and GLP-1 co-agonist compounds, issued on October 25, 2016 to Bokvist et al. of Eli Lilly & Co. (" Bokvist").

[0263] U. S. Patent No. 8,129,343 B2, Acylated GLP-1 compounds, issued on March 6, 2012 to Lau et al. of Novo Nordisk A / S (“Lau”).

[0264] Wegovy® (semaglutide) injection, for subcutaneous use, prescribing information, as of October 14, 2025 (“Wegovy Label”).

[0265] Zepbound® (tirzepatide) injection, for subcutaneous use, prescribing information, as of February 7, 2025 (" Zepbound Label").

Claims

Claims1. A polypeptide, PT-X’n-PTA-Hm(1), or pharmaceutically acceptable salts thereof, whereinPT is a peptide transport or fragment thereof;X’nis linker comprising a triazole moiety, n = 0 or 1;PTA is a peptide therapeutic agent selected from the group consisting of a modified semaglutide, a modified tirzepatide, caplacizumab, ozoralizumab; andHmis a histidine tag where m is 0 to 6.

2. The polypeptide of claim 1, wherein PT comprises:Xaa01 Xaa02 Xaa03 Xaa04 Xaa05 Xaa06 Xaa07 Xaa08 Xaa09 Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Xaa30 Xaa31 Xaa32 Xaa33 Xaa34 Xaa35 Xaa36 Xaa37 Xaa38 Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 whereinXaa01 = 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 = G, A, S, N;Xaa21 = N, G, A, Q;Xaa22 = R, K, Q;Xaa23 = G, A, S;Xaa24 = G, A, S;Xaa25 = F, Y, W, L, H; Xaa26 = R, K, Q;Xaa27 = G, A, S;Xaa28 = G, A, S;Xaa29 = F, Y, W, L, M; Xaa30 = G, A, S;Xaa31 = S, A, T, N;Xaa32 = G, S, N;Xaa33 = I, V, M;Xaa34 = R, K, Q;Xaa35 = G, A, S, N;Xaa36 = R, K, Q;Xaa37 = G, A, S, N;Xaa38 = R, K, Q;Xaa39 = G, A, S, N;Xaa40 = R, K, Q;Xaa41 = G, A, S, N;Xaa42 = R, K, Q;Xaa43 = G, A, S, N;Xaa44 = R, K, Q;Xaa45 = G, A, S, N;Xaa46 = R, K, Q;Xaa47 = G, A, S, N;Xaa48 = R, K, Q;Xaa49 = G, A, S, N; and Xaa50 = A, G, V, L.

3. The polypeptide of claim 1, wherein PT comprises PT(a-co), where a = 1-37 and co =49-50.

4. The polypeptide of claim 1, wherein PT comprises PT(1-50) and / or a fragment thereof selected from the group consisting of PT(1-49), PT(19-49), PT(20-49), PT(19-50), and PT(20-50); and wherein the PTA is a modified semaglutide or wherein PT-X’n-PTA-Hm(1) comprises any one of SEQ ID NOs: 9-10.

5. A composition for oral delivery comprising the polypeptide of claim 4 and an acceptable carrier.

6. A method of treating a condition in a subject in need thereof the method comprising administering orally therapeutically effective amount of the composition of claim 5, wherein the condition is type 2 diabetes, obesity, obstructive sleep apnea, or a combination thereof.

7. The polypeptide of claim 1, wherein PT comprises PT(1-50) and / or a fragment thereof selected from the group consisting of PT(1-49), PT(19-49), PT(20-49), PT(19-50), and PT(20-50); and wherein the PTA is a modified tirzepatide; or wherein PT-X’n-PTA-Hm(1) comprises SEQ ID NO: 12.

8. A composition for oral delivery comprising the polypeptide of claim 7 and an acceptable carrier.

9. A method of treating a condition in a subject in need thereof comprising administering orally a therapeutically effective amount of the composition of claim 8, wherein the condition is type 2 diabetes, obesity, obstructive sleep apnea, or a combination thereof.

10. The polypeptide of claim 1, wherein PT comprises PT(1-50) and / or a fragment thereof selected from the group consisting of PT(1-50), PT(1-49), PT(19-49), PT(20-49), PT( 19-50), and PT(20-50); wherein n = 0; and wherein the PTA is caplacizumab; or wherein PT-X’n-PTA-Hm(1 ) comprises any one of SEQ ID NOs: 13-16.

11. A composition for oral delivery comprising the polypeptide of claim 10 and an acceptable carrier.

12. A method of treating acquired Thrombotic Thrombocytopenic Purpura (aTTP) in a subject in need thereof the method comprising administering orally to the subject a therapeutically effective amount of the composition of claim 11.

13. A method of inhibiting von Willibrand factor in a subject in need thereof comprising administering orally to the subject a therapeutically effective amount of the composition of claim 11.

14. The polypeptide of claim 1, wherein PT comprises PT(1-50) and / or a fragment thereof selected from the group consisting of PT(1-50), PT(1-49), PT(19-49), PT(20-49), PT(19-50), and PT(20-50); wherein n = 0; and wherein the PTA is ozoralizumab; or wherein PT-X’n-PTA-Hm(1) comprises any one of SEQ ID NOs: 17-20.

15. A composition for oral delivery comprising the polypeptide of claim 14 and an acceptable carrier.

16. A method of treating inflammation by targeting TNF-alpha in a subject in need thereof the method comprising administering orally a composition comprising a therapeutically effective amount of the composition of claim 15.

17. A method of treating an autoimmune disorder characterized by inflammation, such as rheumatoid arthritis, in a subject in need thereof, the method comprising administering orally a composition comprising a therapeutically effective amount of the composition of claim 15.

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