Alpha-v beta-6 integrin ligands and uses thereof

Abstract

To provide integrin ligands having serum stability and affinity for αvβ6 integrins.SOLUTION: Disclosed are peptide-based alpha-v beta-6 (αvβ6) integrin ligands useful in targeting αvβ6 integrin and / or targeting cells that express αvβ6 integrin. The αvβ6 integrin ligands can be conjugated to one or more cargo molecules to facilitate the delivery of the cargo molecules to cells expressing αvβ6 integrin, such as epithelial cells.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 415,752, filed on November 1, 2016, the content of which is incorporated herein by reference.

[0002] Field of the Invention Disclosed are peptide - based alpha - v - beta - 6 (αvβ6) integrin ligands useful in targeting αvβ6 integrin and / or targeting cells expressing αvβ6 integrin. The αvβ6 integrin ligands can be conjugated to one or more cargo molecules to facilitate delivery of the cargo molecules to cells expressing αvβ6 integrin, such as epithelial cells.

Background Art

[0003] Background Integrin alpha - v - beta - 6 (αvβ6), which is expressed in various epithelial cells, is a receptor for the latent - associated peptide (LAP) of TGF - β and for the (ECM) proteins fibronectin, vitronectin, and tenascin.

[0004] Although hardly detectable in normal healthy adult epithelium, αvβ6 integrin is up - regulated during wound healing and in various cancers (e.g., colon cancer, ovarian cancer, endometrial cancer, and gastric cancer), and is often associated with poor cancer prognosis. αvβ6 integrin has been shown to promote cell invasion and migration in metastasis and to inhibit apoptosis. αvβ6 integrin can also control the expression of matrix metalloproteinases (MMPs) and activate TGF - β1. Evidence suggesting that αvβ6 integrin can promote cancer progression is increasing, mainly from in vitro studies. Thus, integrin αvβ6 is attractive as a tumor biomarker and potential therapeutic target, and is attractive because of its role in the expression of matrix metalloproteinases (MMPs) and the activation of TGF - β1.

Summary of the Invention

[0005] Summary Described herein is a novel, engineered, non-naturally occurring peptide-based αvβ6 integrin ligand (also referred to as an αvβ6 ligand). The αvβ6 integrin ligand disclosed herein is stable in serum, has an affinity for αvβ6 integrin, and is capable of specifically binding to αvβ6 integrin. Further described herein are compositions containing the αvβ6 integrin ligand, as well as methods of using the αvβ6 integrin ligand and compositions described herein.

[0006] The αvβ6 integrin ligand described herein has improved stability compared to other known αvβ6 integrin-binding peptides, such as the native peptide RGDLATLRQL (SEQ ID NO: 1). While the serum stability is increased, the novel αvβ6 ligand described herein retains its binding to αvβ6 integrin (affinity for αvβ6 integrin).

[0007] In a first aspect, the disclosure provides an engineered, non-naturally occurring αvβ6 integrin ligand. In some embodiments, the αvβ6 integrin ligand has the general formula: Z-RG 1 DLXaa 1 Xaa 2 L (SEQ ID NO: 85) (Formula I), wherein Z is an amine-terminal cap (e.g., any of the amine-terminal caps described herein or known in the art); R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); and Xaa 2 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art).

[0008] In some embodiments, the αvβ6 integrin ligand has the general formula: Z-RG 1 DLXaa 1 Xaa 2 L-J-R 1 (SEQ ID NO: 86) (Formula II), wherein Z is an amine-terminal cap (e.g., any of the amine-terminal caps described herein or known in the art); R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 2is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); J is optional and, when present, is one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30) of L-α-amino acids (e.g., any of the L-α-amino acids described herein or known in the art), L-β-amino acids (e.g., any of the L-β-amino acids described herein or known in the art), or α,α-disubstituted amino acids (e.g., any of the α,α-disubstituted amino acids described herein or known in the art), or combinations thereof; and R 1 is optional and, when present, contains polyethylene glycol (PEG) and / or a linking group.

[0009] In some embodiments, the αvβ6 integrin ligands disclosed herein can include a reactive group or a protected reactive group, and the general formula: Z-RG 1 DLXaa 1 Xaa 2 L-J-R 1 -R 2 (SEQ ID NO: 87) (Formula III), wherein Z is an amine-terminal cap (e.g., any of the amine-terminal caps described herein or known in the art); R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa1 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 2 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); J is optional and, when present, is one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30) of L-α-amino acids (e.g., any of the L-α-amino acids described herein or known in the art), L-β-amino acids (e.g., any of the L-β-amino acids described herein or known in the art), or α,α-disubstituted amino acids (e.g., any of the α,α-disubstituted amino acids described herein or known in the art), or combinations thereof; R 1 is optional and, when present, contains polyethylene glycol (PEG) and / or a linking group; and R 2 contains a reactive group or a protected reactive group.

[0010] In some embodiments, the αvβ6 integrin ligand can be conjugated to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30) cargo molecules (e.g., any of the cargo molecules described herein or known in the art), where the αvβ6 integrin ligand has the general formula: (Z-RG 1 DLXaa 1 Xaa 2 L-J-R 1 ) n -R 3 (SEQ ID NO: 88) (Formula IV), wherein Z is an amine-terminal cap (e.g., any of the amine-terminal caps described herein or known in the art); R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 2is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); J is optional and, when present, is one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30) of L-α amino acids (e.g., any of the L-α amino acids described herein or known in the art), L-β amino acids (e.g., any of the L-β amino acids described herein or known in the art), or α,α-disubstituted amino acids (e.g., any of the α,α-disubstituted amino acids described herein or known in the art), or combinations thereof; R 1 is optional and, when present, contains polyethylene glycol (PEG) and / or a linking group; n is an integer greater than 0 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30); and R 3 contains one or more cargo molecules. In some embodiments, R 3 contains one cargo molecule. In some embodiments, R 3 contains more than one cargo molecule.

[0011] In some embodiments, the αvβ6 integrin ligand can be conjugated to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30) cargo molecules (e.g., any of the cargo molecules described herein or known in the art), where the αvβ6 integrin ligand has the general formula: (Z-RG 1 DLXaa 1 Xaa 2 L-J-R 1 ) n -R 4 -(R 3 ) p (SEQ ID NO: 89) (Formula V), wherein Z is an amino-terminal cap (e.g., any of the amino-terminal caps described herein or known in the art); R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 2is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); J is optional and, when present, is one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30) of L-α amino acids (e.g., any of the L-α amino acids described herein or known in the art), L-β amino acids (e.g., any of the L-β amino acids described herein or known in the art), or α,α-disubstituted amino acids (e.g., any of the α,α-disubstituted amino acids described herein or known in the art), or combinations thereof; R 1 is optional and, when present, contains polyethylene glycol (PEG) and / or a linking group; n is an integer greater than 0 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30); R 3contains one or more cargo molecules; p is an integer greater than 0 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 - 30, 1 - 25, 1 - 20, 1 - 15, 1 - 10, 1 - 5, 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 30, 15 - 25, 15 - 20, 20 - 30, 20 - 25, or 25 - 30); and, R 4 is optional and, if present, contains a scaffold and / or a linker, such scaffold and / or linker containing at least one attachment point for each ligand and at least one attachment point for each cargo molecule. In some embodiments, R 3 contains one cargo molecule. In some embodiments, R 3 contains more than one cargo molecule.

[0012] As used herein, an “amine terminal cap” (shown as “Z” in the formulas herein) includes a chemical moiety that can increase and / or otherwise improve the protease resistance and / or serum stability properties of an RGDLATL native peptide. Such improvements can be determined, for example, using methods generally known in the art, such methods including, but not limited to, determining the half-life of an αvβ6 integrin ligand, an αvβ6 integrin ligand-cargo molecule conjugate, or an αvβ6 integrin ligand-containing composition, in vivo and / or in vitro. In some embodiments, Z includes protease-resistant acylation, sulfonylation, or alkylation of the N-terminal amine of an αvβ6 integrin ligand disclosed herein. In some embodiments, the amine terminal cap Z can be an alkyl-CO, ArCO, alkyl-SO2, ArSO2, alkyl or aryl group. In some embodiments, the alkyl group can be either a straight-chain or branched-chain aliphatic alkyl group, and the aryl group can be either an aromatic or heteroaromatic group. In some embodiments, the amine terminal cap Z is, but not limited to, CH3CO, CH3CH2CO, CH3(CH2)2CO, (CH3)2CHCO, CH3(CH2)3CO, (CH3)2CHCH2CO, CH3CH2CH(CH3)CO, (CH3)3CCO, CH3(CH2)4CO, CH3SO2, CH3CH2SO2, CH3(CH2)2SO2, (CH3)2CHSO2, CH3(CH2)3SO2, (CH3)2CHCH2SO2, CH3CH2CH(CH3)SO2, (CH3)3CSO2, PhCO, PhSO2, an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, methyl, ethyl, propyl, butyl, pentyl, NH2NH, PEG, guanidyl, CH3OCH2CH2OCH2CH2CO, CH3O(CH2CH2O)2CH2CH2CO, CH3O(CH2CH2O)3CH2CH2CO, CH3O(CH2CH2O)4CH2CH2CO, CH3O(CH2CH2O)5CH2CH2CO, CH3OCH2CH2OCH2CO, CH3O(CH2CH2O)2CH2CO, CH3O(CH2CH2O)3CH2CO,It may be CH3O(CH2CH2O)4CH2CO, CH3O(CH2CH2O)5CH2CO, CH3OCH2CH2OCO, CH3O(CH2CH2O)2CO, CH3O(CH2CH2O)3CO, CH3O(CH2CH2O)4CO, CH3O(CH2CH2O)5CO, HOCH2CH2OCH2CH2CO, HO(CH2CH2O)2CH2CH2CO, HO(CH2CH2O)3CH2CH2CO, HO(CH2CH2O)4CH2CH2CO, HO(CH2CH2O)5CH2CH2CO, HOCH2CH2OCH2CO, HO(CH2CH2O)2CH2CO, HO(CH2CH2O)3CH2CO, HO(CH2CH2O)4CH2CO, HO(CH2CH2O)5CH2CO, HOCH2CH2OCO, HO(CH2CH2O)2CO, HO(CH2CH2O)3CO, HO(CH2CH2O)4CO, HO(CH2CH2O)5CO, CH3CH2OCH2CH2OCH2CH2CO, CH3CH2O(CH2CH2O)2CH2CH2CO, CH3CH2O(CH2CH2O)3CH2CH2CO, CH3CH2O(CH2CH2O)4CH2CH2CO, CH3CH2O(CH2CH2O)5CH2CH2CO, CH3CH2OCH2CH2OCH2CO, CH3CH2O(CH2CH2O)2CH2CO, CH3CH2O(CH2CH2O)3CH2CO, CH3CH2O(CH2CH2O)4CH2CO, CH3CH2O(CH2CH2O)5CH2CO, CH3CH2OCH2CH2OCO, CH3CH2O(CH2CH2O)2CO, CH3CH2O(CH2CH2O)3CO, CH3CH2O(CH2CH2O)4CO, CH3CH2O(CH2CH2O)5CO, CH3OCH2CH2CO, HOCH2CH2CO, or CH3CH2OCH2CH2CO.

[0013] In some embodiments, the amine - terminal cap Z is CH3CO. In some embodiments, the amine - terminal cap Z is CH3CH2CO. In some embodiments, the amine - terminal cap Z is CH3(CH2)2CO. In some embodiments, the amine - terminal cap Z is CH3(CH2)3CO. In some embodiments, the amine - terminal cap Z is CH3(CH2)4CO.

[0014] In some embodiments, the αvβ6 integrin ligand has the general formula: Z-RG 1 DLAXaa u L (SEQ ID NO: 90) (Formula Ic), wherein Z, R, G 1 , D, and L are each as defined for Formula I herein; A is L-alanine; and Xaa u is a non-standard amino acid.

[0015] In some embodiments, the αvβ6 integrin ligand has the general formula: Z-RG 1 DLAAbuL (SEQ ID NO: 91) (Formula Id), wherein Z, R, G 1 , D, and L are each as defined for Formula I herein; A is L-alanine; and Abu is L-α-amino-butyric acid (2-aminobutyric acid).

[0016] In some embodiments, the αvβ6 integrin ligand has the general formula: Z-RG 1 DLXaa 1 Xaa 2 L-Xaa 3 Xaa 4 L-R 1 (SEQ ID NO: 92) (Formula VI), wherein Z is an amine-terminal cap (e.g., any of the amine-terminal caps described herein or known in the art); R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 2 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 3 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 4 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); and R 1 is optional and, when present, contains polyethylene glycol (PEG) and / or a linking group.

[0017] In some embodiments, the αvβ6 integrin ligand has the general formula: Z-RG 1 DLAXaa u L-Xaa 3 Xaa 4 L-R 1 (SEQ ID NO: 93) (Formula VIb), wherein Z, R, G 1 , D, L, and R 1are as defined for Formula VI herein; A is L-alanine; Xaa u is a non-standard amino acid; Xaa 3 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); and Xaa 4 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art).

[0018] In some embodiments, the αvβ6 integrin ligand has the general formula: Z-RG 1 DLAAbuL-Xaa 3 Xaa 4 L-R 1 (SEQ ID NO: 94) (Formula VIc), wherein Z, R, G 1 , D, L, and R 1 are as defined for Formula VI herein; A is L-alanine; Abu is L-α-aminobutyric acid (2-aminobutyric acid); Xaa 3 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); and Xaa 4is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art).

[0019] In some embodiments, the αvβ6 integrin ligand has the general formula: Z-RG 1 DLAXaa u L-Xaa u Xaa u L-R 1 (SEQ ID NO: 95) (Formula VId), wherein Z, R, G 1 , D, L, and R 1 are each as defined for Formula VI herein; A is L-alanine; and Xaa u is a non-standard amino acid.

[0020] In some embodiments, Z-R in any of the formulas or ligands herein is replaced by R´, where R´ is Dap(guanidino):

[0021]

Chemical formula

[0022] In some embodiments, the αvβ6 integrin ligand has the general formula: RG 1 DLXaa 1 Xaa 2 L-Xaa 3 Xaa 4 L-R 1 (SEQ ID NO: 96) (Formula VIII), wherein R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 2 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 3 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 4 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); R 1 is optional and, when present, contains polyethylene glycol (PEG) and / or a linking group; and Xaa 1 , Xaa 2 , Xaa 3 , and Xaa 4 and at least one of Xaa

[0023] In some embodiments, the αvβ6 integrin ligand has the general formula: RG 1 DLXaa 1 Xaa 2 L-Xaa 3 Xaa 4L-R 1 (SEQ ID NO: 96) (Formula VIII), wherein Xaa 1 , Xaa 2 , Xaa 3 , and Xaa 4 at least two of which are non-standard amino acids. In some embodiments, the αvβ6 integrin ligand has the general formula: RG 1 DLXaa 1 Xaa 2 L-Xaa 3 Xaa 4 L-R 1 (SEQ ID NO: 96) (Formula VIII), wherein Xaa 1 , Xaa 2 , Xaa 3 , and Xaa 4 at least three of which are non-standard amino acids.

[0024] In some embodiments, the αvβ6 integrin ligand has the general formula: RG 1 DLAAbuL-CitAibL-R 1 (SEQ ID NO: 97) (Formula VIIIa), wherein R, G 1 , D, L, and R 1 are each as defined for Formula VIII herein; A is L-alanine; Abu is L-α-aminobutyric acid (2-aminobutyric acid); Cit is citrulline; and Aib is α-aminoisobutyric acid (2-aminoisobutyric acid).

[0025] In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of the structure of Figure 1.

[0026] In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of the structure of Figure 2.

[0027] In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of the structure of Figure 3.

[0028] In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of the structure of FIG. 4.

[0029] In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of the structure of FIG. 5.

[0030] In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of the structure of FIG. 6.

[0031] In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of the structure of FIG. 7.

[0032] In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of the structure of FIG. 8.

[0033] In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of the structure of FIG. 9.

[0034] In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of the structure of FIG. 10.

[0035] In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of the structure of FIG. 11.

[0036] In some embodiments, any of the αvβ6 integrin ligands disclosed herein can be linked to a cargo molecule, a reactive group, and / or a protected reactive group. The reactive group can be used to facilitate the conjugation of the αvβ6 integrin ligand to a molecule, such as one or more cargo molecules (e.g., any of the cargo molecules described herein or known in the art). The αvβ6 integrin ligands disclosed herein can increase the targeting of cargo molecules to αvβ6 integrin or cells that express αvβ6 integrin. The cargo molecule can be, but is not limited to, a pharmaceutically active ingredient or compound, a pharmaceutical, a prodrug, or a therapeutically effective substance. In some embodiments, the cargo molecule can be, but is not limited to, a small molecule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified nucleic acid or polynucleotide (e.g., an oligomeric compound, such as an antisense oligonucleotide or an RNAi agent), a peptide, an aptamer, a polymer, a polyamine, a protein, a toxin, a vitamin, polyethylene glycol, a hapten, digoxigenin, biotin, a radioactive atom or molecule, or a fluorophore. In some embodiments, the cargo molecule comprises a pharmaceutically active ingredient, a pharmaceutical, or a prodrug. In some embodiments, the cargo molecule comprises an oligomeric compound as a pharmaceutically active ingredient. In some embodiments, the cargo molecule comprises an RNAi agent as a pharmaceutically active ingredient.

[0037] Described herein is the use of the described αvβ6 ligand for targeting a cargo molecule to αvβ6-expressing cells. The cells can be in vitro, in situ, ex vivo, or in vivo.

[0038] In another aspect, the disclosure provides a composition comprising one or more of the engineered, non-naturally occurring αvβ6 ligands described herein. For example, in some embodiments, a composition comprising one or more of the αvβ6 integrin ligands disclosed herein comprises one or more oligomeric compounds, such as one or more RNAi agents, that are to be delivered to cells in vivo. In some embodiments, described herein is a composition for delivering an RNAi agent to cells in vivo, where the RNAi agent is conjugated to one or more αvβ6 ligands.

[0039] Compositions comprising one or more αvβ6 ligands are described. In some embodiments, the composition comprises a pharmaceutically acceptable excipient. In some embodiments, a composition comprising one or more αvβ6 ligands comprises one or more other pharmaceutical substances or pharmaceutically active ingredients or compounds.

[0040] In other embodiments, the composition comprises a medicament comprising one or more of the αvβ6 ligands described herein. In some embodiments, the medicament further comprises a pharmaceutically acceptable excipient.

[0041] Compositions comprising one or more of the αvβ6 integrin ligands disclosed herein can be delivered in vivo or in vitro to, for example, type I and type II alveolar epithelial cells, goblet cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells, dermal epithelial cells, bile duct cells, intestinal cells, ductal epithelial cells, glandular epithelial cells, renal tubules, and epithelial tumors (cancers).

[0042] In another aspect, the disclosure provides methods comprising the use of one or more of the αvβ6 ligands and / or compositions described herein, and, if desired, methods of formulating the disclosed αvβ6 ligands and / or compositions in a form suitable for administration as a medicament. In other embodiments, the disclosure provides methods of manufacturing the ligands and compositions described herein, such as medicaments.

[0043] A composition containing one or more αvβ6 integrin ligands can be administered in vivo to a subject using an administration route known in the art to be suitable for such administration, taking into account the cargo molecule(s) to be administered. Such administration routes include, for example, intravenous, subcutaneous, intraperitoneal, intradermal, transdermal, oral, sublingual, topical, intratumoral, intranasal, or inhalation (aerosol or dry powder formulation) administration. In some embodiments, a composition containing one or more αvβ6 integrin ligands can be administered for systemic delivery, for example, by intravenous or subcutaneous administration. In some embodiments, a composition containing one or more αvβ6 integrin ligands can be administered for local delivery, for example, by inhalation delivery using a dry powder inhaler or nebulizer. In some embodiments, a composition containing one or more αvβ6 integrin ligands can be administered for local delivery by topical administration.

[0044] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to type I alveolar epithelial cells in vivo, the method comprising administering to the subject one or more αvβ6 integrin ligands conjugated to one or more cargo molecules.

[0045] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to type II alveolar epithelial cells in vivo, the method comprising administering to the subject one or more αvβ6 integrin ligands conjugated to one or more cargo molecules.

[0046] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to goblet cells in vivo, the method comprising administering to the subject one or more αvβ6 integrin ligands conjugated to one or more cargo molecules.

[0047] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to secretory epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more cargo molecules.

[0048] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to ciliated epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more cargo molecules.

[0049] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to corneal epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more cargo molecules.

[0050] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to conjunctival epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more cargo molecules.

[0051] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to dermal epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more cargo molecules.

[0052] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to bile duct cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more cargo molecules.

[0053] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to intestinal cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more cargo molecules.

[0054] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to mammary duct epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more cargo molecules.

[0055] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to glandular epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more cargo molecules.

[0056] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to renal tubules in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more cargo molecules.

[0057] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to epithelial tumors (cancers) in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more cargo molecules.

[0058] In some embodiments, disclosed herein is a method of delivering an oligomeric compound to type I alveolar epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein is a method of delivering an RNAi agent to type I alveolar epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method of inhibiting the expression of a target gene in type I alveolar epithelial cells in vivo, the method comprising administering to a subject an RNAi agent conjugated to one or more ligands having an affinity for αvβ6 integrin.

[0059] In some embodiments, disclosed herein is a method of delivering an oligomeric compound to type II alveolar epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein is a method of delivering an RNAi agent to type II alveolar epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method of inhibiting the expression of a target gene in type II alveolar epithelial cells in vivo, the method comprising administering to a subject an RNAi agent conjugated to one or more ligands having an affinity for αvβ6 integrin.

[0060] In some embodiments, disclosed herein is a method of delivering an oligomeric compound to goblet cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein is a method of delivering an RNAi agent to goblet cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method of inhibiting the expression of a target gene in goblet cells in vivo, the method comprising administering to a subject an RNAi agent conjugated to one or more ligands having an affinity for αvβ6 integrin.

[0061] In some embodiments, disclosed herein is a method of delivering an oligomeric compound to secretory epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein is a method of delivering an RNAi agent to secretory epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method of inhibiting the expression of a target gene in secretory epithelial cells in vivo, the method comprising administering to a subject an RNAi agent conjugated to one or more ligands having an affinity for αvβ6 integrin.

[0062] In some embodiments, disclosed herein is a method of delivering an oligomeric compound to ciliated epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein is a method of delivering an RNAi agent to ciliated epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method of inhibiting the expression of a target gene in ciliated epithelial cells in vivo, the method comprising administering to a subject an RNAi agent conjugated to one or more ligands having an affinity for αvβ6 integrin.

[0063] In some embodiments, disclosed herein is a method of delivering an oligomeric compound to corneal epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein is a method of delivering an RNAi agent to corneal epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method of inhibiting the expression of a target gene in corneal epithelial cells in vivo, the method comprising administering to a subject an RNAi agent conjugated to one or more ligands having an affinity for αvβ6 integrin.

[0064] In some embodiments, disclosed herein is a method for delivering an oligomeric compound to conjunctival epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein is a method for delivering an RNAi agent to conjunctival epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method for inhibiting the expression of a target gene in conjunctival epithelial cells in vivo, the method comprising administering to a subject an RNAi agent conjugated to one or more ligands having an affinity for αvβ6 integrin.

[0065] In some embodiments, disclosed herein is a method for delivering an oligomeric compound to dermal epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein is a method for delivering an RNAi agent to dermal epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method for inhibiting the expression of a target gene in dermal epithelial cells in vivo, the method comprising administering to a subject an RNAi agent conjugated to one or more ligands having an affinity for αvβ6 integrin.

[0066] In some embodiments, disclosed herein is a method of delivering an oligomeric compound to bile duct cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein is a method of delivering an RNAi agent to bile duct cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method of inhibiting the expression of a target gene in bile duct cells in vivo, the method comprising administering to a subject an RNAi agent conjugated to one or more ligands having an affinity for αvβ6 integrin.

[0067] In some embodiments, disclosed herein is a method of delivering an oligomeric compound to intestinal cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein is a method of delivering an RNAi agent to intestinal cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method of inhibiting the expression of a target gene in intestinal cells in vivo, the method comprising administering to a subject an RNAi agent conjugated to one or more ligands having an affinity for αvβ6 integrin.

[0068] In some embodiments, disclosed herein is a method of delivering an oligomeric compound to mammary duct epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein is a method of delivering an RNAi agent to mammary duct epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method of inhibiting the expression of a target gene in mammary duct epithelial cells in vivo, the method comprising administering to a subject an RNAi agent conjugated to one or more ligands having an affinity for αvβ6 integrin.

[0069] In some embodiments, disclosed herein is a method of delivering an oligomeric compound to glandular epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein is a method of delivering an RNAi agent to glandular epithelial cells in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method of inhibiting the expression of a target gene in glandular epithelial cells in vivo, the method comprising administering to a subject an RNAi agent conjugated to one or more ligands having an affinity for αvβ6 integrin.

[0070] In some embodiments, disclosed herein is a method of delivering an oligomeric compound to renal tubules in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein is a method of delivering an RNAi agent to renal tubules in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method of inhibiting the expression of a target gene in renal tubules in vivo, the method comprising administering to a subject an RNAi agent conjugated to one or more ligands having an affinity for αvβ6 integrin.

[0071] In some embodiments, disclosed herein is a method of delivering an oligomeric compound to an epithelial tumor (cancer) in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein is a method of delivering an RNAi agent to an epithelial tumor (cancer) in vivo, the method comprising administering to a subject one or more αvβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method of inhibiting the expression of a target gene in an epithelial tumor (cancer) in vivo, the method comprising administering to a subject an RNAi agent conjugated to one or more ligands having an affinity for αvβ6 integrin.

[0072] As used herein, the term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group having from 1 to 10 carbon atoms, unless otherwise specified. For example, "C1-C6 alkyl" includes alkyl groups having 1, 2, 3, 4, 5, or 6 carbons in a straight or branched chain arrangement. Non-limiting examples of alkyl groups include methyl, ethyl, isopropyl, tert-butyl, and n-hexyl. As used herein, the term "aminoalkyl" refers to an alkyl group as defined above substituted at any position with one or more amino groups as permitted by normal valency. The amino group may be unsubstituted, monosubstituted, or disubstituted. Non-limiting examples of aminoalkyl groups include aminomethyl, dimethylaminomethyl, and 2-aminoprop-1-yl.

[0073] As used herein, the term "cycloalkyl" means a saturated or unsaturated non-aromatic hydrocarbon ring group having from 3 to 14 carbon atoms, unless otherwise specified. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, and cyclohexyl. Cycloalkyl may include multiple spiro- or fused rings. The cycloalkyl group is optionally mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valency.

[0074] As used herein, the term "alkenyl", unless otherwise specified, refers to a straight-chain or branched non-aromatic hydrocarbon group containing at least one carbon-carbon double bond and having 2 to 10 carbon atoms. Up to 5 carbon-carbon double bonds may be present in such a group. For example, "C2-C6" alkenyl is defined as an alkenyl group having 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, and cyclohexenyl. The straight-chain, branched-chain, or cyclic portion of the alkenyl group may contain a double bond and may optionally be mono-substituted, di-substituted, tri-substituted, tetra-substituted, or penta-substituted at any position as permitted by normal valences. The term "cycloalkenyl" means a monocyclic hydrocarbon group having a specific number of carbon atoms and at least one carbon-carbon double bond.

[0075] As used herein, the term "alkynyl", unless otherwise specified, refers to a straight-chain or branched hydrocarbon group containing 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Up to 5 carbon-carbon triple bonds may be included. Thus, "C2-C6 alkynyl" means an alkynyl group having 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl. The straight-chain or branched-chain portion of the alkynyl group may optionally be mono-substituted, di-substituted, tri-substituted, tetra-substituted, or penta-substituted at any position as permitted by normal valences.

[0076] As used herein, "alkoxyl" or "alkoxy" refers to an -O-alkyl group having the indicated number of carbon atoms. For example, C1-C6 alkoxy is intended to include C1, C2, C3, C4, C5, and C6 alkoxy groups. For example, C1-C8 alkoxy is intended to include C1, C2, C3, C4, C5, C6, C7, and C8 alkoxy groups. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, s-pentoxy, n-heptoxy, and n-octoxy.

[0077] As used herein, "keto" refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or aryl group as defined herein attached via a carbonyl bridge. Examples of keto groups include, but are not limited to, alkanoyl (e.g., acetyl, propionyl, butanoyl, pentanoyl, or hexanoyl), alkenoyl (e.g., acryloyl), alkynoyl (e.g., ethynoyl, propynoyl, butynoyl, pentynoyl, or hexynoyl), aroyl (e.g., benzoyl), heteroaryloyl (e.g., pyrroloyl, imidazoloyl, quinolinoyl, or pyridinoyl).

[0078] As used herein, "alkoxycarbonyl" refers to any alkoxy group as defined above attached via a carbonyl bridge (i.e., -C(O)O-alkyl). Examples of alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-propoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, or n-pentoxycarbonyl.

[0079] As used herein, "aryloxycarbonyl" refers to an aryl group as defined herein bonded via an oxycarbonyl bridge (i.e., -C(O)O-aryl). Examples of aryloxycarbonyl groups include, but are not limited to, phenoxycarbonyl and naphthyloxycarbonyl.

[0080] As used herein, "heteroaryloxycarbonyl" refers to any heteroaryl group as defined herein bonded via an oxycarbonyl bridge (i.e., -C(O)O-heteroaryl). Examples of heteroaryloxycarbonyl groups include, but are not limited to, 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyl-oxycarbonyl.

[0081] As used herein, "aryl" or "aromatic" means a stable monocyclic or polycyclic carbocyclic ring having up to 6 atoms in each ring, wherein at least one ring is aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl, and biphenyl. When an aryl substitution is bicyclic and one ring is non-aromatic, the bond is understood to be through the aromatic ring. The aryl group is optionally mono-substituted, di-substituted, tri-substituted, tetra-substituted, or penta-substituted at any position as permitted by normal valency.

[0082] As used herein, the term "heteroaryl" represents a stable monocyclic or polycyclic ring having up to 7 atoms in each ring, wherein at least one ring is aromatic and contains 1 to 4 heteroatoms selected from the group consisting of O, N, and S. Examples of heteroaryl groups include, but are not limited to, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl, isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl, isoindolonyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. "Heteroaryl" is also understood to include N-oxide derivatives of any nitrogen-containing heteroaryl. When the heteroaryl substitution is bicyclic and one ring is non-aromatic or contains no heteroatoms, the bond is understood to be through the aromatic ring or through the heteroatom-containing ring. The heteroaryl group is optionally mono-substituted, di-substituted, tri-substituted, tetra-substituted, or penta-substituted at any position as permitted by normal valency.

[0083] As used herein, the terms "heterocyclic", "heterocyclic ring", or "heterocyclyl" mean a 3- to 14-membered aromatic or non-aromatic heterocyclic ring containing 1 to 4 heteroatoms selected from the group consisting of O, N, and S, including polycyclic groups. As used herein, the term "heterocyclic ring" is also considered to be synonymous with the terms "heterocyclic" and "heterocyclyl" and is understood to have the same definitions described herein. "Heterocyclyl" includes the above-described heteroaryl, as well as its dihydro and tetrahydro analogs.Examples of heterocyclyl groups include, but are not limited to, azetidinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indradinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthopyridinyl, oxadiazolyl, oxooxazolidinyl, oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyridinonyl, pyrimidinyl, pyrimidinonyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxidothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and their N-oxides. The bond of the heterocyclyl substitution can occur via a carbon atom or via a heteroatom.The heterocyclic group is optionally mono-substituted, di-substituted, tri-substituted, tetra-substituted, or penta-substituted at any position as permitted by normal valency.

[0084] As used herein, terms such as "treating" and "treatment" mean a method or step taken to provide a reduction or alleviation in the number, severity, and / or frequency of one or more symptoms of a disease or condition in a subject.

[0085] Unless otherwise specified, as used herein, the use of the following symbols means that any group or groups may be linked as defined by the scope of the invention described herein. [Chemical formula]

[0086] As used herein, the term "isomer" refers to compounds that have the same molecular formula but differ in the nature or order of bonding of their atoms or in the spatial arrangement of their atoms. Isomers that differ in the spatial arrangement of their atoms are called "stereoisomers". Stereoisomers that are not mirror images of each other are called "diastereoisomers", and stereoisomers that are non-superimposable mirror images are called "enantiomers" or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is called a "chiral center". As used herein, a linking group is one or more atoms, and such one or more atoms connect one molecule or part of a molecule to another second molecule or second part of a molecule. In the art, the terms linking group and spacer are sometimes used interchangeably. Similarly, as used in the art, the term scaffold is sometimes used interchangeably with linking group. In some embodiments, the linking group may include a peptide-cleavable linking group. In some embodiments, the linking group may comprise or consist of the peptide FCitFP (SEQ ID NO: 131).

[0087] As used herein, the term "linked," when referring to a connection between two molecules, means that the two molecules are joined by a covalent bond or that the two molecules are associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond). In some examples where the term "linked" refers to an association between two molecules via a non-covalent bond, the association between the two different molecules has a KD of less than 1x10 -4 M (e.g., less than 1x10 -5 M, less than 1x10 -6 M, or less than 1x10 -7 M). Unless otherwise described, as used herein, the term linked may refer to a connection between a first compound and a second compound, regardless of the presence or absence of any intervening atom or atomic group.

[0088] As used herein, "standard amino acids" or "natural amino acids" include alanine, cysteine, aspartic acid (aspartate), glutamic acid (glutamate), phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine.

[0089] As used herein, "non-standard amino acids" includes, but is not limited to, selenocysteine, pyrrolidine, N-formylmethionine, hydroxyproline, selenomethionine, α-amino-isobutyric acid (Aib), L-α-aminobutyric acid (Abu), α,γ-diaminobutyric acid, dehydroalanine, norleucine, alloisoleucine, t-leucine, α-amino-n-heptanoic acid, α,β-diaminopropionic acid, β-N-oxalyl-α,β-diaminopropionic acid, allothreonine, homocysteine, homoserine, β-homo-alanine (β3-hΑ), isovaline, norvaline (Nva), citrulline (Cit), ornithine, α-methyl-aspartate (αMeD), α-methyl-leucine (αMeL), N-methylalanine, N-methyl-glycine (N Me G), N-methylleucine (N Me L), β-cyclohexyl-alanine (Cha), N-ethylalanine, Ν,Ν-ε-dimethyllysine (K( Me )2), dimethylarginine (R(Me)2), Dap(Ac), n-alkylated L-α amino acids, and other amino acid analogs or amino acid mimetics that function in a manner similar to naturally occurring amino acids.

[0090] As used herein, unless specifically identified in a structure having a particular stereochemistry, each structure has an asymmetric center and thus gives rise to the formation of enantiomers, diastereomers, or other stereoisomers, and each structure disclosed herein is intended to represent all such possible isomers (including their optically pure forms and racemates). For example, the structures disclosed herein are intended to encompass mixtures of diastereomers as well as single stereoisomers.

[0091] Those skilled in the art will readily understand and recognize that the compounds and compositions disclosed herein may contain certain atoms (e.g., N, O, or S atoms) in protonated or deprotonated states depending on the environment in which the compound or composition is placed. Accordingly, as used herein, the structures disclosed herein are assumed to account for the possibility that certain functional groups, such as OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to encompass the disclosed compounds and compositions regardless of their states of protonation based on the pH of the environment, as will be readily understood by those skilled in the art.

[0092] As used in the claims of the present application, the phrase "consisting of" excludes any element, step, or component not specified in the claim. As used in the claims of the present application, the phrase "consisting essentially of" limits the scope of the claim to a particular material or step and those that do not materially affect the basic and novel characteristics (s) of the invention described in the claim.

[0093] Unless otherwise defined, all technical and chemical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0094] Pharmacokinetics is a common concern for peptide-based pharmaceuticals and pharmaceutical compositions containing peptides. Many peptides do not circulate in the blood for more than a few minutes, for example, due to enzymatic degradation. This often significantly reduces or even prevents their usefulness as therapeutic agents or as components of pharmaceuticals.

[0095] Stability tests in various serum preparations (e.g., measurement of in vitro degradation of peptides in serum and / or plasma) are important screening assays in peptide-based drug development. In particular, as shown by such tests, the αvβ6 integrin ligands disclosed herein are stable in serum and have an affinity for or can bind to αvβ6 integrin.

[0096] Other features and advantages of the invention will become apparent from the following detailed description and claims.

Brief Description of the Drawings

[0097]

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Figure 10

Figure 11

Figure 12

DETAILED DESCRIPTION OF THE INVENTION

[0098] Detailed Description Disclosed herein is a novel, engineered, non-naturally occurring peptide-based αvβ6 integrin ligand that has serum stability and affinity for αvβ6 integrin. The αvβ6 integrin ligand can be used to target αvβ6 integrin-expressing cells in vitro, in situ, ex vivo, and / or in vivo. In some embodiments, the αvβ6 integrin ligand is conjugated to one or more cargo molecules to direct the cargo molecules to αvβ6 integrin-expressing cells in vitro, in situ, ex vivo, and / or in vivo. In some embodiments, the cargo molecule comprises or consists of a pharmaceutically active compound. In some embodiments, the αvβ6 integrin ligands disclosed herein are conjugated to cargo molecules to direct the cargo molecules to epithelial cells in vivo.

[0099] In some embodiments, the αvβ6 integrin ligand is as follows: Z-RG 1 DLXaa 1 Xaa 2 L (SEQ ID NO: 85) (Formula I) comprising, wherein Z is an amine-terminal cap (e.g., any of the amine-terminal caps described herein or known in the art); R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); and Xaa2 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art).

[0100] In some embodiments, the amine-terminal cap (Z) in Formula I comprises CH3CO (also referred to herein as "Ac"). In some embodiments, the amine-terminal cap (Z) in Formula I is CH3CO.

[0101] In some embodiments, the αvβ6 integrin ligand is as follows: R´G 1 DLXaa 1 Xaa 2 L (SEQ ID NO: 98) (Formula Ia) and wherein R´ is Dap (guanidino); and G 1 , D, L, Xaa 1 , and Xaa 2 are each as defined for Formula I herein.

[0102] In some embodiments, the αvβ6 integrin ligand has the general formula: Z-RG 1 DLXaa 1 Xaa u L (SEQ ID NO: 99) (Formula Ib) and wherein Z, R, G 1 , D, L, and Xaa 1 are each as defined for Formula I herein; and Xaa u is a non-standard amino acid.

[0103] In some embodiments, the αvβ6 integrin ligand has the general formula: Z-RG 1DLAXaa u L (SEQ ID NO: 90) (Formula Ic) comprising, wherein Z, R, G 1 , D, and L are each as defined for Formula I herein; A is L-alanine; and Xaa u is a non-standard amino acid.

[0104] In some embodiments, αvβ6 integrin ligands are described, as follows: Z-RG 1 DLXaa 1 Xaa 2 L-J-R 1 (SEQ ID NO: 86) (Formula II) comprising, wherein Z is an amine-terminal cap (e.g., any of the amine-terminal caps described herein or known in the art); R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 2 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); J is optional and, when present, comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 - 30, 1 - 25, 1 - 20, 1 - 15, 1 - 10, 1 - 5, 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 30, 15 - 25, 15 - 20, 20 - 30, 20 - 25, or 25 - 30) of L-α-amino acids (e.g., any of the L-α-amino acids described herein or known in the art), L-β-amino acids (e.g., any of the L-β-amino acids described herein or known in the art), or α,α-disubstituted amino acids (e.g., any of the α,α-disubstituted amino acids described herein or known in the art), or combinations thereof; and, R 1 is optional and, when present, comprises PEG and / or a linking group.

[0105] In some embodiments, L is linked to J via an amide bond.

[0106] In some embodiments, ανβ6 integrin ligands are described as follows: R´G 1 DLXaa 1 Xaa 2 L-J-R 1 (SEQ ID NO: 100) (Formula IIa) comprising, wherein, R´ is Dap(guanidino); and, G 1 D, L, Xaa 1 Xaa 2 J, and R 1 are each as defined for Formula II herein.

[0107] In some embodiments, L is linked to J via an amide bond.

[0108] In some embodiments, the αvβ6 integrin ligand can comprise a reactive group or a protected reactive group, as follows: Z-RG 1 DLXaa 1 Xaa 2 L-J-R 1 -R 2 (SEQ ID NO: 87) (Formula III) and wherein Z is an amine terminal cap (e.g., any of the amine terminal caps described herein or known in the art); R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 2 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); J is optional and, when present, comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 - 30, 1 - 25, 1 - 20, 1 - 15, 1 - 10, 1 - 5, 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 30, 15 - 25, 15 - 20, 20 - 30, 20 - 25, or 25 - 30) L-α-amino acids (e.g., any of the L-α-amino acids described herein or known in the art), L-β-amino acids (e.g., any of the L-β-amino acids described herein or known in the art), or α,α-disubstituted amino acids (e.g., any of the α,α-disubstituted amino acids described herein or known in the art), or combinations thereof; R 1 is optional and, when present, comprises PEG and / or a linking group, and R 2 comprises a reactive group or a protected reactive group.

[0109] The reactive group or protected reactive group can be used to attach the αvβ6 integrin ligand to the molecule of interest, i.e., the cargo molecule. In some embodiments, L is linked to J via an amide bond.

[0110] In some embodiments, the αvβ6 integrin ligand is synthesized to have a reactive group or a protected reactive group and has the following formula: R´G 1 DLXaa 1 Xaa 2 L-J-R 1 -R 2 (SEQ ID NO: 101) (Formula IIIa) comprises, wherein R´ is Dap(guanidino); and G 1 , D, L, Xaa 1 , Xaa 2 , J, R 1 , and R2 are as defined for Formula III herein, respectively.

[0111] In some embodiments, one or more αvβ6 integrin ligands can conjugate to one or more cargo molecules, as follows: (Z-RG 1 DLXaa 1 Xaa 2 L-J-R 1 ) n -R 3 (SEQ ID NO: 88) (Formula IV) comprising, wherein Z is an amine terminal cap (e.g., any of the amine terminal caps described herein or known in the art); R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 2 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); J is optional and, when present, comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 - 30, 1 - 25, 1 - 20, 1 - 15, 1 - 10, 1 - 5, 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 30, 15 - 25, 15 - 20, 20 - 30, 20 - 25, or 25 - 30) of L-α-amino acids (e.g., any of the L-α-amino acids described herein or known in the art), L-β-amino acids (e.g., any of the L-β-amino acids described herein or known in the art), or α,α-disubstituted amino acids (e.g., any of the α,α-disubstituted amino acids described herein or known in the art), or combinations thereof; R 1 is optional and, when present, comprises polyethylene glycol (PEG) and / or a linking group; n is an integer greater than 0 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 - 30, 1 - 25, 1 - 20, 1 - 15, 1 - 10, 1 - 5, 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 30, 15 - 25, 15 - 20, 20 - 30, 20 - 25, or 25 - 30); and R 3 comprises a cargo molecule.

[0112] In some embodiments, L is linked to J via an amide bond. In some embodiments, the cargo molecule can be any molecule that is desirably targeted to αvβ6 integrin-expressing cells. In some embodiments, n is an integer from 1 to 4. In some embodiments, n is 1. In some embodiments, n is 3. When n is 1, the αvβ6 integrin ligand can be referred to herein as a "monodentate" αvβ6 integrin ligand. When n is 3, the αvβ6 integrin ligand can be referred to herein as a "tridentate" αvβ6 integrin ligand. When n is 2, the αvβ6 integrin ligand can be referred to herein as a "bidentate" αvβ6 integrin ligand. When n is 4, the αvβ6 integrin ligand can be referred to herein as a "tetradentate" αvβ6 integrin ligand.

[0113] In some embodiments, one or more αvβ6 integrin ligands can conjugate to one or more cargo molecules, as follows: (R´G 1 DLXaa 1 Xaa 2 L-J-R 1 ) n -R 3 (SEQ ID NO: 102) (Formula IVa) comprising, wherein R´ is Dap(guanidino); and G 1 , D, L, Xaa 1 , Xaa 2 , J, R 1 , n, and R 3 are each as defined for Formula IV herein.

[0114] In some embodiments, L is linked to J via an amide bond. In some embodiments, n is an integer from 1 to 4. In some embodiments, n is 3. In some embodiments, the cargo molecule can be any molecule that is desirably targeted to αvβ6 integrin-expressing cells.

[0115] In some embodiments, one or more αvβ6 integrin ligands can conjugate to one or more cargo molecules as follows: (Z-RG 1 DLXaa 1 Xaa 2 L-J-R 1 ) n -R 4 -(R 3 ) p (SEQ ID NO: 89) (Formula V) comprising, wherein Z is an amine-terminal cap (e.g., any of the amine-terminal caps described herein or known in the art); R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 2is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); J is optional and, when present, contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 - 30, 1 - 25, 1 - 20, 1 - 15, 1 - 10, 1 - 5, 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 30, 15 - 25, 15 - 20, 20 - 30, 20 - 25, or 25 - 30) of L-α amino acids (e.g., any of the L-α amino acids described herein or known in the art), L-β amino acids (e.g., any of the L-β amino acids described herein or known in the art), or α,α-disubstituted amino acids (e.g., any of the α,α-disubstituted amino acids described herein or known in the art), or combinations thereof; R 1 is optional and, when present, contains PEG and / or a linking group; n is an integer greater than 0 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 - 30, 1 - 25, 1 - 20, 1 - 15, 1 - 10, 1 - 5, 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 30, 15 - 25, 15 - 20, 20 - 30, 20 - 25, or 25 - 30); R 4is optional and, if present, includes a scaffold and / or a linking group, said scaffold and / or linking group including at least one binding site for each ligand present (i.e., a number of binding sites equal to at least n) and at least one binding site for each cargo molecule present (i.e., a number of binding sites equal to at least p); p is an integer greater than 0 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 - 30, 1 - 25, 1 - 20, 1 - 15, 1 - 10, 1 - 5, 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 30, 15 - 25, 15 - 20, 20 - 30, 20 - 25, or 25 - 30); and, R 3 includes one or more cargo molecules.

[0116] In some embodiments, L is linked to J via an amide bond. In some embodiments, the cargo molecule can be any molecule that is desirably targeted to αvβ6 integrin-expressing cells.

[0117] The αvβ6 integrin ligands disclosed herein can include one or more scaffolds. Scaffolds, sometimes also referred to in the art as linking groups or linkers, can be used to facilitate the attachment of one or more cargo molecules to one or more of the αvβ6 integrin ligands disclosed herein. Useful scaffolds compatible with the ligands disclosed herein are generally known in the art. Non-limiting examples of scaffolds that can be used with the αvβ6 integrin ligands disclosed herein include, but are not limited to, polymers (e.g., polyacrylate polymers, polyvinyl ester polymers, etc.), amino acid polymers (e.g., bis-glutamic acid, bis-lysine, poly-L-lysine (PLL), etc.), and cysteine. In some embodiments, the scaffold can provide additional desired properties in addition to simply functioning as a linker, such as enhancing pharmacokinetic (PK) properties, for example.

[0118] In some embodiments, one or more αvβ6 integrin ligands can conjugate to one or more cargo molecules, as follows: (R´G 1 DLXaa 1 Xaa 2 L-J-R 1 ) n -R 4 -(R 3 ) p (SEQ ID NO: 103) (Formula Va) comprising, wherein R´ is Dap (guanidino); and G 1 , D, L, Xaa 1 , Xaa 2 , J, R 1 , n, R 4 , p, and R 3 are each as defined for Formula V herein.

[0119] In some embodiments, L is linked to J via an amide bond. The cargo molecule can be any molecule that desirably can be targeted to αvβ6 integrin-expressing cells.

[0120] In some embodiments, J in any of the formulas herein includes one, two, three, or more than three L-α-amino acids, L-β-amino acids, or α,α-disubstituted amino acids. One, two, three, or more than three amino acids are independently naturally occurring L-α-amino acids, naturally occurring proteinogenic amino acids, naturally occurring standard amino acids (i.e., the 20 amino acids directly encoded by the codons of the universal genetic code, also referred to as the encoded amino acids of canonical amino acids), or non-standard amino acids (also referred to as non-natural, non-encoded, or non-canonical amino acids).

[0121] Standard or natural amino acids include alanine, cysteine, aspartic acid (aspartate), glutamic acid (glutamate), phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine.

[0122] Non-standard amino acids include, but are not limited to, selenocysteine, pyrrolidine, N-formylmethionine, hydroxyproline, selenomethionine, α-amino-isobutyric acid (Aib), L-α-aminobutyric acid (Abu), α,γ-diaminobutyric acid, dehydroalanine, norleucine, alloisoleucine, t-leucine, α-amino-n-heptanoic acid, α,β-diaminopropionic acid, β-N-oxalyl-α,β-diaminopropionic acid, allothreonine, homocysteine, homoserine, β-homo-alanine (β3-hΑ), isovaline, norvaline (Nva), citrulline (Cit), ornithine, α-methyl-aspartate (αMeD), α-methyl-leucine (αMeL), N-methylalanine, N-methyl-glycine (N Me G), N-methylleucine (N MeL), β-cyclohexyl-alanine (Cha), N-ethylalanine, Ν,Ν-ε-dimethyllysine (K( Me Me)2), dimethylarginine (R(Me)2), Dap(Ac), n-alkylated L-α amino acids, and other amino acid analogs or amino acid mimetics that function similarly to naturally occurring amino acids.

[0123] In some embodiments, J comprises at least one non-standard amino acid. In some embodiments, J is Aib, Cit, CitAib, CitAibL, CitE, CitF, CitG, CitK, CitP, CitQ, CitQL, EAib, FAib, KAib, PAib, QAib, RabuL, RAibL, RCitL, RDap(Ac)L, RLQ, or RNvaL or comprises any of these.

[0124] In some embodiments, J in any of the formulas described herein is Xaa 3 Xaa 4 comprising or consisting of L, where L is L-leucine; Xaa 3 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); and Xaa 4 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art).

[0125] In some embodiments, J is Xaa 3 Xaa 4 or comprises it, where Xaa 3is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); and Xaa 4 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art).

[0126] In some embodiments, the αvβ6 integrin ligand is as follows: Z-RG 1 DLXaa 1 Xaa 2 LXaa 3 Xaa 4 L-R 1 (SEQ ID NO: 92) (Formula VI) comprising, wherein Z is an amine-terminal cap (e.g., any of the amine-terminal caps described herein or known in the art); R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 2is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 3 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 4 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); and R 1 is optional and, when present, contains PEG and / or a linking group.

[0127] In some embodiments, Xaa 1 is an L-α-amino acid, an L-β-amino acid, or an α,α-disubstituted amino acid. Xaa 1 is not limited to these, and can be a naturally occurring L-α-amino acid, a naturally occurring proteinogenic amino acid, a naturally occurring standard (i.e., the 20 amino acids directly encoded by the codons of the universal genetic code table, also referred to as the canonical amino acids), or a non-standard (also referred to as non-natural, non-coded, or non-canonical) amino acid.

[0128] In some embodiments, Xaa 2 is an L-α-amino acid, an L-β-amino acid, or an α,α-disubstituted amino acid. Xaa2 can be, but is not limited to, a naturally occurring L-α-amino acid, a naturally occurring proteinogenic amino acid, a naturally occurring standard (i.e., one of the 20 amino acids directly encoded by the codons of the universal genetic code table, also referred to as the canonical amino acids) amino acid, or a non-standard (also referred to as non-natural, non-coded, or non-canonical) amino acid.

[0129] In some embodiments, Xaa 3 is an L-α-amino acid, an L-β-amino acid, or an α,α-disubstituted amino acid. Xaa 3 can be, but is not limited to, a naturally occurring L-α-amino acid, a naturally occurring proteinogenic amino acid, a naturally occurring standard (i.e., one of the 20 amino acids directly encoded by the codons of the universal genetic code table, also referred to as the canonical amino acids) amino acid, or a non-standard (also referred to as non-natural, non-coded, or non-canonical) amino acid.

[0130] In some embodiments, Xaa 4 is an L-α-amino acid, an L-β-amino acid, or an α,α-disubstituted amino acid. Xaa 4 can be, but is not limited to, a naturally occurring L-α-amino acid, a naturally occurring proteinogenic amino acid, a naturally occurring standard (i.e., one of the 20 amino acids directly encoded by the codons of the universal genetic code table, also referred to as the canonical amino acids) amino acid, or a non-standard (also referred to as non-natural, non-coded, or non-canonical) amino acid.

[0131] In some embodiments, Xaa 1 or Xaa 2 is a non-standard amino acid. In some embodiments, Xaa 1 is a non-standard amino acid. In some embodiments, Xaa 2 is a non-standard amino acid. In some embodiments, Xaa 1 and Xaa 2 are both non-standard amino acids.

[0132] In some embodiments, Xaa 1 and / or Xaa 2 is Abu. In some embodiments, Xaa 1 is Abu. In some embodiments, Xaa 2 is Abu.

[0133] In some embodiments, Xaa 1 or Xaa 2 is uncharged. In some embodiments, Xaa 1 is uncharged. In some embodiments, Xaa 2 is uncharged. In some embodiments, Xaa 1 and Xaa 2 are uncharged.

[0134] In some embodiments, Xaa 1 is uncharged and Xaa 2 is a non-standard amino acid. In some embodiments, Xaa 2 is uncharged and Xaa 1 is a non-standard amino acid. In some embodiments, Xaa 1 is uncharged and Xaa 2 is Abu. In some embodiments, Xaa 2 is uncharged and Xaa 1 is Abu.

[0135] In some embodiments, Xaa 1 Xaa 2 is AAbu, K Abu, E Abu, F Abu, Q Abu, G Abu, P Abu, AK, AE, AF, AQ, AG, and AP, where A is L-alanine, Abu is L-α-aminobutyric acid, K is L-lysine, E is L-glutamic acid (glutamate), F is L-phenylalanine, Q is L-glutamine, G is L-glycine, and P is L-proline.

[0136] In some embodiments, RG 1DLXaa 1 Xaa 2 L (SEQ ID NO: 117) is selected from the group consisting of: RGDLAAbuL (SEQ ID NO: 118), RGDLKAbuL (SEQ ID NO: 119), RGDLEAbuL (SEQ ID NO: 120), RGDLFAbuL (SEQ ID NO: 121), RGDLQAbuL (SEQ ID NO: 122), RGDLGAbuL (SEQ ID NO: 123), RGDLPAbuL (SEQ ID NO: 124), RGDLAKL (SEQ ID NO: 125), RGDLAEL (SEQ ID NO: 126), RGDLAFL (SEQ ID NO: 127), RGDLAQL (SEQ ID NO: 128), RGDLAGL (SEQ ID NO: 129), and RGDLAPL (SEQ ID NO: 130); wherein R is L-arginine; G is L-glycine; D is L-aspartic acid (aspartate); L is L-leucine; A is L-alanine; Abu is L-α-aminobutyric acid; K is L-lysine; E is L-glutamic acid (glutamate); F is L-phenylalanine; Q is L-glutamine; and P is L-proline. In some embodiments, G (L-glycine) in any of the above formulas is replaced by MeGly (N-methylglycine).

[0137] In some embodiments, Xaa 3 or Xaa 4 is a non-standard amino acid. In some embodiments, Xaa 3 is a non-standard amino acid. In some embodiments, Xaa 4 is a non-standard amino acid. In some embodiments, Xaa 3 and Xaa 4 are both non-standard amino acids.

[0138] In some embodiments, Xaa 3 and / or Xaa 4 is Cit. In some embodiments, Xaa 3 is Cit. In some embodiments, Xaa 4 is Cit.

[0139] In some embodiments, Xaa 3or Xaa 4 is uncharged. In some embodiments, Xaa 3 is uncharged. In some embodiments, Xaa 4 is uncharged. In some embodiments, Xaa 3 and Xaa 4 are uncharged.

[0140] In some embodiments, Xaa 3 is uncharged and Xaa 4 is a non-standard amino acid. In some embodiments, Xaa 3 is uncharged and Xaa 4 is a non-standard amino acid. In some embodiments, Xaa 3 is Aib. In some embodiments, Xaa 4 is Aib.

[0141] In some embodiments, Xaa 3 Xaa 4 is CitAib, CitE, CitF, CitG, CitK, CitP, CitQ, EAib, FAib, KAib, PAib, or QAib, where Cit is citrulline, Aib is amino isobutyric acid (α-methylalanine), K is L-lysine, E is L-glutamic acid (glutamate), F is L-phenylalanine, Q is L-glutamine, G is L-glycine, and P is L-proline.

[0142] In some embodiments, the αvβ6 integrin ligand is the following: R´G 1 DLXaa 1 Xaa 2 LXaa 3 Xaa 4 L-R 1 (SEQ ID NO: 104) (Formula VIa) comprising, wherein R´ is Dap (guanidino); and G 1 , D, L, Xaa 1 , Xaa 2 , Xaa3 , Xaa 4 , and R 1 are each as defined for Formula VI herein.

[0143] In some embodiments, the αvβ6 integrin ligand comprises a reactive group or a protected reactive group and is as follows: (Z-RG 1 DLXaa 1 Xaa 2 L-J-R 1 ) n -R 4 -(R 2 ) p (SEQ ID NO: 105) (Formula VII) and wherein Z is an amine terminal cap (e.g., any of the amine terminal caps described herein or known in the art); R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 2 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); J is optional and, when present, comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 - 30, 1 - 25, 1 - 20, 1 - 15, 1 - 10, 1 - 5, 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 30, 15 - 25, 15 - 20, 20 - 30, 20 - 25, or 25 - 30) L-α-amino acids (e.g., any of the L-α-amino acids described herein or known in the art), L-β-amino acids (e.g., any of the L-β-amino acids described herein or known in the art), or α,α-disubstituted amino acids (e.g., any of the α,α-disubstituted amino acids described herein or known in the art), or combinations thereof; R 1 R is optional and, when present, comprises polyethylene glycol (PEG) and / or a linking group; n is an integer greater than 0 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 - 30, 1 - 25, 1 - 20, 1 - 15, 1 - 10, 1 - 5, 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 30, 15 - 25, 15 - 20, 20 - 30, 20 - 25, or 25 - 30); R 4 R is optional and, when present, comprises a scaffold and / or a linking group, such scaffold and / or linking group comprising at least one attachment point for each ligand and at least one attachment point for each cargo molecule; p is an integer greater than 0 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 - 30, 1 - 25, 1 - 20, 1 - 15, 1 - 10, 1 - 5, 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 30, 15 - 25, 15 - 20, 20 - 30, 20 - 25, or 25 - 30); and, R 2 comprises a reactive group or a protected reactive group.

[0144] In some embodiments, L is linked to J via an amide bond. In some embodiments, an αvβ6 integrin ligand comprising one or more reactive groups or protected reactive groups can react with a cargo molecule to form an αvβ6 integrin ligand - cargo molecule conjugate.

[0145] In some embodiments, the αvβ6 integrin ligand comprises a reactive group or a protected reactive group and is as follows: (R´G 1 DLXaa 1 Xaa 2 L - J - R 1 ) n -R 4 -(R 2 ) p (SEQ ID NO: 106) (Formula VIIa) and in the formula, R´ is Dap(guanidino); and G 1 , D, L, Xaa 1 , Xaa 2 , J, R 1 , n, R 4 , p, and R 2 are each as defined for Formula VII herein.

[0146] In some embodiments, L is linked to J via an amide bond. The cargo molecule can be any molecule that is desirably targeted to αvβ6 integrin-expressing cells.

[0147] In some embodiments, particularly when only local delivery (e.g., by inhalation or insufflation of a powder or aerosol, including those by nebulizer, intratracheal, intranasal administration, or by topical administration) is desired, the αvβ6 integrin ligand is synthesized without an amine-terminal cap, provided that at least one or more of the amino acids are non-standard amino acids.

[0148] In some embodiments, the αvβ6 integrin ligand is as follows: RG 1 DLXaa 1 Xaa 2 L-Xaa 3 Xaa 4 L-R 1 (SEQ ID NO: 96) (Formula VIII) and wherein, R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1 is an L-α amino acid (e.g., any of the L-α amino acids described herein or known in the art), an L-β amino acid (e.g., any of the L-β amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 2is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 3 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); Xaa 4 is an L-α-amino acid (e.g., any of the L-α-amino acids described herein or known in the art), an L-β-amino acid (e.g., any of the L-β-amino acids described herein or known in the art), or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein or known in the art); R 1 is optional and, when present, contains PEG and / or a linking group; and Xaa 1 , Xaa 2 , Xaa 3 , and Xaa 4 at least one of is a non-standard amino acid.

[0149] In some embodiments, the αvβ6 integrin ligand is as follows: RG 1 DLXaa 1 Xaa 2 L-Xaa 3 Xaa 4 L-R 1 (SEQ ID NO: 96) (Formula VIII) comprises, wherein each variable is defined above for Formula VIII, wherein Xaa 1 , Xaa 2 , Xaa 3, and Xaa 4 At least two of them are non-standard amino acids.

[0150] In some embodiments, the αvβ6 integrin ligand is as follows: RG 1 DLXaa 1 Xaa 2 L-Xaa 3 Xaa 4 L-R 1 (SEQ ID NO: 96) (Formula VIII) comprising, each variable being defined above for Formula VIII, wherein Xaa 1 , Xaa 2 , Xaa 3 , and Xaa 4 At least three of them are non-standard amino acids.

[0151] In some embodiments, the αvβ6 integrin ligand is as follows: RG 1 DLXaa 1 Xaa 2 L-Xaa 3 Xaa 4 L-R 1 (SEQ ID NO: 96) (Formula VIII) comprising, each variable being defined above for Formula VIII, wherein Xaa 2 , Xaa 3 , and Xaa 4 are non-standard amino acids.

[0152] In some embodiments, the αvβ6 integrin ligand is as follows: RG 1 DLAAbuLCitAibL-R 1 (SEQ ID NO: 97) (Formula VIIIa) comprising, wherein R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; A is L-alanine; Abu is L-α-aminobutyric acid; Cit is citrulline; Aib is α-amino-isobutyric acid; and, R 1 is optional and, when present, comprises PEG and / or a linking group.

[0153] In some embodiments, the αvβ6 integrin ligand comprises a reactive group or a protected reactive group and: (RG 1 DLAAbuLCitAibL-R 1 ) n -R 4 -(R 2 ) p (SEQ ID NO: 107) (Formula VIIIb) comprising, wherein, R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; A is L-alanine; Abu is L-α-aminobutyric acid; Cit is citrulline; Aib is α-amino-isobutyric acid; R 1 is optional and, when present, comprises PEG and / or a linking group; n is an integer greater than 0 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30); R 4is optional and, if present, includes a scaffold or a linking group, such scaffold or linking group including at least one binding point for each ligand and at least a binding point for each cargo molecule; p is an integer greater than 0 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30); and R 2 includes a reactive group or a protected reactive group.

[0154] In some embodiments, one or more cargo molecules are conjugated to one or more αvβ6 integrin ligands, and the following: (RG 1 DLAAbuLCitAibL-R 1 ) n -R 4 -(R 3 ) p (SEQ ID NO: 108) (Formula VIIIc) is included, wherein R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; R 1 is optional and, if present, includes PEG and / or a linking group; n is an integer greater than 0 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 - 30, 1 - 25, 1 - 20, 1 - 15, 1 - 10, 1 - 5, 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 30, 15 - 25, 15 - 20, 20 - 30, 20 - 25, or 25 - 30); R 4 is optional and, if present, includes a scaffold or a linking group, such scaffold or linking group including at least one binding point for each ligand and at least one binding point for each cargo molecule; p is an integer greater than 0 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1 - 30, 1 - 25, 1 - 20, 1 - 15, 1 - 10, 1 - 5, 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 30, 15 - 25, 15 - 20, 20 - 30, 20 - 25, or 25 - 30); and, R 3 includes one or more cargo molecules.

[0155] One or more cargo molecules can be any molecule that is desirably targeted to αvβ6 integrin expressing cells.

[0156] As used herein, in some embodiments, R 1exists and contains a PEG group, and such a PEG group contains from 1 to 100 ethylene oxide (CH2-CH2-O) units (for example, from 1 to 90, from 1 to 80, from 1 to 70, from 1 to 60, from 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 10, from 1 to 5, from 2 to 100, from 2 to 90, from 2 to 80, from 2 to 70, from 2 to 60, from 2 to 50, from 2 to 40, from 2 to 30, from 2 to 20, from 2 to 10, from 2 to 5, from 5 to 100, from 5 to 90, from 5 to 80, from 5 to 70, from 5 to 60, from 5 to 50, from 5 to 40, from 5 to 30, from 5 to 20, from 5 to 10, from 10 to 100, from 10 to 90, from 10 to 80, from 10 to 70, from 10 to 60, from 10 to 50, from 10 to 40, from 10 to 30, from 10 to 20, from 20 to 100, from 20 to 90, from 20 to 80, from 20 to 70, from 20 to 60, from 20 to 50, from 20 to 40, from 20 to 30, from 30 to 100, from 30 to 90, from 30 to 80, from 30 to 70, from 30 to 60, from 30 to 50, from 30 to 40, from 40 to 100, from 40 to 90, from 40 to 80, from 40 to 70, from 40 to 60, from 40 to 50, from 50 to 100, from 50 to 90, from 50 to 80, from 50 to 70, from 50 to 60, from 60 to 100, from 60 to 90, from 60 to 80, from 60 to 70, from 70 to 100, from 70 to 90, from 70 to 80, from 80 to 100, from 80 to 90, or from 90 to 100 ethylene oxide units). In some embodiments, R 1 exists and contains a PEG group having from 2 to 30 ethylene oxide units. In some embodiments, R 1 exists and contains a PEG group having from 2 to 20 ethylene oxide units. In some embodiments, R 1 exists and contains a PEG group having from 2 to 10 ethylene oxide units. In some embodiments, R 1 exists and contains a PEG group having from 5 to 20 ethylene oxide units. In some embodiments, R 1 exists and contains a PEG group having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ethylene oxide units.

[0157] Reactive groups are well known in the art and provide for the formation of covalent bonds between two molecules or reactants. Reactive groups suitable for use within the scope of the invention herein include, but are not limited to: amino groups, amide groups, carboxylic acid groups, azides, alkynes, propargyl groups, BCN (bicyclo[6.1.0]nonyne), DBCO (dibenzocyclooctyne) thiol, maleimide groups, aminooxy groups, N-hydroxysuccinimide (NHS) or other activated esters (e.g., PNP, TFP, PFP), bromo groups, aldehydes, carbonates, tosylates, tetrazines, trans-cyclooctene (TCO), hydrazides, hydroxyl groups, disulfides, and orthopyridyldisulfide groups.

[0158] The incorporation of reactive groups can facilitate the conjugation of the αvβ6 integrin ligands disclosed herein to cargo molecules. Conjugation reactions are well known in the art and provide for the formation of covalent bonds between two molecules or reactants. Conjugation reactions suitable for use within the scope of the invention herein include, but are not limited to, amide coupling reactions, Michael addition reactions, hydrazone formation reactions, and click chemistry addition cyclization reactions.

[0159] In some embodiments, the αvβ6 integrin-targeting ligands disclosed herein are synthesized as tetrafluorophenyl (TFP) esters, which can be substituted by reactive amino groups and bound to cargo molecules.

[0160] Protected reactive groups are also commonly used in the art. A protecting group provides a temporary chemical conversion of a reactive group to a group that does not react under conditions in which the unprotected group would react, and provides, for example, chemoselectivity in subsequent chemical reactions. Protected reactive groups suitable for use in the scope of the invention herein include, but are not limited to, BOC group (t-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), carboxybenzyl (CBZ) group, benzyl ester, and PBF (2,2,4,6,7-pentamethylpentamethyldihydrobenzofuran-5-sulfonyl).

[0161] A cargo molecule is any molecule for which targeting to αvβ6 integrin or cells expressing αvβ6 integrin may be desired. A cargo molecule can be, but is not limited to, a pharmaceutical ingredient, a drug, a prodrug, a therapeutically valuable substance, a small molecule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified nucleic acid or polynucleotide, a peptide, a polymer, a polyamine, a protein, an aptamer, a toxin, a vitamin, PEG, a hapten, digoxigenin, biotin, a radioactive atom or molecule, or a fluorophore. In some embodiments, one or more cargo molecules (e.g., the same or different cargo molecules) are linked to one or more αvβ6 integrin ligands to target the cargo molecule to cells expressing αvβ6 integrin.

[0162] In some embodiments, one or more cargo molecules are pharmaceutical ingredients or pharmaceutical compositions. In some embodiments, one or more cargo molecules are oligomeric compounds. As used herein, "oligomeric compound" refers to a compound having about 10 to 50 (e.g., 10 to 48, 10 to 46, 10 to 44, 10 to 42, 10 to 40, 10 to 38, 10 to 36, 10 to 34, 10 to 32, 10 to 30, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 50, 12 to 48, 12 to 46, 12 to 44, 12 to 42, 12 to 40, 12 to 38, 12 to 36, 12 to 34, 12 to 32, 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 50, 14 to 48, 14 to 46, 14 to 44, 14 to 42, 14 to 40, 14 to 38, 14 to 36, 14 to 34, 14 to 32, 14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 16 to 50, 16 to 48, 16 to 46, 16 to 44, 16 to 42, 16 to 40, 16 to 38, 16 to 36, 16 to 34, 16 to 32, 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to 50, 18 to 48, 18 to 46, 18 to 44, 18 to 42, 18 to 40, 18 to 38, 18 to 36, 18 to 34, 18 to 32, 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, 18 to 20, 20 to 50, 20 to 48, 20 to 46, 20 to 44, 20 to 42, 20 to 40, 20 to 38, 20 to 36, 20 to 34, 20 to 32, 20 to 30, 20 to 28, 20 to 26, 20 to 24, 20 to 22, 22 to 50, 22 to 48, 22 to 46, 22 to 44, 22 to 42, 22 to 40, 22 to 38, 22 to 36, 22 to 34, 22 to 32, 22 to 30, 22 to 28, 22 to 26, 22 to 24, 24 to 50, 24 to 48, 24 to 46, 24 to 44, 24 to 42, 24 to 40, 24 to 38, 24 to 36, 24 to 34, 24 to 32, 24 to 30, 24 to 28, 24 to 26, 26 to 50, 26 to 48, 26 to 46, 26 to 44, 26 to 42, 26 to 40, 26 to 38, 26 to 36, 26 to 34, 26 to 32, 26 to 30, 26 to 28, 28 to 50, 28 to 48, 28 to 46, 28 to 44,(28 to 42, 28 to 40, 28 to 38, 28 to 36, 28 to 34, 28 to 32, 28 to 30, 30 to 50, 30 to 48, 30 to 46, 30 to 44, 30 to 42, 30 to 40, 30 to 38, 30 to 36, 30 to 34, 30 to 32, 32 to 50, 32 to 48, 32 to 46, 32 to 44, 32 to 42, 32 to 40, 32 to 38, 32 to 36, 32 to 34, 34 to 50, 34 to 48, 34 to 46, 34 to 44, 34 to 42, 34 to 40, 34 to 38, 34 to 36, 36 to 50, 36 to 48, 36 to 46, 36 to 44, 36 to 42, 36 to 40, 36 to 38, 38 to 50, 38 to 48, 38 to 46, 38 to 44, 38 to 42, 38 to 40, 40 to 50, 40 to 48, 40 to 46, 40 to 44, 40 to 42, 42 to 50, 42 to 48, 42 to 46, 42 to 44, 44 to 50, 44 to 48, 44 to 46, 46 to 50, 46 to 48, or 48 to 50) nucleotides or a nucleotide sequence comprising nucleotide base pairs. In some embodiments, the oligomeric compound has a nucleobase sequence that is at least partially complementary to the coding sequence in the target nucleic acid or target gene expressed in the cell. In some embodiments, the oligomeric compound can inhibit the expression of the underlying gene upon delivery to a cell that expresses the gene, and is herein referred to as an "expression-inhibiting oligomeric compound". Gene expression can be inhibited in vitro or in vivo.,

[0163] "Oligomeric compound" includes, but is not limited to: oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, small interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), ribozymes, interfering RNA molecules, and dicer substrates. In some embodiments, the oligomeric compound is a single-stranded oligomeric compound. In some embodiments, the oligomeric compound is a double-stranded oligomeric compound.

[0164] In some embodiments, one or more cargo molecules are “RNAi agents,” and as defined herein, an RNAi agent is an agent that includes an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can sequence specifically inhibit the degradation of messenger RNA (mRNA) transcripts of a target mRNA or its translation. As used herein, an RNAi agent can operate via the RNA interference mechanism (i.e., induce RNA interference through interaction with the RNA interference pathway machinery of mammalian cells (RNA-induced silencing complex or RISC)), or by any alternative mechanism or pathway. When the term is used herein, an RNAi agent is considered to operate primarily via the RNA interference mechanism, but the disclosed RNAi agents are not restricted or limited to any particular pathway or mechanism of action. RNAi agents include, but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, small interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), and dicer substrates.

[0165] Typically, an RNAi agent can be composed of at least a sense strand (also called a passenger strand) containing a first sequence and an antisense strand (also called a guide strand) containing a second sequence. The lengths of the sense and antisense strands of the RNAi agent can each be 16 to 49 nucleotides in length. In some embodiments, the sense and antisense strands of the RNAi agent are independently 17 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 19 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21 to 24 nucleotides in length. The sense and antisense strands may be of the same length or of different lengths. The RNAi agent contains an antisense strand sequence that is at least partially complementary to a sequence in the target gene, and upon delivery to a cell expressing the target, the RNAi agent can inhibit the expression of one or more target genes in vivo or in vitro.

[0166] Oligomeric compounds can generally, and RNAi agents in particular, be composed of modified nucleotides and / or one or more non-phosphodiester linkages. As used herein, "modified nucleotide" is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2'-modified nucleotides, 3' to 3' linkages (inverted) nucleotides, non-natural base-containing nucleotides, bridged nucleotides, peptide nucleic acids, 2',3'-seco nucleotide mimics (unlocked nucleobase analogs, locked nucleotides, 3'-O-methoxy (2' nucleoside internucleotide linkage) nucleotides), 2'-F-arabinonucleotides, 5'-Me,2'-fluoronucleotides, morpholino nucleotides, vinyl phosphonate deoxyribonucleotides, vinyl phosphonate-containing nucleotides, and cyclopropyl phosphonate-containing nucleotides. 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoronucleotides, 2'-deoxynucleotides, 2'-methoxyethyl (2'-O-2-methoxylethyl) nucleotides, 2'-aminonucleotides, and 2'-alkyl nucleotides.

[0167] In addition, one or more nucleotides of an oligomeric compound, such as an RNAi agent, can be linked by non-standard linkages or backbones (i.e., modified internucleotide linkages or modified backbones). Modified internucleotide linkages can be non-phosphate-containing covalent internucleotide linkages. Modified internucleotide linkages or backbones include, but are not limited to, 5'-phosphorothioate groups, chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkylphosphonates (e.g., methylphosphonate or 3'-alkylene phosphonate), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidate, aminoalkylphosphoramidate, or thionophosphoramidate), thionoalkyl-phosphonate, thionoalkylphosphotriester, morpholino linkages, boranophosphates having a normal 3'-5' linkage, 2'-5' linkage analogs of boranophosphates, or boranophosphates having an inverted polarity in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0168] Not all positions of a given compound need to be uniformly modified. Conversely, two or more modifications can be incorporated into a single oligomeric compound or even into its single nucleotides.

[0169] The sense and antisense strands of an RNAi agent can be synthesized and / or modified by methods known in the art. For example, a disclosure of an RNAi agent for inhibition of alpha-ENaC expression can be found, for example, in International Publication No. WO 2008 / 152131, which is incorporated herein by reference in its entirety. Additional disclosures regarding RNAi agents can be found, for example, in disclosures of modifications, for example, in International Patent Application No. PCT / US2017 / 0455446 of Arrowhead Pharmaceuticals, Inc., which is also incorporated herein by reference in its entirety.

[0170] In some embodiments, one or more cargo molecules can comprise or consist of a PEG moiety that can act as a pharmacokinetic (PK) modulator. In some embodiments, one or more cargo molecules can comprise a PEG moiety, and such PEG moiety can have from about 20 to 900 ethylene oxide (CH2-CH2-O) units (e.g., 20 to 850, 20 to 800, 20 to 750, 20 to 700, 20 to 650, 20 to 600, 20 to 550, 20 to 500, 20 to 450, 20 to 400, 20 to 350, 20 to 300, 20 to 250, 20 to 200, 20 to 150, 20 to 100, 20 to 75, 20 to 50, 100 to 850, 100 to 800, 100 to 750, 100 to 700, 100 to 650, 100 to 600, 100 to 550, 100 to 500, 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 200 to 850, 200 to 800, 200 to 750, 200 to 700, 200 to 650, 200 to 600, 200 to 550, 200 to 500, 200 to 450, 200 to 400, 200 to 350, 200 to 300, 250 to 900, 250 to 850, 250 to 800, 250 to 750, 250 to 700, 250 to 650, 250 to 600, 250 to 550, 250 to 500, 250 to 450, 250 to 400, 250 to 350, 250 to 300, 300 to 900, 300 to 850, 300 to 800, 300 to 750, 300 to 700, 300 to 650, 300 to 600, 300 to 550, 300 to 500, 300 to 450, 300 to 400, 300 to 350, 350 to 900, 350 to 850, 350 to 800, 350 to 750, 350 to 700, 350 to 650, 350 to 600, 350 to 550, 350 to 500, 350 to 450, 350 to 400, 400 to 900, 400 to 850, 400 to 800, 400 to 750, 400 to 700, 400 to 650, 400 to 600, 400 to 550, 400 to 500, 400 to 450, 450 to 900, 450 to 850, 450 to 800, 450 to 750, 450 to 700, 450 to 650, 450 to 600, 450 to 550, 450 to 500, 500 to 900, 500 to 850, 500 to 800, 500 to 750, 500 to 700, 500 to 650, 500 to 600,having from 500 to 550, 550 to 900, 550 to 850, 550 to 800, 550 to 750, 550 to 700, 550 to 650, 550 to 600, 600 to 900, 600 to 850, 600 to 800, 600 to 750, 600 to 700, 600 to 650, 650 to 900, 650 to 850, 650 to 800, 650 to 750, 650 to 700, 700 to 900, 700 to 850, 700 to 800, 700 to 750, 750 to 900, 750 to 850, 750 to 800, 800 to 900, 850 to 900, or 850 to 900 ethylene oxide units). In some embodiments, one or more cargo molecules consist of a PEG moiety having about 455 ethylene oxide units (a molecular weight of about 20 kilodaltons (kDa)). In some embodiments, the PEG moiety has a molecular weight of about 2 kilodaltons. In some embodiments, the PEG moiety has a molecular weight of about 20 kilodaltons. In some embodiments, the PEG moiety has a molecular weight of about 40 kilodaltons. The PEG moieties described herein can be linear or branched. The PEG moiety can be discrete (monodisperse) or non-discrete (polydisperse). PEG moieties for use as PK enhancing cargo molecules are commercially available. In some embodiments, one or more cargo molecules include a PEG moiety that can act as a PK modulator or enhancer, as well as different cargo molecules, such as a pharmaceutically active ingredient or compound.,

[0171] The αvβ6 integrin ligands described include salts or solvates. Solvates of the αvβ6 ligand are interpreted to mean the addition of inert solvent molecules to the αvβ6 integrin ligand formed by their mutual attraction. Solvates are, for example, monohydrates or dihydrates, or addition compounds with alcohols such as methanol or ethanol.

[0172] Free amino or hydroxyl groups can be provided as substituents of the αvβ6 integrin ligand having the corresponding protecting groups.

[0173] The ανβ6 integrin ligand also includes, for example, derivatives, i.e., ανβ6 integrin ligands modified with, for example, an alkyl or acyl group, a sugar, or an oligopeptide, which are cleaved either in vitro or in vivo.

[0174] In some embodiments, the ανβ6 integrin ligands disclosed herein facilitate the delivery of cargo molecules to the cytosol of cells presenting ανβ6 integrin on their surface, either via ligand-mediated endocytosis, pinocytosis, or by any other means. In some embodiments, the ανβ6 integrin ligands disclosed herein facilitate the delivery of cargo molecules to the plasma membrane of cells presenting ανβ6 integrin.

[0175] In some embodiments, the ανβ6 integrin ligand comprises a structure represented by:

[0176] [Chemical formula] (SEQ ID NO: 109) (Formula IX)

[0177] Wherein Z comprises an amine-terminal cap (e.g., any of the amine-terminal caps described herein or known in the art), and R 5 and R 6 are the side chains of amino acids Xaa 1 and Xaa 2 respectively.

[0178] In some embodiments, the ανβ6 integrin ligand has the following structure, wherein Z and R 1 are as defined herein for Formulas III and IV, and R 7 is OH, J, J-R 1 , J-Rx-R 2 , or Y-Rx-R 3 (each of which is as defined herein for Formulas III and IV):

[0179] [Chemistry] (SEQ ID NO: 110) (Formula X) [Chemistry] (SEQ ID NO: 111) (Formula XI) [Chemistry] (SEQ ID NO: 112) (Formula XII) [Chemistry] (SEQ ID NO: 113) (Formula XIII) [Chemistry] (SEQ ID NO: 114) (Formula XIV) [Chemistry] (SEQ ID NO: 115) (Formula XV) [Chemistry] (SEQ ID NO: 116) (Formula XVI)

[0180] In some embodiments, the described αvβ6 ligand showed increased serum stability compared to the naturally occurring αvβ6 integrin-binding peptide RGDLATLRQL (SEQ ID NO: 1). As shown in the examples herein, only about 5% of the naturally occurring peptide RGDLATLRQL (SEQ ID NO: 1) was detectable in mouse plasma after incubation at 37° C. for 4 hours. The peptide RGDLATLRQL (SEQ ID NO: 1) was not detectable in mouse plasma after 8 hours at 37° C. In some embodiments, the αvβ6 integrin ligands disclosed herein have shown that more than 20% of the ligand remains detectable by HPLC in mouse plasma after incubation at 37° C. for 12 hours. Having increased serum stability from the native peptide, the described αvβ6 integrin ligands retained binding to the αvβ6 integrin (affinity for the αvβ6 integrin).

[0181] Pharmaceutical composition In some embodiments, the present disclosure provides a pharmaceutical composition comprising, consisting of, or consisting essentially of one or more of the αvβ6 integrin ligands disclosed herein.

[0182] As used herein, a "pharmaceutical composition" contains a pharmacologically effective amount of an active pharmaceutical ingredient (API) and optionally one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than the active pharmaceutical ingredient (API, therapeutic product) intentionally included in the drug delivery system. An excipient does not exert, or is not intended to exert, a therapeutic effect at the intended dosage. Excipients assist in a) processing the drug delivery system during manufacture, b) protecting, supporting, or enhancing the stability, bioavailability, or patient acceptability of the API, c) assisting in product identification, and / or d) enhancing any other attribute of the overall safety and effectiveness of the delivery of the API during storage or use. Pharmaceutically acceptable excipients may or may not be inert substances.

[0183] Excipients include, but are not limited to: absorption enhancers, antiadhesion agents, antifoaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, flow promoters, humectants, lubricants, oils, polymers, preservatives, saline solutions, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickeners, isotonic agents, vehicles, water repellents, and wetting agents.

[0184] The pharmaceutical compositions described herein can include other additional components commonly found in pharmaceutical compositions. In some embodiments, the additional component is a pharmaceutically active substance. Pharmaceutically active substances include, but are not limited to: anti-itch agents, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.), small molecule drugs, antibodies, antibody fragments, aptamers, and / or vaccines.

[0185] The pharmaceutical compositions can also include preservatives, solubilizing agents, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for changing osmotic pressure, buffers, coating agents, or antioxidants. They can also include other therapeutically valuable agents.

[0186] The pharmaceutical composition can be administered in many ways depending on whether local or systemic treatment is desired and depending on the area to be treated. Administration can be by any method generally known in the art, for example, but not limited to, topically (e.g., by transdermal patch), intranasally (e.g., by inhalation or ventilation of a powder or aerosol including those by nebulizer, intratracheal, intranasal), epidermally, transdermally, orally or parenterally. Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous (e.g., via an implantable device), intracranial, intrasubstantial, intrathecal, and intraventricular administration. In some embodiments, the pharmaceutical composition described herein is administered by subcutaneous injection. The pharmaceutical composition can be administered orally, for example, in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions. Administration can also be rectally, for example, using suppositories; topically or transdermally, for example, using ointments, creams, gels, or solutions; or parenterally, for example, using injectable solutions.

[0187] A pharmaceutical composition suitable for injectable use includes a sterile aqueous solution (if water-soluble), a dispersion, and a sterile powder for the immediate preparation of a sterile injectable solution or dispersion. In the case of intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), or phosphate-buffered saline. It should be stable under the conditions of manufacture and storage and should be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion, and by the use of surfactants. In many cases, it will be preferable to include in the composition isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of injectable compositions is brought about by including in the composition agents that delay absorption, for example, aluminum monostearate and gelatin.

[0188] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in an appropriate solvent with one or a combination of the ingredients enumerated above, and, as required, followed by sterile filtration. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle that includes a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and freeze-drying, whereby a powder of the active ingredient plus any additional desired ingredients is obtained from its previously sterile-filtered solution.

[0189] Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of any of the ligands described herein, which can be in the form of a microcrystalline form, for example, an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems can also be used to present any of the ligands described herein for both intra-articular and ophthalmic administration.

[0190] The active compound can be prepared with a carrier that will protect the compound from rapid excretion from the body, such as, for example, in a controlled release formulation including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, as described, for example, in U.S. Patent No. 4,522,811.

[0191] The pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to: antipruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). As used herein, "pharmacologically effective amount", "therapeutically effective amount", or simply "effective amount" refers to the amount of a pharmaceutically active agent to bring about a pharmacological, therapeutic, or prophylactic result.

[0192] Pharmaceuticals containing the αvβ6 ligand are also an object of the present invention, as are methods for manufacturing such pharmaceuticals, which include formulating one or more compounds containing the αvβ6 ligand, and, if desired, one or more other therapeutically valuable substances, into a dosage form suitable for administration to a human subject.

[0193] Cells, tissues, and non-human organisms Cells, tissues, and non-human organisms containing at least one of the αvβ6 ligands described herein are contemplated. The cells, tissues, or non-human organisms are made by delivering the αvβ6 ligand to the cells, tissues, or non-human organisms by any means available in the art. In some embodiments, the cells are mammalian cells including, but not limited to, human cells.

[0194] The αvβ6 ligands described herein, and pharmaceutical compositions containing the disclosed αvβ6 ligands, may be packaged or contained in a kit, container, pack, or dispenser. The αvβ6 ligands and pharmaceutical compositions containing the αvβ6 ligands may be packaged in pre-filled syringes or vials.

[0195] The embodiments and items provided above are, from here on, illustrated by the following non-limiting examples.

Example

[0196] Example Example 1. Synthesis of αvβ6 Ligand for Serum Stability Test The Chem-Matrix Rink Amide resin was placed in a fritted polypropylene syringe and stirred in DCM for 30 minutes before use. The following standard solid-phase peptide synthesis conditions were used. Fmoc deprotection was carried out by immersing the resin in 40 ml of piperidine:DMF solution (20:80 v / v) per 1 mmol of resin for 20 minutes. Amide coupling was performed by immersing the resin in 4 molar equivalents of Fmoc-amino acid, 4 molar equivalents of HBTU, and 10 molar equivalents of diisopropylethylamine in DMF at a 0.1 M concentration of Fmoc-amino acid in DMF for 40 minutes. Fmoc-Dap(DNP)-OH was used to attach the DNP chromophore to the resin and the peptide was synthesized from the Dap α-amino group. Cleavage from the resin was carried out in trifluoroacetic acid solution for 2 hours. The solvent was reduced to 10% of the original volume via pressurized air and precipitated with Et2O. The identity of the product was confirmed by micro-cleavage via TFA and analytical HPLC-MS. Next, the peptide was purified to >95% purity on a preparative scale Shimadzu HPLC using a Supelco "Discovery BIO" wide pore C18 column (25 cm × 21 mm, 10 μm particles) eluting at a linear gradient of approximately 1 ml / min. The purity was evaluated over 50 minutes using an analytical Shimadzu HPLC equipped with a Waters XBridge BEH130 C18 column (250 mm × 6.6 mm, 5 μm particles) using 10 - 90% B solvent. Solvent A represents H2O:F3CCO2H 100:0.1 v / v and solvent B represents CH3CN:F3CCO2H 100:0.1 v / v.

[0197]

Chem.

[0198] Example 2. Serum stability of the αvβ6 ligand The serum stability of the αvβ6 ligand was tested by incubating the αvβ6 ligand in mouse serum and analyzing the percentage of undigested peptide at various time points. The undigested αvβ6 ligand was determined by analytical HPLC. Individual stock solutions of the αvβ6 ligand were prepared by dissolving the peptide in H2O at a concentration >10 mg / ml. The concentration of the αvβ6 ligand was evaluated using UV / Vis absorption (DNP: λ = 365, ε = 17300 M -1 Cm -1 ). The αvβ6 ligand was diluted to 1 mg / ml ligand in 90% mouse plasma and placed in an incubator at 37 °C. At given time points (4, 8, 12, and 24 hours), samples were injected onto an analytical HPLC (Shimadzu HPLC) equipped with a Waters XBridge BEH130 C18 column (250 mm × 6.6 mm, 5 μm particles) using a gradient of 10 - 90% solvent B over 50 minutes. Solvent A represents H2O:F3CCO2H 100:0.1 v / v; solvent B represents CH3CN:F3CCO2H 100:0.1 v / v.

[0199] The percent of ligand remaining after serum incubation was calculated using the following formula:

[0200]

Equation

[0201] Where the area at t = 0 was the peak area under the ligand immediately after dilution of the ligand in plasma, and the area at t = x was the peak area under the peptide at time = x.

[0202] Each peptide was covalently linked to the cargo molecule PEG8 - Dap(DNP). Next, PEG8 - Dap(DNP) was used to facilitate the analysis.

[0203]

Chemical Structure

[0204] The peptide derivative was conjugated to the cargo molecule PEG8-Dap(DNP) and incubated in mouse serum at 37 °C for 4, 8, 12, or 24 hours. The stability of the peptide derivative was measured by HPLC. The data are shown in Table 1 below (the amino-terminal cap and Xaa 1 Xaa 2 is underlined):

[0205]

Table 1

[0206] As shown here, the presence of the amino-terminal cap (Z) can provide increased serum stability. Furthermore, as shown here, Xaa in the formula disclosed herein 1 Xaa 2 and / or J (e.g., Xaa 3 and Xaa 4 ) also provides increased serum stability compared to the native peptide of SEQ ID NO: 1.

[0207] Example 3. Integrin Binding of αvβ6 Ligand A. αvβ6 Ligand-Cargo Molecule Conjugation Each αvβ6 ligand was conjugated to a reversibly modified 1170-100B polymer cargo molecule. The 1170-100B polymer cargo molecule (a 56:44 ethoxyethylamine acrylate:propyl acrylate copolymer having an MW of about 45000) was labeled with Cy5 (NHS linker) and, in 50 mM HEPES pH 9.0 buffer for 1 hour at room temperature, with aldehyde-PEG 24 -ACit, in a weight ratio of 2:1 (polymer:aldehyde-PEG 24 -ACit), to form (aldehyde-PEG 24 -ACit) n -1170-100B (where n is an integer greater than 0). Typically, n was about 10.

[0208] [Chemical formula] Aldehyde-PEG 24 -ACit(n = 24)

[0209] Next, the aldehyde-PEG 24 -ACit-modified polymer was reacted with PEG 12 -ACit at a weight ratio of 1:8 (polymer:PEG 12 -ACit) in 50 mM HEPES, pH 9.0 buffer at room temperature for 1 hour to form (aldehyde-PEG 24 -ACit) n -1170-100B-(CitA-PEG 12 ) m (where m is an integer greater than 0).

[0210] [Chemical formula] PEG12-ACit(n = 11)

[0211] Next, the modified polymer was purified using a sephadex G-50 spin column and the concentration was determined:

[0212] [Mathematics]

[0213] Each αvβ6 ligand was modified with HyNic to facilitate conjugation to cargo molecules. The purified polymer was combined with αvβ6 ligand-(PEG)8-K-HyNic in 50 mM NaOAC-HOAc, pH 5.0 buffer at room temperature overnight at a weight ratio of 1:1.9 (polymer:αvβ6 ligand) to form an αvβ6 ligand-polymer conjugate. The αvβ6 ligand-polymer conjugate was purified using a sephadex G-50 spin column.

[0214] [Chemical formula] (PEG)8-K-HyNic

Chem.

[0215] For the bis-arylhydrazone bond, the absorbance of the αvβ6-polymer conjugate at 354 nm was measured using an extinction coefficient of 2.9×10 1 M -1 cm -1 to quantify the conjugation efficiency.

[0216]

Math.

[0217] The αvβ6-polymer conjugate was diluted to the desired concentration with an isotonic glucose solution for further analysis.

[0218] B. αvβ6 Ligand Binding (Flow Cytometry Analysis) To evaluate the specificity of the binding of an ανβ6 ligand to ανβ6 integrin, each ανβ6 ligand or negative control peptide was conjugated to a Cy5-labeled polymer (as described above) and evaluated for binding to cells. HUH7 (human stem cells) and SKOV3 (human ovarian cancer) cells were determined to exhibit very low ανβ6 cell surface expression and were used as negative control cell lines. H2009 (human lung adenocarcinoma) and CAPAN-2 (human pancreatic adenocarcinoma) cells functioned as ανβ6 positive control cell lines. Cells were detached from culture flasks using Accutase, washed with PBS, and seeded at 200,000 cells in 200 μl of complete medium (culture medium containing additives and fetal bovine serum) in 5 ml polystyrene round-bottom tubes. ανβ6 ligand-polymer conjugates or polymer conjugates without ligand were added to the cells at 5 μg / ml (polymer-Cy5 concentration), mixed, and incubated at 37 °C for 3 hours. Incubation at 37 °C promotes ligand / receptor interactions, which can result in static binding to the extracellular cell surface and / or internalized ligand / receptor complexes. The mixtures were resuspended at 1-hour intervals. After 3 hours of incubation, the cells were washed twice with 4 ml of cold buffer (PBS-2% FCS) and resuspended in 200 μl of buffer containing 10 μΜ SYTOX blue staining for live / dead cell gating. Samples were analyzed on a BD Biosciences Canto II cytometer equipped with violet (405 nm), blue (488 nm), and red (633 nm) lasers.

[0219] Live cells were first gated as a SYTOX blue-negative population using the violet laser on the C detector. These live cells were then evaluated for conjugate binding / uptake using the red laser as the mean fluorescence intensity (MFI) of the Cy5 fluorophore. Data analysis was performed using FlowJo v10.1 software. The specific MFI Ratio (sMFIr) for each ανβ6 ligand was determined by the following equation:

[0220] [Number]

[0221] As shown in Table 2 below, only the peptide Ac-RGDLAAbuLCitAibL showed moderate binding to SKOV3 cells. This peptide did not show significant binding to HUH7 cells. Therefore, none of the tested peptides showed specific binding. The RGD-free negative control peptides AcRGαMeDLAAbuLCitAib, AcRGDαMeLAAbuLCitAib, RGELATLRQL, AcCitGDLATLCitQL, AcK(Me)2GDLATLRQL, and AcR(Me)2GDLATLRQL did not show significant binding to the αvβ6 integrin-positive control cells of H2009 or CAPAN-2, indicating a lack of affinity for αvβ6 integrin. Most of the other peptides showed binding to H2009 or CAPAN-2 cells, and such binding was equal to or greater than that of the native peptides RGDLATLRQL and RGDLATL, indicating good affinity for αvβ6 integrin.

[0222]

Table 2-1

Table 2-2

Table 2-3

[0223] The specific abbreviations of the non-standard amino acids and other chemical groups identified in the above table have the following chemical structures:

[0224]

Chemistry

Chemistry

[0225] Example 4. In Vivo Intratracheal Administration of an RNAi Agent Targeting Alpha-ENaC Conjugated to an αvβ6 Integrin Ligand in Rats A double-stranded oligonucleotide composition (i.e., a type of RNAi agent) containing a sense strand and an antisense strand each less than 26 nucleotides was synthesized by the phosphoramidite technique on a solid phase according to general procedures known in the art and commonly used in oligonucleotide synthesis. The synthesis of the RNAi agents herein was performed on a commercially available solid support made of controlled pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston, PA, USA) using either MerMade96E® (Bioautomation), MerMadel2® (Bioautomation), or OP Pilot 100 (GE Healthcare) depending on the scale for synthesis. All RNA and 2'-modified RNA phosphoramidites were purchased commercially (Thermo Fisher Scientific (Milwaukee, WI, USA)). For cleavage and deprotection, after completion of solid-phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt% methylamine in water and 28% ammonium hydroxide solution (Aldrich) at 30 °C for 1.5 hours. The solution was evaporated and the solid residue was reconstituted with water. For purification, the crude oligomers were purified by anion-exchange HPLC using a TSKgel SuperQ-5PW 13 μm column and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20% acetonitrile. Buffer B was the same as Buffer A with the addition of 1.5 M sodium chloride. A UV trace at 260 nm was recorded. Appropriate fractions were pooled and then subjected to size-exclusion HPLC using a GE Healthcare XK 26 / 40 column packed with Sephadex G-25 fine in a running buffer of 100 mM ammonium bicarbonate, pH 6.7 and 20% acetonitrile. For annealing, the complementary strands were mixed by combining equimolar RNA solutions (sense and antisense) in 1× PBS (phosphate-buffered saline, 1×, Corning, Cellgro) to form the RNAi agents. Some of the RNAi agents were lyophilized and stored at -15 to -25 °C.The double-stranded concentration was measured by measuring the absorbance of the solution with a UV-Vis spectrometer in 1×PBS. Next, the double-stranded concentration was determined by multiplying the solution absorbance at 260 nm by the conversion coefficient and the dilution coefficient. Unless otherwise specified, all conversion coefficients were 0.037 mg / (mL·cm). For some experiments, the conversion coefficient was calculated from the experimentally determined extinction coefficient.

[0226] The RNAi agent synthesized for Example 4 included an antisense strand, and such antisense strand had a nucleobase sequence that was at least partially complementary to the gene that expresses the alpha subunit of the amiloride-sensitive epithelial sodium channel (commonly called alpha-ENaC or SCNN1A). The RNAi agent for alpha-ENaC was designed to be able to degrade or inhibit the translation of the messenger RNA (mRNA) transcript of alpha-ENaC in a sequence-specific manner, thereby inhibiting the expression of the alpha-ENaC gene. The RNAi agent was composed of modified nucleotides and two or more non-phosphodiester bonds.

[0227] On the first and second days of the test, male Sprague-Dawley rats were intratracheally administered a dose of 200 microliters via a microsyringe device (Perm Century, Philadelphia, PA), which included the following treatment groups: (1) 5% dextrose in water vehicle (D5W); (2) an alpha-ENaC RNAi agent without a ligand (a "naked RNAi agent") formulated in 5% dextrose and containing 1.5 mg / kg; (3) an alpha-ENaC RNAi agent conjugated to the alpha v beta 6 integrin ligand of Figure 3 (having an alpha v beta 6 integrin ligand conjugated at the 5' end of the sense strand) formulated in 5% dextrose and containing 1.5 mg / kg; or (4) 1.5 mg / kg of the structure Ac-RG to function as a negative control ligand EAn alpha-ENaC RNAi agent conjugated to an inactivated αvβ6 integrin ligand having LAAbuL-CitAibL (SEQ ID NO: 132). The aspartic acid (D) in the "RGD" motif is thought to be necessary for the ligand to bind to the alpha-v integrin receptor, and a ligand having a substitution of glutamic acid (E) has a significantly reduced av integrin binding affinity. The same alpha-ENaC RNAi agent was used in groups 2, 3, and 4.

[0228] The αvβ6 integrin ligand used was synthesized as a TFP ester (as shown in Figure 1) using general peptide synthesis techniques similar to those described in Example 1 of this specification, which are well known in the art, except that resin cleavage was achieved using 20% HFIP (hexafluoroisopropanol) in DCM (dichloromethane) for 30 minutes to 1 hour instead of TFA cleavage. The 5´ end of the sense strand of the RNAi agent was modified with C6 amine (-NH2). Next, at room temperature, using 3 equivalents of the TFP-ester αvβ6 integrin ligand in DMSO:water 9:1 and an excess of triethylamine as a base, the TFP-ester αvβ6 integrin ligand was conjugated to the amino group located at the 5´ end of the modified sense strand of the RNAi agent. Purification was performed by adding ACN to the solution, precipitating the product, and drying it under high vacuum.

[0229] Four rats per group were dosed. The rats were euthanized on the 5th day of the test, and total RNA was isolated after collection and homogenization from both lungs. The mRNA abundance of alpha-ENaC was quantified by probe-based quantitative PCR, normalized to GAPDH expression, and expressed as a ratio to the vehicle control group (geometric mean, + / - 95% confidence interval).

[0230]

Table 3

[0231] As shown in Table 3 above, the αvβ6 ligand of Figure 3 conjugated to the alpha-ENaC RNAi agent showed an increased relative knockdown of alpha-ENaC mRNA (approximately 78% knockdown) in vivo compared to the naked RNAi agent (46% knockdown) and the RNAi agent conjugated to the RGE-control ligand of the alpha-ENaC lung target (57% knockdown).

[0232] Example 5. In Vivo Intratracheal Administration of an Alpha-ENaC Targeting RNAi Agent Conjugated to an Avβ6 Integrin Ligand in Rats An alpha-ENaC RNAi agent similar to that described in Example 4 was synthesized according to the same synthetic procedure. On test days 1 and 2, male Sprague-Dawley rats were administered a 200 microliter dose via a microspray device (Penn Century, Philadelphia, PA), which included the following dosing groups: (1) D5W vehicle; (2) an RNAi agent without ligand (a "naked RNAi agent") formulated in D5W at 1.5 mg / kg; (3) an alpha-ENaC RNAi agent conjugated to the alpha v beta 6 integrin ligand of FIG. 3 at 1.5 mg / kg in D5W; or (4) an alpha-ENaC RNAi agent conjugated to a trispecific alpha v beta 6 integrin ligand having the structure shown in FIG. 11 at 1.5 mg / kg. The RNAi agent was designed to inhibit the expression of the alpha-ENaC gene. The same alpha-ENaC RNAi agent was used in groups 2, 3, and 4. The 5' end of the sense strand of the RNAi agent was modified with C6 amine (-NH2) as in Example 4. The alpha v beta 6 integrin ligand of FIG. 3 was synthesized according to the same procedure as in Example 4 and conjugated to the alpha-ENaC RNAi agent. When conjugating to the avb6 ligand shown in FIG. 11, the alpha-ENaC-RNAi agent was first functionalized with DBCO-PEG5-NHS ester by conjugating to the 5' amine-functionalized end of the sense strand using triethylamine as the base. The trispecific avb6 integrin ligand was synthesized to have a PEG-azide reactive group as shown in FIG. 10. After precipitation in a phosphate buffered saline / acetonitrile solvent system, the trispecific avb6 integrin ligand was conjugated to the RNAi agent using copper-free click chemistry.

[0233] Five rats per group were dosed. The rats were euthanized on test day 5, and total RNA was isolated after collection and homogenization from both lungs. The target mRNA abundance was quantified by probe-based quantitative PCR, normalized to GAPDH expression, and expressed as a ratio to the vehicle control group (geometric mean, + / - 95% confidence interval).

[0234]

Table 4

[0235] As shown in Table 4 above and as shown in FIG. 11, by conjugating three αvβ6 ligands (forming a "three-seat" ligand) to an alpha-ENaC RNAi agent, in vivo, compared to an RNAi agent conjugated to one αvβ6 ligand (FIG. 3) (71%) and a naked RNAi agent (66%), an increased relative knockdown (about 79%) was shown.

[0236] Example 6. In Vivo Intratracheal Administration of an Alpha-ENaC Targeting RNAi Agent Conjugated to an Αvβ6 Integrin Ligand in Rats An alpha-ENaC RNAi agent similar to that described in Example 4 was synthesized according to the same synthetic procedure. On test days 1 and 2, male Sprague-Dawley rats were administered a 200 microliter dose via a microsyringe device (Perm Century, Philadelphia, PA), which included the following treatment groups: (1) D5W vehicle; (2) an RNAi agent without ligand (3 mg / kg) formulated in D5W ("naked RNAi agent"); or (3) an alpha-ENaC RNAi agent conjugated to the αvβ6 integrin ligand of FIG. 3 (3.0 mg / kg) formulated in D5W. The alpha-ENaC RNAi agent and the αvβ6 integrin ligand were synthesized and conjugated according to the same procedure described in Example 4.

[0237] Five rats per group were dosed. The rats were euthanized on test day 5, and total RNA was isolated after collection and homogenization from both lungs. The target mRNA abundance was quantified by probe-based quantitative PCR, normalized to GAPDH expression, and expressed as a ratio to the vehicle control group (geometric mean, + / - 95% confidence interval).

[0238]

Table 5

[0239] As shown in Table 5 above, the αvβ6 ligand of Figure 3 conjugated to the RNAi agent showed an increased relative knockdown (about 83% knockdown) in vivo compared to the naked RNAi agent targeting the lung (about 51% knockdown).

[0240] Example 7. In Vivo Oropharyngeal Aspiration of an RNAi Agent Targeting a Gene Expressed in the Lung Conjugated to an αvβ6 Integrin Ligand in Rats An alpha-ENaC RNAi agent similar to that described in Example 4 was synthesized according to the same synthetic procedure. On Day 1 of the study, male Sprague-Dawley rats were administered a 200 microliter dose by oropharyngeal aspiration, which included the following treatment groups: (1) isotonic saline; (2) an RNAi agent targeting alpha-ENaC conjugated to 0.5 mg / kg of the αvβ6 integrin ligand of Figure 3 formulated in isotonic saline; or (3) an RNAi agent targeting alpha-ENaC conjugated to 0.5 mg / kg of the αvβ6 integrin ligand of Figure 5 formulated in isotonic saline. The same alpha-ENaC RNAi agent was used in Groups 2 and 3. The 5´ end of the sense strand of the RNAi agent was modified with C6 amine (-NH2) as described in Example 4. The alpha-ENaC RNAi agent and αvβ6 integrin ligand of Figure 3 were synthesized and conjugated according to the same procedure described in Example 4. The αvβ6 integrin ligand of Figure 5 was synthesized as the TFP-ester and the N-terminus of the αvβ6 integrin ligand was protected with an fmoc group. Next, conjugation to the RNAi agent was performed in the same manner as described in Example 4, followed by fmoc deprotection using triethylamine as the base.

[0241] Five rats per group were dosed. Rats were euthanized on day 9 of the study, and total RNA was isolated after collection and homogenization from both lungs. The amount of target mRNA was quantified by probe-based quantitative PCR, normalized to GAPDH expression, and expressed as a ratio to the vehicle control group (geometric mean, + / - 95% confidence interval).

[0242]

Table 6

[0243] As shown in Table 6 above, both the αvβ6 ligands of FIGS. 3 and 5 conjugated to the RNAi agent showed knockdown of the alpha-ENaC lung target in vivo.

[0244] Example 8. In Vivo Intratracheal Administration of an RNAi Agent Targeting Alpha-ENaC Conjugated to an αvβ6 Integrin Ligand and a Poly-L-Lysine Scaffold in Rats An α-ENaC RNAi agent similar to that described in Example 4 was synthesized according to the same synthetic procedure. On test days 1 and 2, male Sprague-Dawley rats were administered one of the following 200 microliter doses via a microspray device (Penn Century, Philadelphia, PA): (1) D5W (5% dextrose in water); (2) an RNAi agent without a ligand (a "naked RNAi agent") containing 0.5 mg / kg formulated in D5W; (3) a naked RNAi agent containing 1.5 mg / kg formulated in D5W; (4) a naked RNAi agent containing 5 mg / kg formulated in D5W; (5) an α-ENaC RNAi agent conjugated via a poly-L-lysine (PLL) scaffold to the αvβ6 integrin ligand of FIG. 4 at 0.5 mg / kg formulated in D5W; (6) an α-ENaC RNAi agent conjugated via a PLL scaffold to the αvβ6 integrin ligand of FIG. 4 at 1.5 mg / kg formulated in D5W; or (7) an α-ENaC RNAi agent conjugated via a PLL scaffold to the αvβ6 integrin ligand of FIG. 4 at 5 mg / kg formulated in D5W. The RNAi agent was designed to inhibit the expression of the α-ENaC gene. The same α-ENaC RNAi agent was used for groups 2 to 7. The αvβ6 integrin ligand was first synthesized as the TFP-ester (shown in FIG. 2). The PLL scaffolds used in groups 5, 6, and 7 of Example 8 were approximately 100 L-lysine monomer units (approximately 12 kilodaltons). The poly-L-lysine polymer was modified with 3 equivalents of SMPT (4-succinimidyl oxycarbonyl-α-methyl-α(2-pyridyldithio)toluene), and the 5'-amine of the sense strand of the RNAi agent (C6 amine modification) was modified with SATA (N-succinimidyl S-acetylthioacetate). Next, the αvβ6 integrin ligand of FIG. 2 (15 equivalents) was added as a solid and stirred for 1 hour. Next, a protease-cleavable functionalized alanine-citrulline-PEG 12(10 equivalents; functionalization with para-nitrophenyl carbonate) was added. After 15 minutes, the SATA-modified RNAi agent (1 equivalent) was added dropwise while maintaining the pH at 8.6. The remaining lysine groups were functionalized with protease-cleavable functionalized alanine-citrulline-PEG 12 The product was purified by tangential flow filtration.

[0245] Five rats per group were dosed. The rats were euthanized on day 5 of the study, and total RNA was isolated after collection and homogenization from both lungs. The target mRNA abundance was quantified by probe-based quantitative PCR, normalized to GAPDH expression, and expressed as a ratio to the vehicle control group (geometric mean, + / - 95% confidence interval). The data are reported in the graph of Figure 12.

[0246] As shown in Figure 12, the αvβ6 ligand of Figure 4 conjugated to a poly-L-lysine scaffold and an RNAi agent showed increased relative knockdown at all three dose levels compared to the naked RNAi agent (68% knockdown vs. 47% knockdown at the 0.5 mg / kg dose, 78% knockdown vs. 47% knockdown at the 1.5 mg / kg dose, and 86% knockdown vs. 75% knockdown at the 5 mg / kg dose).

[0247] Example 9. Selective Uptake of Labeled αvβ6 Ligand Conjugates by Primary Epithelial Cells In Vitro Primary human lung epithelial cells, endothelial cells, and smooth muscle cells were cultured and exposed for 24 hours to: (1) a Cy3-labeled (red) polyacrylate polymer scaffold without ligand (ligand-free-conjugate); or (2) a Cy3-labeled (red) polyacrylate polymer scaffold conjugated to the αvβ6 integrin ligand of Figure 7 (αvβ6 ligand-conjugate). The cells were stained with FITC-phalloidin (F-actin, green) and Hoechst stain (DNA, blue) and imaged by fluorescence microscopy.

[0248] Fluorescent microscope images were prepared using standard methods known in the art. The fluorescent images showed that a Cy3-labeled conjugate without an αvβ6 integrin ligand was not internalized by any cell type. However, the Cy3 conjugate with the αvβ6 ligand in FIG. 7 was internalized by primary lung epithelial cells (as shown by the accumulation of the red signal within the endosomal compartment of primary lung epithelial cells in the image), but not by primary endothelial and smooth muscle cells. This indicates that the αvβ6 integrin ligand disclosed herein can be selectively internalized by epithelial cells expressing αvβ6 integrin.

[0249] Example 10. Selective Uptake of Labeled αvβ6 Ligand Conjugate by Epithelial Tissue In Vivo C57bl / 6 mice were injected with the following at an intravenous dose of 120 micrograms: (1) a Cy3-labeled (red) polyacrylate polymer scaffold without a ligand (ligand-free conjugate) or (2) a Cy3-labeled (red) polyacrylate polymer scaffold conjugated to the αvβ6 integrin ligand in FIG. 7 (αvβ6 ligand-conjugate) or (3) the structure Ac-RG E A Cy3-labeled (red) polyacrylate polymer scaffold conjugated to an inactivated αvβ6 integrin ligand having LAAbuL-CitAibL (SEQ ID NO: 132), such inactivated αvβ6 integrin ligand being used as a negative control ligand as previously described in Example 4. Twenty-four hours after injection, the mice were sacrificed and tissues were harvested, fixed, processed, and sectioned. Tissue sections were stained with FITC-phalloidin (F-actin, green) and Hoechst stain (DNA, blue) and imaged by fluorescence microscopy.

[0250] A. Pulmonary Bronchial Epithelial Cells Fluorescence microscopy images of lung bronchial epithelial cells from the mice of Example 10 were prepared using standard methods. From these images, the Cy3-labeled conjugate with the αvβ6 ligand of FIG. 7 (indicated by the red marks in the images) was selectively internalized into the endosomal compartment by lung bronchial epithelial cells in vivo, while no epithelial internalization was essentially observed in constructs containing ligand-free or RGE-ligand control conjugates (i.e., no red color was seen in these images).

[0251] B. Renal epithelial tissue Fluorescence microscopy images of renal tubular epithelial tissue from the mice of Example 10 were prepared using standard methods. From these images, the Cy3-labeled conjugate with the αvβ6 ligand of FIG. 7 was selectively internalized into the endosomal compartment by renal tubular epithelial cells in vivo (indicated by the red marks in the images), while no epithelial internalization was essentially observed in the ligand-free control conjugate.

[0252] C. Gastrointestinal tract epithelial cells Fluorescence microscopy images of rental epithelial tissue from the mice of Example 10 were prepared using standard methods. The Cy3-labeled conjugate with the αvβ6 ligand of FIG. 7 was selectively internalized into the endosomal compartment by GI tract epithelial cells in vivo in both the small intestine and the gallbladder (indicated by the red marks in the images), while no epithelial internalization was essentially observed in the ligand-free control conjugate.

[0253] Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative and not to limit the scope of the invention defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Claim 1 an αvβ6 integrin ligand, comprising the following: Z-RG 1 DLXaa 1 Xaa 2 L (SEQ ID NO: 85) (Formula I); or Z-RG 1 DLXaa 1 Xaa 2 L-J-R 1 (SEQ ID NO: 86) (Formula II); or Z-RG 1 DLXaa 1 Xaa 2 L-J-R 1 -R 2 (SEQ ID NO: 87) (Formula III); or (Z-RG 1 DLXaa 1 Xaa 2 L-J-R 1 ) n -R 3 (SEQ ID NO: 88) (Formula IV); or (Z-RG 1 DLXaa 1 Xaa 2 L-J-R 1 ) n -R 4 -(R 3 ) p (SEQ ID NO: 89) (Formula V); or Z-RG 1 DLXaa 1 Xaa 2 L-Xaa 3 Xaa 4 L-R 1 (SEQ ID NO: 92) (Formula VI); or (Z-RG 1 DLXaa 1 Xaa 2 L-J-R 1 ) n -R 4 -(R 2 ) p (SEQ ID NO: 105) (Formula VII); wherein, in the formula Z is an amino-terminal cap; R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1 is an L-α-amino acid, an L-β-amino acid, or an α,α-disubstituted amino acid; Xaa 2 is an L-α-amino acid, an L-β-amino acid, or an α,α-disubstituted amino acid; Xaa 3 is an L-α-amino acid, an L-β-amino acid, or an α,α-disubstituted amino acid; Xaa 4 is an L-α amino acid, an L-β amino acid, or an α,α-disubstituted amino acid; R 1 is optional and, if present, contains PEG and / or a linking group; J is optional and, when present, comprises one or more L-α amino acids, L-β amino acids, α,α-disubstituted amino acids, or combinations thereof; R 2 contains a reactive group or a protected reactive group; R 3 contains cargo molecules; n is an integer greater than 0; p is an integer greater than 0; and R 4 is optional and, when present, comprises a scaffold and / or a linking group, said scaffold and / or linking group comprising at least one binding site for each ligand present and at least one binding site for each cargo molecule present, an αvβ6 integrin ligand.

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