EA042923B1 - ALPHA-V BETA-6 INTEGRIN LIGANDS AND OPTIONS FOR THEIR APPLICATIONS - Google Patents

Description

Link to related applications

The present application claims priority under U.S. Provisional Application No. 62/415,752, filed Nov. 1, 2016, the contents of which are incorporated herein by reference.

The field of technology to which the invention belongs

Peptide ligands of alpha-v beta-6 (αvβ6) integrin are disclosed that are capable of targeting αγβ6 integrin and/or targeting cells that express αvβ6 integrin. The αvβ6 integrin ligands can be conjugated to one or more cargo molecules to facilitate delivery of cargo molecules to cells expressing the αγβ6 integrin, such as epithelial cells.

State of the art

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

Difficult to detect in the epithelium of normal healthy adults, αvβ6 integrin is overexpressed during wound healing and in various cancers (eg, colon, ovarian, endometrial, and gastric cancers) and is often associated with poor cancer prognosis. It has been shown that integrin αvβ6 can stimulate cell invasion and migration during metastasis and inhibit apoptosis. The αvβ6 integrin can also regulate the expression of matrix metalloproteinases (MMPs) and activate TGF-β 1. There is increasing evidence, primarily from in vitro studies, that suggests that the αvβ6 integrin may promote cancer. Thus, the αvβ6 integrin is attractive both as a tumor biomarker and potential therapeutic target and because of its role in matrix metalloproteinase (MMP) expression and TGF-P1 activation.

The essence of the invention

Described herein are novel, engineered, non-naturally occurring αvβ6 integrin peptide ligands (also referred to as αvβ6 ligands). The αvβ6 integrin ligands disclosed herein are stable in serum and have affinity for and can specifically bind to αvβ6 integrins. In addition, compositions that contain αvβ6 integrin ligands and methods of using the αvβ6 integrin ligands and the compositions described herein are described herein.

The αvβ6 integrin ligands described herein have improved stability compared to other known αvβ6 integrin binding peptides, such as the natural peptide RGDLATLRQL (SEQ ID NO: 1). While having increased serum stability, the novel αvβ6 ligands described herein retain binding (affinity for) the αvβ6 integrin.

In a first aspect, the present disclosure provides engineered, non-naturally occurring αvβ6 integrin ligands. In some embodiments, αvβ6 integrin ligands have the general formula

Z-RG ¹ DLXaa ¹ Xaa ² L (SEQ ID NO: 85) (Formula I) wherein Z is an amino terminal cap (eg, 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 represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art); A

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art).

In some embodiments, αvβ6 integrin ligands have the general formula Z-RG ¹ DLXaa ¹ Xaa ² LJR ¹ (SEQ ID NO: 86) (Formula II), wherein Z is an amino terminal cap (e.g., any of the amino terminal caps described herein or known in the art);

- 1 042923

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 represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

J is optional and, if present, contains one or more , 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 (for example, any of the L-α amino acids described herein or known in the art), L-β amino acids (for example, any of the L-β amino acids described herein document or known in the art), or α,α-disubstituted amino acids (eg, any of the α,α-disubstituted amino acids described herein or known in the art), or a combination thereof; And

R ¹ is optional and, if present, contains polyethylene glycol (PEG) and/or a linking group.

In some embodiments, the αvβ6 integrin ligands disclosed herein may contain a reactive group or a protected reactive group and have the general formula

Z-RG ¹ DLXaa ¹ Xaa ² LJR^R ² (SEQ ID NO:87) (Formula III) wherein Z is an amino terminal cap (eg, 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 represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

J is optional and, if present, contains one or more , 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 (for example, any of the L-α amino acids described herein or known in the art), L-β amino acids (for example, any of the L-β amino acids described herein document or known in the art), or α,α-disubstituted amino acids (eg, any of the α,α-disubstituted amino acids described herein or known in the art), or a combination thereof;

R ¹ is optional, and if present, contains polyethylene glycol (PEG) and/or a linking group; And

R ² contains a reactive group or a protected reactive group.

In some embodiments, the αγβ6 integrin ligand may be conjugated to one or more , 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 (eg any of the cargo molecules described herein or known in the art),

- 2 042923 wherein the αvβ6 integrin ligand has the general formula (Z-RG ¹ DLXaa ¹ Xaa ² LJ-R ¹ ) _n -R ³ (SEQ ID NO: 88) (Formula IV), in which Z represents an amino terminal cap (for example, 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 represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

J is optional and, if present, contains one or more , 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 (for example, any of the L-α amino acids described herein or known in the art), L-β amino acids (for example, any of the L-β amino acids described herein document or known in the art), or α,α-disubstituted amino acids (eg, any of the α,α-disubstituted amino acids described herein or known in the art), or a combination thereof;

R ¹ is optional, and if present, contains polyethylene glycol (PEG) and/or a linking group; n represents a number greater than 0 (for example, 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, 515, 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.

In some embodiments, the αvβ6 integrin ligand may be conjugated to one or more , 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 (for example, with any of the cargo molecules described herein or known in the art), while the αvβ6 integrin ligand has the general formula (Z-RG ¹ DLXaa ¹ Xaa ² LJR ¹ )nR ⁴ -(R ³ )P (SEQ ID NO: 89) (Formula V) _r wherein Z is an amino terminal cap (eg, 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 represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

J is optional and, if present, contains one or more , 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 (for example, any of the L-α amino acids described herein or known

- 3 042923 in this field), L-β amino acids (for example, any of the L-β amino acids described herein or known in the field) or α,α-disubstituted amino acids (for example, any of the α,α-disubstituted amino acids described herein or known in the art), or a combination thereof;

R ¹ is optional, and if present, contains polyethylene glycol (PEG) and/or a linking group; n represents a number greater than 0 (for example, 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 -thirty);

R ³ contains one or more cargo molecules;

p represents a number greater than 0 (for example, 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 -thirty); And

R ⁴ is optional and, if present, contains a backbone and/or a linking group that contains 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.

Within the scope of the invention, the amino terminal cap (shown as Z in the formulas herein) contains a chemical moiety that is capable of increasing and/or otherwise improving the protease resistance and/or serum stability characteristics of the natural RGDLATL peptide. Such improvements can be determined, for example, using methods widely known in the art, including without limitation, for example, by 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 provides for a protease-resistant acylation, sulfonylation, or alkylation of the N-terminal amine of the ανβ6 integrin ligand disclosed herein. In some embodiments, the amino terminal cap Z may be alkyl-CO, ArCO, alkyl-SO ₂ , ArSO ₂ , alkyl or aryl groups. In some embodiments, the alkyl groups may be either linear or branched aliphatic alkyl groups, and the aryl groups may be either aromatic or heteroaromatic groups. In some embodiments, the amino terminal cap Z can be, without limitation, CH3CO, CH ₃ CH ₂ CO, CH ₃ (CH ₂ ) ₂ CO, (CH ₃ ) ₂ CHCO, CH ₃ (CH ₂ ) ₃ CO, (CH ₃ ) ₂ CHCH ₂ CO, CH ₃ CH ₂ CH (CH ₃ )CO, (CH ₃ ) ₃ CCO, CH ₃ (CH ₂ ) ₄ CO, CH ₃ SO ₂ , CH ₃ CH ₂ SO ₂ , CH ₃ (CH ₂ ) ₂ SO ₂ , (CH ₃ ) ₂ CHSO ₂ , CH ₃ (CH ₂ ) ₃ SO ₂ , (CH ₃ ) ₂ CHCH ₂ SO ₂ , CH ₃ CH ₂ CH(CH ₃ )SO ₂ , (CH ₃ ) ₃ CSO ₂ , PhCO, PhSO ₂ , alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, methyl, ethyl, propyl, butyl, pentyl, NH2NH, PEG, guanidinyl, CH3OCH2CH2OCH2CH2CO, CH3O (CH2CH2O)2CH2CH2CO, CH3O(CH2CH2O)3CH2CH2CO, CH ₃ O (CH ₂ CH ₂ O) ₄ CH ₂ CH ₂ CO, CH ₃ O (CH ₂ CH ₂ O) ₅ CH ₂ CH ₂ CO, CH ₃ OCH ₂ CH ₂ OCH ₂ CO, CH ₃ O (CH ₂ CH ₂ O) ₂ CH ₂ CO, CH ₃ O (CH ₂ CH ₂ O) ₃ CH ₂ CO, CH ₃ O (CH ₂ CH ₂ O) ₄ CH ₂ CO, CH ₃ O (CH ₂ CH ₂ O) ₅ CH ₂ CO, CH ₃ OCH ₂ CH ₂ OCO, 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, CH3 CH2O(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.

In some embodiments, the amino terminal cap of Z is CH3CO. In some embodiments, the amino terminal cap of Z is CH ₃ CH ₂ CO. In some embodiments, the amino terminal cap of Z is CH ₃ (CH ₂ ) ₂ CO. In some embodiments, the amino terminal cap of Z is CH ₃ (CH ₂ ) ₃ CO. In some embodiments, the amino terminal cap of Z is CH3(CH2)4CO.

In some embodiments, the ανβ6 integrin ligands have 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 represents L-alanine; And

Xaa ^U is a non-standard amino acid.

In some embodiments, the ανβ6 integrin ligands have the general formula Z-RG'DLAAbuL (SEQ ID NO: 91) (Formula Id), wherein Z, R, G ¹ , D, and L are each as defined for Formula I herein.

- 4 042923 Research Institute;

A represents L-alanine; And

Abu is L-α-aminoα-hydrochloric acid (2-aminobutyric acid).

In some embodiments, αvβ6 integrin ligands have the general formula Z-RG ¹ DLXaa ¹ Xaa ² L-Xaa ³ Xaa ⁴ LR ¹ (SEQ ID NO: 92) (Formula VI), wherein Z represents 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 represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ³ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ⁴ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art); And

R ¹ is optional and, if present, contains polyethylene glycol (PEG) and/or a linking group.

In some embodiments, the αvβ6 integrin ligands have the general formula Z-RG ¹ DLAXaa ^u L-Xaa ³ Xaa ⁴ LR ¹ (SEQ ID NO: 93) (Formula VIb) in which Z, R, G ¹ , D, L and R ¹ each corresponds to the definition for formula VI in the present description;

A represents L-alanine;

Xaa ^u represents a non-standard amino acid;

Xaa ³ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art); And

Xaa ⁴ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art).

In some embodiments, the αvβ6 integrin ligands have the general formula Z-RG ¹ DLAAbuL-Xaa ³ Xaa ⁴ LR ¹ (SEQ ID NO: 94) (Formula Vic) wherein Z, R, G ¹ , D, L, and R ¹ are each corresponds to the definition for formula VI in the present description;

A represents L-alanine;

Abu is L-α-aminoα-hydrochloric acid (2-aminobutyric acid);

Xaa ³ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein

- 5 042923 or known in this field); And

Xaa ⁴ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art).

In some embodiments, αvβ6 integrin ligands have the general formula Z-RGAAXaaUL-XaaAaaA-R ¹ (SEQ ID NO: 95) (Formula Vid) wherein Z, R, G ¹ , D, L, and R ¹ each correspond to the definition for formula VI in the present description;

A represents L-alanine; And

Xaa ^U is a non-standard amino acid.

In some embodiments, Z-R in any of the formulas or ligands herein is replaced by R', wherein R' is Dap(guanidine)

In some embodiments, the αvβ6 integrin ligands have the general formula RG ¹ DLXaa ¹ Xaa ² L-Xaa ³ Xaa ⁴ LR 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 ¹ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ³ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ⁴ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

R ¹ is optional, and if present, contains polyethylene glycol (PEG) and/or a linking group; and at least one of Xaa ¹ , Xaa ² , Xaa ³ and Xaa ⁴ is a non-standard amino acid.

In some embodiments, the αγβ6 integrin ligands have the general formula RG ¹ DLXaa ¹ Xaa ² L-XaaAaaA-R ¹ (SEQ ID NO: 96) (Formula VIII) wherein at least two of Xaa ¹ , Xaa ² , Xaa ³ and Xaa ⁴ are non-standard amino acids.

In some embodiments, the αγβ6 integrin ligands have the general formula RG ¹ DLXaa ¹ Xaa ² L-Xaa ³ Xaa ⁴ LR ¹ (SEQ ID NO: 96) (Formula VIII) wherein at least three of Xaa ¹ , Xaa ² , Xaa ³ and Xaa ⁴ represent non-standard amino acids.

In some embodiments, αvβ6 integrin ligands have the general formula

RG^LAAbuL-CitAibL-R ¹ (SEQ ID NO: 97) (Formula Villa) wherein R, G ¹ , D, L, and R ¹ are each as defined for formula VIII in the present specification;

A represents L-alanine;

Abu is L-a-aminobutyric acid (2-aminobutyric acid);

Cit is citrulline and Ab is α-aminoisobutyric acid (2-aminoisobutyric acid).

In some embodiments, the αΎβ6 integrin ligand comprises, consists of, or essentially the structure consists of the structure of FIG. 1. In some embodiments, the αΎβ6 integrin ligand comprises, consists of, or essentially the structure consists of the structure of FIG. 2. In some embodiments, the αΎβ6 integrin ligand comprises, consists of, or essentially the structure consists of the structure of FIG. 3. In some embodiments, the αΎβ6 integrin ligand comprises, consists of, or essentially the structure consists of the structure of FIG. 4. In some embodiments, the αΎβ6 integrin ligand comprises, consists of, or essentially the structure consists of the structure of FIG. 5. In some embodiments, the αΎβ6 integrin ligand comprises, consists of, or essentially the structure consists of the structure of FIG. 6. In some embodiments, the αΎβ6 integrin ligand comprises, consists of, or essentially the structure consists of the structure of FIG. 7. In some embodiments, the αΎβ6 integrin ligand comprises, consists of, or essentially the structure consists of the structure of FIG. 8. In some embodiments, the αΎβ6 integrin ligand comprises, consists of, or essentially the structure consists of the structure of FIG. 9. In some embodiments, the αΎβ6 integrin ligand comprises, consists of, or essentially the structure consists of the structure of FIG. 10. In some embodiments, the αΎβ6 integrin ligand comprises, consists of, or essentially

the structure consists of the structure of FIG. eleven.

In some embodiments, any of the αΎβ6 integrin ligands disclosed herein may be linked to a cargo molecule, a reactive group, and/or a protected reactive group. The reactive group can be used to facilitate conjugation of the αΎβ6 integrin ligand to a molecule, such as one or more cargo molecules (eg, any of the cargo molecules described herein or known in the art). The αΎβ6 integrin ligands disclosed herein can increase the targeting of the cargo molecule to the αΎβ6 integrin or an αΎβ6 integrin expressing cell. A cargo molecule can be, without limitation, a pharmaceutically active ingredient or compound, a drug, a prodrug, or a substance of therapeutic value. In some embodiments, a cargo molecule can be, without limitation, 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 a polynucleotide (e.g., an oligomeric compound, e.g., an antisense oligonucleotide or an RNAi agent). ), peptide, aptamer, polymer, polyamine, protein, toxin, vitamin, polyethylene glycol, hapten, digoxigenin, biotin, radioactive atom or molecule, or fluorophore. In some embodiments, the implementation of the cargo molecule contains a pharmaceutically active ingredient, drug or prodrug. In some embodiments, the implementation of the cargo molecule as a pharmaceutically active ingredient contains an oligomeric compound. In some embodiments, the implementation of the cargo molecule as a pharmaceutically active ingredient contains an RNAi agent.

The present disclosure describes the use of the disclosed αΎβ6 ligands to target a cargo molecule to an αΎβ6 expressing cell. The cell may be in vitro, in situ, ex vivo, or in vivo.

In another aspect, the present disclosure provides compositions that contain one or more engineered, non-naturally occurring αΎβ6 ligands described herein. For example, in some embodiments, compositions comprising one or more of the αΎβ6 integrin ligands disclosed herein comprise one or more oligomeric compound(s), such as one or more RNAi agent(s), to be delivered to a cell in vivo. In some embodiments, the present document describes compositions for delivering an RNAi agent into a cell in vivo, wherein the RNAi agent is conjugated to one or more αΎβ6 ligands.

Compositions are described that contain one or more αΎβ6 ligands. In some embodiments, the composition contains a pharmaceutically acceptable adjuvant. In some embodiments, a composition that contains one or more αΎβ6 ligands contains

- 7 042923 one or more other pharmaceutical substances or pharmaceutically active ingredients or compounds.

In other embodiments, the implementation of the compositions contain drugs that contain one or more αΎβ6 ligands, which are described in the present description. In some embodiments, the drug product further comprises a pharmaceutically acceptable adjuvant.

Compositions that contain one or more of the ave6 integrin ligands disclosed herein can be delivered in vivo or in vitro to, for example, type I and II alveolar epithelial cells, goblet cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells , epithelial cells of the skin, cholangiocytes, enterocytes, epithelial cells of the ducts, glandular epithelial cells, renal tubules and epithelial tumors (carcinomas).

In another aspect, the present disclosure provides methods comprising administering one or more αΎβ6 ligands and/or compositions as described herein, and optionally delivering the disclosed αΎβ6 ligands and/or compositions in a form suitable for administration as a pharmaceutical product. In other embodiments, the disclosure provides methods for making ligands and compositions, such as drugs, as described herein.

Compositions that contain one or more αΎβ6 integrin ligands can be administered to subjects in vivo using routes of administration known in the art, suitable for such administration, taking into account the cargo molecule to be administered, including, for example, intravenous, subcutaneous, intraperitoneal, intradermal , transdermal, oral, sublingual, topical, intratumoral, intranasal or inhalation (ready-made forms in the form of an aerosol or dry powder) administration. In some embodiments, compositions that contain one or more αΎβ6 integrin ligands can be administered for systemic delivery, such as by intravenous or subcutaneous administration. In some embodiments, compositions that contain one or more αΎβ6 integrin ligands can be administered for localized delivery, such as by inhalation delivery via an inhaler or dry powder nebulizer. In some embodiments, compositions that contain one or more αΎβ6 integrin ligands can be administered for localized delivery by topical administration.

In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a type I alveolar epithelial cell in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more cargo molecules.

In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a type II alveolar epithelial cell in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more cargo molecules.

In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a goblet cell in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more cargo molecules.

In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a secretory epithelial cell in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more cargo molecules. In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a ciliated epithelial cell in vivo, the methods comprising administering to a subject one or more ave6 integrin ligands conjugated to one or more cargo molecules.

In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a corneal epithelial cell in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more cargo molecules.

In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a conjunctival epithelial cell in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more cargo molecules.

In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a skin epithelial cell in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more cargo molecules.

- 8 042923

In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to a cholangiocyte in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more cargo molecules.

In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to an enterocyte in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more cargo molecules.

In some embodiments, provided herein are methods for delivering one or more desired cargo molecule(s) to a ductal epithelial cell in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more cargo molecules.

In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to glandular epithelial cells in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more cargo molecules.

In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to the renal tubule in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more cargo molecules.

In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to an epithelial tumor (carcinoma) in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more cargo molecules.

In some embodiments, disclosed herein are methods for delivering an oligomeric compound to a type I alveolar epithelial cell in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to a Type I alveolar epithelial cell in vivo, the methods comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting expression of a target gene in a type I alveolar epithelial cell in vivo, the methods comprising administering to a subject an RNAi agent conjugated to one or more ligands having affinity for αΎβ6 integrin.

In some embodiments, disclosed herein are methods for delivering an oligomeric compound to a type II alveolar epithelial cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to a type II alveolar epithelial cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting expression of a target gene in a type II alveolar epithelial cell in vivo, the methods comprising administering to a subject an RNAi agent conjugated to one or more ligands having affinity for αΎβ6 integrin.

In some embodiments, disclosed herein are methods for delivering an oligomeric compound to a goblet cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to a goblet cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting expression of a target gene in a goblet cell in vivo, the methods comprising administering to a subject an RNAi agent conjugated to one or more ligands having affinity for αΎβ6 integrin.

In some embodiments, disclosed herein are methods for delivering an oligomeric compound to a secretory epithelial cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to a secretory epithelial cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting expression of a target gene in a secretory epithelial cell in vivo, the methods comprising administering to a subject an RNAi agent,

- 9 042923 conjugated with one or more ligands with affinity for the integrin ανβ6.

In some embodiments, disclosed herein are methods for delivering an oligomeric compound to a ciliated epithelial cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to a ciliary epithelial cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting expression of a target gene in a ciliary epithelial cell in vivo, the methods comprising administering to a subject an RNAi agent conjugated to one or more ligands having affinity for αΎβ6 integrin.

In some embodiments, disclosed herein are methods for delivering an oligomeric compound to a corneal epithelial cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to a corneal epithelial cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting expression of a target gene in a corneal epithelial cell in vivo, the methods comprising administering to a subject an RNAi agent conjugated to one or more ligands having affinity for αΎβ6 integrin.

In some embodiments, provided herein are methods for delivering an oligomeric compound to a conjunctival epithelial cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to a conjunctival epithelial cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting expression of a target gene in a conjunctival epithelial cell in vivo, the methods comprising administering to a subject an RNAi agent conjugated to one or more ligands having affinity for αΎβ6 integrin.

In some embodiments, disclosed herein are methods for delivering an oligomeric compound to a skin epithelial cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to a skin epithelial cell in vivo, comprising administering to a subject one or more ave6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting expression of a target gene in a skin epithelial cell in vivo, the methods comprising administering to a subject an RNAi agent conjugated to one or more αΎβ6 integrin affinity ligands.

In some embodiments, provided herein are methods for delivering an oligomeric compound to a cholangiocyte in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to a cholangiocyte in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting expression of a target gene in a cholangiocyte in vivo, the methods comprising administering to a subject an RNAi agent conjugated to one or more ligands having affinity for αΎβ6 integrin.

In some embodiments, provided herein are methods for delivering an oligomeric compound to an enterocyte in vivo, comprising administering to a subject one or more ave6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to an enterocyte in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting expression of a target gene in an enterocyte in vivo, the methods comprising administering to a subject an RNAi agent conjugated to one or more ligands having affinity for αΎβ6 integrin.

In some embodiments, disclosed herein are methods for delivering an oligomeric compound to a ductal epithelial cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to a ductal epithelial cell in vivo, comprising administering one or more ligands to a subject.

- 10 042923 ανβ6 integrins conjugated to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting expression of a target gene in a ductal epithelial cell in vivo, the methods comprising administering to a subject an RNAi agent conjugated to one or more ligands having affinity for αΎβ6 integrin.

In some embodiments, disclosed herein are methods for delivering an oligomeric compound to a glandular epithelial cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to a glandular epithelial cell in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting expression of a target gene in a glandular epithelial cell in vivo, the methods comprising administering to a subject an RNAi agent conjugated to one or more ligands having affinity for αΎβ6 integrin.

In some embodiments, provided herein are methods for delivering an oligomeric compound to the renal tubule in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to a renal tubule in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting expression of a target gene in the renal tubule in vivo, the methods comprising administering to a subject an RNAi agent conjugated to one or more ligands having affinity for αΎβ6 integrin.

In some embodiments, disclosed herein are methods for delivering an oligomeric compound to an epithelial tumor (carcinoma) in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more oligomeric compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to an epithelial tumor (carcinoma) in vivo, comprising administering to a subject one or more αΎβ6 integrin ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting expression of a target gene in an epithelial tumor (carcinoma) in vivo, the methods comprising administering to a subject an RNAi agent conjugated to one or more ligands having affinity for αΎβ6 integrin.

Within the scope of the invention, the term alkyl refers to a straight or branched saturated aliphatic hydrocarbon group having from 1 to 10 carbon atoms, unless otherwise indicated. For example, C ₁ -C ₆ -alkyl contains alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Non-limiting examples of alkyl groups include methyl, ethyl, isopropyl, t-butyl, n-hexyl. Within the scope of the invention, the term aminoalkyl refers to an alkyl group as defined above, substituted at any position with one or more amino groups that the normal valency allows. Amino groups may be unsubstituted, monosubstituted or disubstituted. Non-limiting examples of aminoalkyl groups include aminomethyl, dimethylaminomethyl, and 2-aminoprop-1-yl.

Within the scope of the invention, the term cycloalkyl means a saturated or unsaturated non-aromatic hydrocarbon ring group having from 3 to 14 carbon atoms, unless otherwise indicated. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, methylcyclopropyl, 2,2-dimethylcyclobutyl, 2-ethylcyclopentyl, and cyclohexyl.

Cycloalkyls may contain a plurality of spiro or articulated rings. Cycloalkyl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted at any position the normal valence allows.

Within the scope of the invention, the term alkenyl refers to a non-aromatic hydrocarbon radical, straight or branched, containing at least one carbon-carbon double bond and having from 2 to 10 carbon atoms, unless otherwise indicated. Such groups may have up to five carbon-carbon double bonds. For example, C ₂ -C ₆ -alkenyl is defined as an alkenyl radical having 2 to 6 carbon atoms. Examples of alkenyl groups include, without limitation, ethenyl, propenyl, butenyl, and cyclohexenyl. The straight, branched, or cyclic moiety of the alkenyl group may contain double bonds, and is optionally mono-, di-, tri-, tetra-, or penta-substituted at any position permitted by the normal valency. The term cycloalkenyl means a monocyclic hydrocarbon group having the specified number of carbon atoms and at least one carbon-carbon double bond.

Within the scope of the invention, the term alkynyl refers to a hydrocarbon radical, straight or branched, containing from 2 to 10 carbon atoms, unless otherwise indicated, and containing at least one carbon-carbon triple bond. There may be up to 5 triple carbon-carbon bonds. Thus, C ₂ -C ₆ alkynyl means alkynyl radical having from 2 to 6

- 11 042923 carbon atoms. Examples of alkynyl groups include, without limitation, ethynyl, 2-propynyl, and 2-butynyl. The straight or branched portion of the alkynyl group may optionally be mono-, di-, tri-, tetra-, or penta-substituted at any position that the normal valency allows.

In the context of the invention, alkoxy or alkoxy refers to an -O-alkyl radical having the number of carbon atoms indicated. For example, _C1 . ₆ -αlkoxy implies the content of C1, C ₂ , C ₃ , C ₄ , C ₅ and C ₆ -alkoxy groups. For example, _C1 . ₈ -alkoxy implies the content of C1, C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C7 and C ₈ -alkoxy groups. Examples of alkoxy include, without limitation, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, s-pentoxy, n-heptoxy, and n-octoxy.

Within the meaning of the invention, keto refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or aryl group as defined herein, connected via a carbonyl bridge. Examples of keto groups include, without limitation, alkanoyl (e.g., acetyl, propionyl, butanoyl, pentanoyl, or hexanoyl), alkenoyl (e.g., acryloyl), alkinoyl (e.g., ethinoyl, propionyl, butynoyl, pentinoyl, or hexinoyl), aryloyl (e.g., benzoyl), heteroaryloyl (for example, pyrroloyl, imidazolyl, quinolinoyl or pyridinoyl).

Within the scope of the invention, alkoxycarbonyl refers to any alkoxy group as defined above, connected via a carbonyl bridge (ie -C(O)O-alkyl). Examples of alkoxycarbonyl groups include, without limitation, methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-propoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, or n-pentoxycarbonyl.

Within the scope of the invention, aryloxycarbonyl refers to any aryl group as defined herein, connected via an oxycarbonyl bridge (ie, -C(O)Oaryl). Examples of aryloxycarbonyl groups include, without limitation, phenoxycarbonyl and naphthyloxycarbonyl.

As used herein, heteroaryloxycarbonyl refers to any heteroaryl group, as defined herein, connected via an oxycarbonyl bridge (ie, -C(O)O-heteroaryl). Examples of heteroaryloxycarbonyl groups include, without limitation, 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

In the context of the invention, aryl or aromatic means any stable monocyclic or polycyclic carbon ring with up to 6 atoms in each ring, at least one ring being aromatic. Examples of aryl groups include, without limitation, phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl, and biphenyl. In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it should be understood that the connection occurs via an aromatic ring. Aryl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted at any position permitted by the normal valency.

Within the scope of the invention, the term heteroaryl represents a stable monocyclic or polycyclic ring of 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 without limitation acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl, isoquinolinyl, dihydroisoindolonyl, imidazopyridine yl, isoindolonyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl and tetrahydroquinoline. Heteroaryl also means the content of the N-oxide derivative of any nitrogen-containing heteroaryl. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain heteroatoms, it should be understood that the connection occurs via an aromatic ring or via a ring-containing heteroatom. Heteroaryl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted at any position the normal valence allows.

In the context of the invention, the term heterocycle, heterocyclic or heterocyclyl means a 3-14 membered aromatic or non-aromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, including polycyclic groups. Within the scope of the invention, the term heterocyclic is also considered to be synonymous with the terms heterocycle and heterocyclyl and is also intended to have the same definitions as set forth herein. Heterocyclyl includes the heteroaryls mentioned above as well as its dihydro and tetrahydro analogues. Examples of heterocyclyl groups include, without limitation, azetidinyl, benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indole yl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxooxazolidinyl, oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl , pyridazinyl, pyridyl, pyridinonyl, pyrimidyl, 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, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetra zolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxidothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl and tetrahydrothienyl and their N-oxides. Attachment of a heterocyclyl substituent may be via a carbon atom or via a heteroatom. Heterocyclyl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted at any position the normal valence allows.

Within the scope of the invention, the terms treat, treatment, and the like are means methods or steps taken to provide relief or reduction in the number, severity and/or frequency of one or more symptoms of a disease or condition in a subject.

Unless otherwise indicated, the use of a symbol as used herein means that any group or groups may be associated with it, which is within the scope of the inventions described herein.

Within the scope of the invention, the term isomers refers to compounds which have identical molecular formulas but which differ in nature or in the binding sequence of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space 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.

Within the scope of the invention, a linking group is one or more atoms that link one molecule or molecule part to another with a second molecule or second molecule part. In the art, the terms linking group and spacers are sometimes used interchangeably. Similarly, as used in the art, the term framework is sometimes used interchangeably with a linking group. In some embodiments, the linking group may contain a peptide-cleavable linking group. In some embodiments, the implementation of the linking group may contain or consist of the peptide FCitFP (SEQ ID NO: 131).

Within the scope of the invention, the term "linked" when referring to a connection between two molecules means that two molecules are connected by a covalent bond, or that two molecules are linked by non-covalent bonds (eg, hydrogen bonds or ionic bonds). In some instances where the term bound refers to an association between two molecules via non-covalent bonds, the association between two different molecules has a K _D of less than 1x10' ⁴ M (e.g., less than 1x10'5 M, less than 1x10'6 M, or less than 1x10' ⁷ M) in a physiologically acceptable buffer (eg phosphate buffered saline). Unless otherwise specified, the term "linked" within the scope of the invention may refer to a bond between a first compound and a second compound, either with or without any intermediate atoms or groups of atoms.

Within the scope of the invention, 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, tyrosine.

Within the scope of the invention, non-standard amino acids include, without limitation, selenocysteine, pyrrolysine, N-formylmethionine, hydroxyproline, selenomethionine, α-aminoisobutyric acid (Aib), L-a-aminobutyric acid (Abu), α,γ-diaminobutyric acid, dehydroalanine, norleucine, alloisoleucine, t -leucine, α-amino-p-heptanoic acid, α,β-diaminopropionic acid, β-N-oxalyl-a,βdiaminopropionic acid, allothreonine, homocysteine, homoserine, β-homo-alanine (v3-hA), isovaline, norvaline (Nva), citrulline (Cit), ornithine, α-methylaspartate (aMeD), α-methylleucine (aMeL),

- 13 042923

N-methylalanine, N-methylglycine (NMeG), N-methylleucine (NMeL), β-cyclohexylalanine (Cha),

N-ethylalanine, N,N-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 similar to naturally occurring amino acids.

Within the scope of the invention, unless specifically stated that a structure has a specific conformation, for each structure that has asymmetric centers and thus results in enantiomers, diastereomers, or other stereoisomeric configurations, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure and racemic forms. For example, the structures disclosed herein are intended to cover mixtures of diastereomers as well as individual stereoisomers.

One of ordinary skill in the art will readily understand and appreciate that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environmental conditions in which the compound is located. or composition. Accordingly, within the scope of the invention, the structures disclosed herein provide that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated. The present disclosure is intended to cover the disclosed compounds and compositions regardless of their state of protonation based on the pH of the surrounding conditions, as will be readily understood by one of ordinary skill in the art.

As used in the claims in this specification, the phrase consisting of excludes any element, step, or ingredient not listed in the claims. When used in the claims in the present description, the phrase consisting essentially of limits the scope of the claims to certain materials or steps and elements that do not materially affect the essential and novel feature(s) of the claimed invention.

Unless otherwise defined, all technical and scientific terms used in the present description have the same meaning, which is usually understood by an ordinary specialist in the field to which the present invention relates. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned in the present description are hereby incorporated by reference in their entirety. In the event of a conflict, the present description, including definitions, will take precedence. In addition, the materials, methods, and examples are illustrative and are not intended to be limiting.

Pharmacokinetics is a common problem for peptide drugs and pharmaceutical compositions that contain peptides. Many peptides, for example, circulate in the blood for no more than a few minutes due to enzymatic degradation. This often greatly reduces or even prevents their usefulness as therapeutic agents or as drug components.

Stability studies in various serum formulations (eg, measurement of in vitro degradation of peptides in serum and/or plasma) have become important screening studies in the development of peptide drugs. The ave6 integrin ligands disclosed herein, among others, have been shown to be stable in serum and have affinity for or can bind to ave6 integrins.

Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

Brief description of the drawings

In FIG. 1 shows the chemical structure of an example of the ave6 integrin ligand disclosed herein, synthesized as a tetrafluorophenyl (TFP) ester. The ave6 integrin ligand contains PEG ₂₀ (twenty (20) ethylene oxide units (CH2-CH2-O)) and a linking group FCitFP.

In FIG. 2 shows the chemical structure of an example of the ave6 integrin ligands disclosed herein, synthesized as a tetrafluorophenyl (TFP) ester. The ave6 integrin ligand contains PEG5 (five (5) ethylene oxide units (CH2-CH2-O)).

In FIG. 3 shows the chemical structure of an example of the ave6 integrin ligand disclosed herein, which contains CF3CO as the amino terminal cap, PEG ₂₀ , and an FCitFP linking group.

In FIG. 4 shows the chemical structure of an example of the ave6 integrin ligand disclosed herein that contains CF3CO as the amino terminal cap and PEG5.

In FIG. 5 shows the chemical structure of an example of the ave6 integrin ligand disclosed herein without the amino terminal cap. The ave6 integrin ligand contains PEG ₂₀ and a FCitFP linking group.

In FIG. 6 shows the chemical structure of an example of a ligand disclosed herein.

- 14 042923 integrin ανβ6 without aminoterminal cap. The ανβ6 integrin ligand contains PEG5.

In FIG. 7 shows the chemical structure of an example of the ave6 integrin ligand disclosed herein, which contains CH ₃ CO as the amino terminal cap.

In FIG. 8 shows the chemical structure of an example of the ave6 integrin ligand disclosed herein, which contains CH ₃ CO as the amino terminal cap, PEG ₂₀ , and an FCitFP linking group. In addition, the structure shows a fragment of PEG 20 kilodaltons (kDa).

In FIG. 9 shows the chemical structure of an example of the αγβ6 integrin ligand disclosed herein, which contains CH ₃ CO as the amino terminal cap, PEG ₂₀ , and an FCitFP linking group. In addition, the structure shows a PEG fragment of 20 kilodaltons (kDa) and shows the structure associated with an oligomeric compound such as an RNAi agent.

In FIG. 10 shows the chemical structure of an example of an αχβ6 integrin tridentate ligand disclosed herein that contains CH ₃ CO as the amino terminal cap and PEG5 linked to a bis-glutamic acid backbone and a PEG-azide reactive group.

In FIG. 11 shows the chemical structure of an example of the ave6 integrin tridentate ligand disclosed herein, which contains CH ₃ CO as the amino terminal cap and PEG ₅ linked to a bis-glutamic acid backbone.

In FIG. 12 is a graph showing the total expression of alpha-ENaC in rat lung in Sprague-Dawley rats with a naked RNAi anti-alpha-ENaC agent without a targeting ligand and with the same anti-alpha-ENaC RNAi agent conjugated to a poly-L-lysine backbone and with ave6 integrin ligand represented by the structure of FIG. 4.

Detailed description

This document describes novel, engineered, non-naturally occurring ave6 integrin peptide ligands having serum stability and affinity for ave6 integrins. The ave6 integrin ligands can be used to target ave6 integrin expressing cells in vitro, in situ, ex vivo and/or in vivo. In some embodiments, ave6 integrin ligands can be conjugated to one or more cargo molecules to target cargo molecules to αγβ6 integrin expressing cells in vitro, in situ, ex vivo, and/or in vivo. In some embodiments, the implementation of cargo molecules contain or consist of pharmaceutically active compounds. In some embodiments, the αγβ6 integrin ligands disclosed herein are conjugated to cargo molecules to target cargo molecules to epithelial cells in vivo.

In some embodiments, the αγβ6 integrin ligands comprise Z-RG ¹ DLXaa ¹ Xaa ² L (SEQ ID NO: 85) (Formula I) wherein Z is an amino-terminal cap (e.g., any of the amino-terminal caps described herein or known herein). areas);

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 represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art); A

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art).

In some embodiments, the amino-terminal cap (Z) in formula I contains CH ₃ CO (also referred to herein as 'Ac'). In some embodiments, the amino-terminal cap (Z) in formula I is CH ₃ CO.

In some embodiments, the αγβ6 integrin ligands comprise

R' oAxaaAaa3 (SEQ ID NO: 98) (Formula la) wherein R' is Dap(guanidine); A

G ¹ , D, L, Xaa ¹ and Xaa ² each corresponds to the definition for formula I in the present description.

In some embodiments, αvβ6 integrin ligands have the general formula Z-RG ¹ DLXaa ¹ Xaa ^u L (SEQ ID NO: 99) (Formula lb), wherein Z, R, G ¹ , D, L, and Xaa ¹ each correspond to the definition for formula I in the present description; And

- 15 042923

Xaa ^u represents a non-standard amino acid.

In some embodiments, αvβ6 integrin ligands have the general formula

Z-RG ¹ DLAXaa ^u L (SEQ ID NO: 90) (Formula Ic) in which Z, R, G ¹ , D and L each corresponds to the definition for formula I in the present description;

A represents L-alanine; A

Xaa ^U is a non-standard amino acid.

In some embodiments, αvβ6 integrin ligands are described comprising Z-RG ¹ DLXaa ¹ Xaa ² LJR ¹ (SEQ ID NO: 86) (Formula II) 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 represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

J is optional and, if present, contains one or more , 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 (for example, any of the L-α amino acids described herein or known in the art), L-β amino acids (for example, any of the L-β amino acids described herein or known in the art) or α,α-disubstituted amino acids (eg, any of the α,α-disubstituted amino acids described herein or known in the art), or a combination thereof; A

R ¹ is optional and, if present, contains PEG and/or a linking group.

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

In some embodiments, αvβ6 integrin ligands are described comprising R'G ¹ DLXaa ¹ Xaa ² LJR ¹ (SEQ ID NO: 100) (Formula Ila) wherein R' is Dap(guanidine); A

G ¹ , D, L, Xaa ¹ , Xaa ² , J and R ¹ each corresponds to the definition for formula II in the present description.

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

In some embodiments, the αvβ6 integrin ligands may contain a reactive group or a protected reactive group containing

Z-RG ¹ DLXaa ¹ Xaa ² LJR^R ² (SEQ ID NO: 87) (Formula III) wherein Z is an amino-terminal cap (eg, 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 represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

- 16 042923

J is optional and, if present, contains one or more , 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 (for example, any of the L-α amino acids described herein or known in the art), L-β amino acids (for example, any of the L-β amino acids described herein or those known in the art), or α,α-disubstituted amino acids (eg, any of the α,α-disubstituted amino acids described herein or known in the art), or a combination thereof;

R ¹ is optional, and if present, contains PEG and/or a linking group; A

R ² contains a reactive group or a protected reactive group.

A reactive group or a protected reactive group can be used to couple an αvβ6 integrin ligand to a molecule of interest, i.e. With a cargo molecule. In some embodiments, L is linked to J via an amide bond.

In some embodiments, ανβ6 integrin ligands are synthesized having a reactive group or a protected reactive group having the formula

R' G ¹ DLXaa ¹ Xaa ² L-J-rA ² (SEQ ID NO: 101) (Formula Illa) wherein R' is Dap(guanidine); A

G ¹ , D, L, Xaa ¹ , Xaa ² , J, R ¹ and R ² each corresponds to the definition for formula III in the present description.

In some embodiments, one or more αvβ6 integrin ligand(s) may be conjugated to one or more cargo molecule(s) containing (Z-RG ¹ DLXaa ¹ Xaa ² LJR ¹ ) _n -R ³ (SEQ ID NO: 88 ) (Formula IV) wherein Z represents an amino terminal cap (eg, 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 represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

J is optional and, if present, contains one or more , 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 (for example, any of the L-α amino acids described herein or known in the art), L-β amino acids (for example, any of the L-β amino acids described herein or known in the art), or α,α-disubstituted amino acids (eg, any of the α,α-disubstituted amino acids described herein or known in the art), or a combination thereof;

R ¹ is optional, and if present, contains polyethylene glycol (PEG) and/or a linking group;

n represents a number greater than 0 (for example, 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 -thirty); A

R ³ contains a cargo molecule.

In some embodiments, L is linked to J via an amide bond. In some embodiments, the implementation of the cargo molecule can be any molecule that you want to target expressing ανβ6 integrin cell. In some embodiments, n is an integer between 1 and 4. In some embodiments, n is 1. In some embodiments, n is 3. When n is 1, the αγβ6 integrin ligand may be referred to herein as the ανβ6 monodentate integrin ligand. When n is 3, the ανβ6 integrin ligand may be referred to herein as the tridentate αγβ6 integrin ligand. When n is 2, the αγβ6 integrin ligand may be referred to herein as a bidentate αγβ6 integrin ligand. When

- 17 042923 n is 4, the ανβ6 integrin ligand may be referred to herein as the ανβ6 integrin tetradentate ligand.

In some embodiments, one or more ανβ6 integrin ligand(s) may be conjugated to one or more cargo molecule(s) containing (R ^T G ¹ DLXaa ¹ Xaa ² L-JR ¹ ) _n -R ³ (SEQ ID NO : 102) (Formula IVa) wherein R ¹ is Dap(guanidine); A

G ¹ , D, L, Xaa ¹ , Xaa ² , J, R ¹ , N and R ³ each corresponds to the definition for formula IV in the present description.

In some embodiments, L is linked to J via an amide bond. In some embodiments, n is an integer between 1 and 4. In some embodiments, n is 3. In some embodiments, the cargo molecule can be any molecule that is desired to be targeted to an ανβ6 integrin expressing cell.

In some embodiments, one or more ανβ6 integrin ligands can be conjugated to one or more cargo molecules containing (Z-RG ¹ DLXaa ¹ Xaa ² LJR ¹ ) _n -R ⁴ -(R ³ ) p (SEQ ID NO: 89 ) (Formula V) wherein Z represents an amino terminal cap (eg, 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 represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

J is optional and, if present, contains one or more , 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 (for example, any of the L-α amino acids described herein or known in the art), L-β amino acids (for example, any of the L-β amino acids described herein or those known in the art), or α,α-disubstituted amino acids (eg, any of the α,α-disubstituted amino acids described herein or known in the art), or a combination thereof;

R ¹ is optional, and if present, contains PEG and/or a linking group;

n represents a number greater than 0 (for example, 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 -thirty);

R ⁴ is optional and, if present, contains a backbone and/or a linking group that contains at least one attachment point for each ligand present (i.e., at least n number of attachment points) and at least one attachments for each available cargo molecule (i.e., at least the number of attachment points equal to p);

p represents a number greater than 0 (for example, 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 -thirty); A

R ³ contains one or more cargo molecules.

In some embodiments, L is linked to J via an amide bond. In some embodiments, the implementation of the cargo molecule can be any molecule that you want to target expressing ανβ6 integrin cell.

The ανβ6 integrin ligands disclosed herein may contain one or more scaffolds. Frameworks, also sometimes referred to in the art as linking groups or linkers, can be used to facilitate the linking of one or more cargo molecules to one or more of the ανβ6 integrin ligands disclosed herein. Suitable frameworks compatible with the ligands disclosed herein are well known in the art. Non-limiting examples of scaffolds that can be used with the ανβ6 integrin ligands disclosed herein include, without limitation, polymers (e.g., polyacrylate polymers, polyvinyl ester polymers, etc.), amino acid polymers (e.g., bis-glutamic acid , bislysine, poly-L-lysine PLL, etc.) and cysteine. In some embodiments, scaffolds may provide additional desired properties in addition to functioning as a linker alone, for example, improving pharmacokinetic (PK) properties.

In some embodiments, one or more ανβ6 integrin ligands can be conjugated to one or more cargo molecules containing (R'G ¹ DLXaa ¹ Xaa ² LJR ¹ ) nR ⁴ -(R ³ ) P (SEQ ID NO: 103) ( Formula Va) wherein R ¹ is Dap(guanidine); a

G ¹ , D, L, Xaa ¹ , Xaa ² , J, R ¹ , n, R ⁴ , p and R ³ each corresponds to the definition for formula V in the present description.

In some embodiments, L is linked to J via an amide bond. The cargo molecule can be any molecule that is desired to be targeted to an ανβ6 integrin expressing cell.

In some embodiments, the implementation of J in any of the formulas of the present description contains 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 that are encoded directly by codons of the universal genetic code, also referred to as encoded amino acids of the canonical amino acids), or non-standard amino acids (also called unnatural, non-encoded or non-canonical amino acids).

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.

Non-standard amino acids include, without limitation, selenocysteine, pyrrolysine, N-formylmethionine, hydroxyproline, selenomethionine, α-aminoisobutyric acid (Aib), La-aminobutyric acid (Abu), α,γ-diaminobutyric acid, dehydroalanine, norleucine, alloisoleucine, t-leucine, a-amino-n-heptanoic acid, α,β-diaminopropionic acid, β-N-oxalyl-α,βdiaminopropionic acid, allothreonine, homocysteine, homoserine, β-homo-alanine ^S-hA), isovaline, norvaline (Nva) , citrulline (Cit), ornithine, α-methylaspartate (aMeD), α-methylleucine (aMeL), N-methylalanine, N-methylglycine ( _NMe G), N-methylleucine ( _NMe L), β-cyclohexylalanine (Cha) , N-ethylalanine, N,N-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 similarly to naturally occurring amino acids.

In some embodiments, J contains at least one non-standard amino acid. In some embodiments, J is or contains 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.

In some embodiments, the implementation of J in any of the formulas described herein contains or consists of Xaa ³ Xaa ⁴ L, and

L is L-leucine;

Xaa ³ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art); And

Xaa ⁴ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art).

In some embodiments, J is or contains Xaa ³ Xaa ⁴ , wherein

Xaa ³ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art); And

Xaa ⁴ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or α,α-disubstituted

- 19 042923 amino acid (for example, any of the α,α-disubstituted amino acids described herein or known in the art).

In some embodiments, the ave6 integrin ligands comprise

Z-RG ¹ DLXaa ¹ Xaa ² LXaa ³ Xaa ⁴ LR ¹ (SEQ ID NO: 92) (Formula VI) wherein Z is an amino terminal cap (eg, 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 represents an L-α amino acid (e.g. any of the La 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 (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ³ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ⁴ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art); A

R ¹ is optional and, if present, contains PEG and/or a linking group.

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 can be, without limitation, a naturally occurring L-α amino acid, a naturally occurring proteinogenic amino acid, a naturally occurring standard amino acid (i.e., the 20 amino acids that are encoded directly by codons of the universal genetic code, also called canonical amino acid coded amino acids), or a non-standard (also called non-natural, non-encoded, or non-canonical) amino acid.

In some embodiments, Xaa ² is an L-α amino acid, an L-β amino acid, or an α,α-disubstituted amino acid. Xaa ² can be, without limitation, a naturally occurring L-α amino acid, a naturally occurring proteinogenic amino acid, a naturally occurring standard amino acid (i.e., the 20 amino acids that are encoded directly by codons of the universal genetic code, also called canonical amino acid coded amino acids), or a non-standard amino acid. (also called non-natural, non-encoded, or non-canonical) amino acid.

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, without limitation, a naturally occurring L-α amino acid, a naturally occurring proteinogenic amino acid, a naturally occurring standard amino acid (i.e., the 20 amino acids that are encoded directly by codons of the universal genetic code, also called canonical amino acid coded amino acids), or a non-standard amino acid. (also called non-natural, non-encoded, or non-canonical) amino acid.

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, without limitation, a naturally occurring L-α amino acid, a naturally occurring proteinogenic amino acid, a naturally occurring standard amino acid (i.e., the 20 amino acids that are encoded directly by codons of the universal genetic code, also called canonical amino acid coded amino acids), or a non-standard amino acid. (also called non-natural, non-encoded, or non-canonical) amino acid.

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, Xa ² is a non-standard amino acid. In some embodiments, both Xaa ¹ and Xaa ² are non-standard amino acids.

In some embodiments, Xaa ¹ and/or Xaa ² are Abu. In some variants

- 20 042923 implementation Haa ¹ represents Abu. In some embodiments, Xaa ² is Abu.

In some embodiments, Xaa ¹ or Xaa ² has no charge. In some embodiments, Xaa ¹ has no charge. In some embodiments, Xaa ² has no charge. In some embodiments, Xaa ¹ and Xaa ² have no charge.

In some embodiments, Xaa ¹ has no charge and Xaa ² is a non-standard amino acid. In some embodiments, Xaa ² has no charge and Xaa ¹ is a non-standard amino acid. In some embodiments, Xaa ¹ has no charge and Xaa ² represents Abu. In some embodiments, Xaa ² has no charge and Xaa ¹ represents Abu.

In some embodiments, Xaa ¹ Xaa ² is AAbu, KAbu, EAbu, FAbu, QAbu, GAbu, PAbu, AK, AE, AF, AQ, AG, and AP, where A is L-alanine, Abu is La-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.

In some embodiments, RG ¹ DLXaa 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 represents L-alanine;

Abu is L-a-aminobutyric acid;

K is L-lysine;

E represents L-glutamic acid (glutamate);

F is L-phenylalanine;

Q is L-glutamine; A

P represents L-proline.

In some embodiments, G (L-glycine) in any of the previous formulas is replaced by MeGly (N-methylglycine).

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, both Xaa ³ and Xaa ⁴ are non-standard amino acids.

In some embodiments, Xaa ³ and/or Xaa ⁴ are Cit. In some embodiments, Xaa ³ is Cit. In some embodiments, Xaa ⁴ is Cit.

In some embodiments, Xaa ³ or Xaa ⁴ has no charge. In some embodiments, Xaa ³ has no charge. In some embodiments, Xaa ⁴ has no charge. In some embodiments, Xaa ³ and Xaa ⁴ have no charge.

In some embodiments, Xaa ³ has no charge and Xaa ⁴ is a non-standard amino acid. In some embodiments, Xaa ³ has no charge and Xaa ⁴ is a non-standard amino acid. In some embodiments, Xaa ³ is Aib. In some embodiments, Xaa ⁴ is Aib.

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 aminoisobutyric acid (a-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.

In some embodiments, the ave6 integrin ligands comprise

R' G ¹ DLXaa ¹ Xaa ² LXaa ³ Xaa ⁴ LR ¹ (SEQ ID NO: 104) (Formula Via) wherein R ¹ is Dap(guanidine); A

G ¹ , D, L, Xaa ¹ , Xaa ² , Xaa ³ , Xaa ⁴ and R ¹ each corresponds to the definition for formula VI in the present description.

In some embodiments, the αγβ6 integrin ligands contain a reactive group or a protected reactive group and contain (Z-RG ¹ DLXaa ¹ Xaa ² LJR ¹ ) _n -R ⁴ -(R ² ) p (SEQ ID NO: 105) (Formula VII) in where Z represents an amino terminal cap (eg, any of the amino terminal caps described herein or known in the art);

R is L-arginine;

- 21 042923

G ¹ is L-glycine or N-methylglycine;

D is L-aspartic acid (L-aspartate);

L is L-leucine;

Xaa ¹ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

J is optional and, if present, contains one or more , 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 (for example, any of the L-α amino acids described herein or known in the art), L-β amino acids (for example, any of the L-β amino acids described herein or known in the art), or α,α-disubstituted amino acids (eg, any of the α,α-disubstituted amino acids described herein or known in the art), or a combination thereof;

R ¹ is optional, and if present, contains polyethylene glycol (PEG) and/or a linking group;

n represents a number greater than 0 (for example, 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 -thirty);

R ⁴ is optional, and if present, contains a backbone and/or a linking group that contains at least one point of attachment for each ligand and at least one point of attachment for each cargo molecule;

p represents a number greater than 0 (for example, 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 -thirty); A

R ² contains a reactive group or a protected reactive group.

In some embodiments, L is linked to J via an amide bond. In some embodiments, αvβ6 integrin ligands that contain one or more reactive groups or protected reactive groups can be reacted with a cargo molecule to form an αvβ6 integrin ligand-cargo molecule conjugate.

In some embodiments, the αvβ6 integrin ligands contain a reactive group or a protected reactive group and contain (R'G ¹ DLXaa ¹ Xaa ² LJR ¹ ) nR ⁴ -(R ² ) P (SEQ ID NO: 106) (Formula Vila) wherein R ' represents Dap(guanidine); A

G ¹ , D, L, Xaa ¹ , Xaa ² , J, R ¹ , n, R ⁴ , p and R ² each corresponds to the definition for formula VII in the present description.

In some embodiments, L is linked to J via an amide bond. The cargo molecule can be any molecule that is desired to be targeted to an αvβ6 integrin expressing cell.

In some embodiments, especially when only localized delivery is required (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, intranasal, or topical administration), αvβ6 integrin ligands can be synthesized without the presence of an aminoterminal cap, provided that at least one or more amino acids are non-standard amino acids. In some embodiments, the αvβ6 integrin ligands comprise

RG ¹ DLXaa ¹ Xaa ² L-Xaa ³ Xaa ⁴ LR 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 ¹ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (e.g., any of the α,α-disubstituted amino acids described herein

- 22 042923 or known in the art);

Xaa ² represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ³ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

Xaa ⁴ represents an L-α amino acid (for example, any of the L-α amino acids described herein or known in the art), an L-β amino acid (for example, any of the L-β amino acids described herein or known in this region) or an α,α-disubstituted amino acid (eg, any of the α,α-disubstituted amino acids described herein or known in the art);

R ¹ is optional, and if present, contains PEG and/or a linking group; and at least one of Xaa ¹ , Xaa ² , Xaa ³ and Xaa ⁴ is a non-standard amino acid.

In some embodiments, the αvβ6 integrin ligands comprise

RG ¹ DLXaa ¹ Xaa ² L-Xaa ³ Xaa ⁴ LR ¹ (SEQ ID NO: 96) (Formula VIII), each variable as defined above for formula VIII, with at least two of Xaa ¹ , Xaa ² , Xaa ³ and Xaa ⁴ represent a non-standard amino acid.

In some embodiments, the αvβ6 integrin ligands comprise

RG ¹ DLXaa ¹ Xaa ² L-Xaa ³ Xaa ⁴ LR ¹ (SEQ ID NO: 96) (Formula VIII), each variable as defined above for formula VIII, with at least three of Xaa ¹ , Xaa ² , Xaa ³ and Xaa ⁴ represent a non-standard amino acid.

In some embodiments, the αvβ6 integrin ligands comprise

RG ¹ DLXaa ¹ Xaa ² L-Xaa ³ Xaa ⁴ LR ¹ (SEQ ID NO: 96) (Formula VIII), each variable as defined above for formula VIII, with Xaa ² , Xaa ³ and Xaa ⁴ representing a non-standard amino acid.

In some embodiments, the αvβ6 integrin ligands comprise

RG ¹ DLAAbuLCitAibL-R ¹ (SEQ ID NO: 97) (Formula Villa) 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 represents L-alanine;

Abu is L-α-aminobutyric acid;

Cit represents citrulline;

Aib is α-aminoisobutyric acid; A

R ¹ is optional and, if present, contains PEG and/or a linking group.

In some embodiments, the αvβ6 integrin ligands contain a reactive group or a protected reactive group and contain (RGYlAAbuLCitAibL-R ¹ ) nR ⁴ -(R ² ) p (SEQ ID NO: 107) (Formula VIIlb) 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 represents L-alanine;

Abu is L-α-aminobutyric acid;

Cit represents citrulline;

Aib is α-aminoisobutyric acid;

R ¹ is optional, and if present, contains PEG and/or a linking group; n represents a number greater than 0 (for example, 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 -thirty);

R ⁴ is optional and, if present, contains a backbone or linking group that contains at least one attachment point for each ligand and at least one attachment point for each cargo molecule;

p represents a number greater than 0 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,

- 23 042923

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); A

R ² contains a reactive group or a protected reactive group.

In some embodiments, one or more cargo molecules are conjugated to one or more ave6 integrin ligands and contain (RG^LAAbuLCitAibL-R ¹ ) nR ⁴ -(R ³ ) p (SEQ ID NO: 108) (Formula VIlie) wherein R represents 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, contains PEG and/or a linking group;

n represents a number greater than 0 (for example, 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 -thirty);

R ⁴ is optional and, if present, contains a backbone or linking group that contains at least one attachment point for each ligand and at least one attachment point for each cargo molecule;

p represents a number greater than 0 (for example, 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 -thirty); A

R ³ contains one or more cargo molecules.

The one or more cargo molecules can be any molecule that is desired to be targeted to an ave6 integrin expressing cell.

Within the scope of the invention, in some embodiments, R ¹ is present and contains a PEG group having 1-100 units of ethylene oxide (CH2-CH2-O) (for example, 1-90, 1-80, 1-70, 1-60, 1-50 , 1-40, 1-30, 1-20, 1-10, 1-5, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2 -30, 2-20, 2-10, 2-5, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20 , 5-10, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20 -80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40 , 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60 -90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90 or 90-100 units of ethylene oxide). In some embodiments, R ¹ is present and contains a PEG group having 2-30 ethylene oxide units. In some embodiments, R ¹ is present and contains a PEG group having 2-20 ethylene oxide units. In some embodiments, R ¹ is present and contains a PEG group having 2-10 ethylene oxide units. In some embodiments, R ¹ is present and contains a PEG group having 5-20 ethylene oxide units. In some embodiments, R ¹ is present 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 units of ethylene oxide.

Reactive groups are well known in the art and provide for the formation of covalent bonds between two molecules or reactants. Suitable reactive groups for use herein include, without limitation, amino groups, amido groups, carboxylic acid groups, azides, alkynes, propargyl groups, BCN(bicyclo[6.1.0]nonine, DBCO(dibenzocyclooctyne)thiols, maleimide groups, aminooxy groups, N -hydroxysuccinimide (NHS) or other activated ester (eg PNP, TFP, PFP), bromine groups, aldehydes, carbonates, tosylates, tetrazines, trans-cyclooctene (TCO), hydrazides, hydroxyl groups, disulfides and orthopyridyl disulfide groups.

The inclusion of reactive groups can facilitate conjugation of the αγβ6 integrin ligand disclosed herein to a cargo molecule. Conjugation reactions are well known in the art and provide for the formation of covalent bonds between two molecules or reactants. Suitable conjugation reactions for use herein include, but are not limited to, amide coupling, Michael addition, hydrazone formation, and click cycloaddition.

In some embodiments, the ave6 integrin targeting ligands disclosed herein are synthesized as a tetrafluorophenyl (TFP) ester that can be displaced with a reactive amino group to attach a cargo molecule.

Protected reactive groups are also widely used in the art. The protecting group provides a temporary chemical transformation of the reactive group into a group that does not react under the conditions under which the unprotected group reacts, for example, to ensure the chemoselectivity of the subsequent chemical reaction. Suitable protected reactive groups for use herein include, but are not limited to, BOC (t-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), carboxybenzyl (CBZ) groups, benzyl esters, and PBF (2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl).

A cargo molecule is any molecule that may require targeted

- 24 042923 effect on the ανβ6 integrin or cell expressing ανβ6 integrin. A cargo molecule can be, without limitation, a pharmaceutical ingredient, a drug, a prodrug, a therapeutic agent, 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. , polyamine, protein, aptamer, toxin, vitamin, PEG, hapten, digoxigenin, biotin, radioactive atom or molecule, or fluorophore. In some embodiments, one or more cargo molecules (eg, the same or different cargo molecules) are linked to one or more αΎβ6 integrin ligands to target the cargo molecules to an αΎβ6 integrin expressing cell.

In some embodiments, one or more cargo molecules is a pharmaceutical ingredient or pharmaceutical composition. In some embodiments, one or more of the cargo molecules is an oligomeric compound. Within the scope of the invention, an oligomeric compound is a nucleotide sequence containing approximately 10-50 32, 10-30, 10-28, 10-26, 10-24, 10-22, 10-20, 10-18,

10-16, 10-14, 10-12, 12-50, 12-48, 12-46, 12-44, 12-42, 12-40, 12-38, 12-36, 12-34, 12- 32, 12-30, 12-28, 12-26,

12-24, 12-22, 12-20, 12-18, 12-16, 12-14, 14-50, 14-48, 14-46, 14-44, 14-40, 14-42, 14- 38, 14-36, 14-34, 14-32,

14-30, 14-28, 14-26, 14-24, 14-22, 14-20, 14-18, 14-16, 16-50, 16-48, 16-46, 16-44, 16- 42, 16-40, 16-38, 16-36,

16-34, 16-32, 16-30, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 18-50, 18-48, 18-46, 18- 44, 18-42, 18-40, 18-38,

18-36, 18-34, 18-32, 18-30, 18-28, 18-26, 18-24 18-22, 18-20, 20-50, 20-48, 20-46, 20-44 , 20-42, 20-40, 20-38,

20-36, 20-34 20-32, 20-30, 20-28, 20-26, 20-24, 20-22, 22-50, 22-48, 22-46, 22-44, 22-42 , 22-40, 22-38, 22-36, 22-34, 22-32, 22-30, 22-28, 22-26, 22-24, 24-50, 24-48, 24-46, 24 -44, 24-42, 24-40, 24-38, 24-36, 24-34, 24-32,

24-30, 24-28, 24-26, 26-50, 26-48, 26-46, 26-44, 26-42, 26-40, 26-38, 26-36, 26-34, 26- 32, 26-30, 26-28, 28-50,

28-48, 28-46, 28-44, 28-42, 28-40, 28-38, 28-36, 28-34, 28-32, 28-30, 30-50, 30-48, 30- 46, 30-44, 30-42, 30-40,

30-38, 30-36, 30-34, 30-32, 32-50, 32-48, 32-46, 32-44, 32-42, 32-40, 32-38, 32-36, 32- 34, 34-50, 34-48, 34-46,

34-44, 34-42, 34-40, 34-38 34-36, 36-50, 36-48, 36-46, 36-44, 36-42, 36-40, 36-38, 38-50 , 38-48, 38-46, 38-44,

38-42, 38-40, 40-50, 40-48 40-46, 40-44, 40-42, 42-50, 42-48, 42-46, 42-44, 44-50, 44-48 , 44-46, 46-50, 46-48 or 48-50) nucleotides or nucleic base pairs. In some embodiments, the oligomeric compound has a nucleobase sequence that is at least partially complementary to the coding sequence of an expressed target nucleic acid or target gene within a cell. In some embodiments, the oligomeric compounds, when delivered to a cell expressing a gene, are capable of inhibiting the expression of the underlying gene and are referred to herein as expression-inhibiting oligomeric compounds. Gene expression can be inhibited in vitro or in vivo.

Oligomeric compounds include, without limitation, oligonucleotides, single stranded oligonucleotides, single stranded antisense oligonucleotides, short interfering RNA (siRNA), double stranded RNA (dsRNA), micro RNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and dicer substrates. In some embodiments, the oligomeric compound is a single chain oligomeric compound. In some embodiments, the oligomeric compound is a double-stranded oligomeric compound.

In some embodiments, one or more cargo molecules is/are an RNAi agent, which, as defined herein, is an agent that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of disrupting or inhibiting the translation of transcripts messenger RNA (mRNA) target mRNA in a sequence-specific manner. Within the scope of the invention, RNAi agents may act via the RNAi mechanism (i.e., inducing RNAi through interaction with the machinery of the RNAi pathway (RNA-induced silencing complex or RISC) of mammalian cells) or via any alternative mechanism(s) or pathway. (paths). Although RNAi agents, as the term is used herein, are believed to operate primarily through the mechanism of RNA interference, the disclosed RNAi agents are not associated with or limited to any particular route or mechanism of action. RNAi tools include, without limitation, single stranded oligonucleotides, single stranded antisense oligonucleotides, short interfering RNA (siRNA), double stranded RNA (dsRNA), micro RNA (miRNA), short hairpin RNA (shRNA), and dicer substrates.

Typically, RNAi agents may consist of at least a sense strand (also referred to as a follower strand) which contains a first sequence and an antisense strand (also referred to as a guide strand) which contains a second sequence. The sense and antisense strands of the RNAi agent can each be 16-49 nucleotides in length. In some embodiments, the sense and antisense strands of the RNAi agent are independently 17-26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 19-26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21-26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21-24 nucleotides in length. The sense and antisense strands can be either the same length or different lengths. The RNAi agents contain an antisense strand sequence that is at least partially complementary to the sequence of the target gene, and when delivered to a cell expressing the target, the RNAi agent can inhibit the expression of one or more target genes in vivo or in vitro.

Oligomeric compounds in general, and RNAi agents specifically, may consist of modified nucleotides and/or one or more non-phosphodiester linkages. Within the scope of the invention, a modified nucleotide is a nucleotide that is not 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 at least 98%, at least 99 or 100%) of the nucleotides are modified nucleotides. As used herein, a modified nucleotide includes, but is not limited to, deoxyribonucleotides, nucleotide mimics, nitrogen base removed nucleotides, 2'-modified nucleotides, 3'-3' linkage (inverted) nucleotides, non-natural base-containing nucleotides, bridging nucleotides, peptidic nucleic acids, 2' ,3'-seco nucleotide mimics (unlocked analogs of nucleic bases, locked nucleotides, nucleotides with 3'-O-methoxy (2' internucleoside bonds), 2'T-arabino nucleotides, 5'-Me, 2'-fluoro nucleotide, morpholino nucleotides, vinyl phosphonate deoxyribonucleotides containing vinyl phosphonate nucleotides and nucleotides containing cyclopropylphosphonate 2'-modified nucleotides (i.e., a nucleotide with a non-hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides, 2'-deoxy2'-fluoro nucleotides, 2'-deoxy nucleotides, 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides, 2'-amino nucleotides, and 2'-alkyl nucleotides.

In addition, one or more nucleotides of an oligomeric compound, such as an RNAi agent, may be linked by non-standard bonds or backbones (ie, modified internucleoside bonds or modified backbones). The modified internucleoside bond may be a phosphate-free covalent internucleoside bond. Modified internucleoside bonds or backbones include, but are not limited to, 5'-phosphorothioate groups, chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotris esters, aminoalkyl phosphotris esters, alkyl phosphonates (e.g., methyl phosphonates or 3'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3' -aminophosphoramidate, aminoalkylphosphoramidates or thionophosphoramidates), thionoalkylphosphonates, thionoalkylphosphoric triesters, morpholine linkages, boranophosphates having normal 3'-5' bonds, 2'-5' linked analogs of boranophosphates, or boranophosphates having a reverse orientation in which adjacent pairs of nucleoside units are linked 3'-5' from 5'-3' or 2'-5' from 5'-2'.

It is not necessary that all positions in a given compound be uniformly modified. Conversely, more than one modification may be included in one oligomeric compound, or even in one of its nucleotides.

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

In some embodiments, one or more cargo molecule(s) may contain or consist of a PEG moiety that may act as a pharmacokinetic (PK) modulator. In some embodiments, one or more cargo molecules may contain a PEG fragment having approximately 20-900 units of ethylene oxide (CH2-CH2-O) (e.g., 20-850, 20-800, 20-750, 20-700, 20- 650, 20-600, 20-550, 20-500, 20-450, 20-400, 20-350, 20-300, 20-250, 20-200, 20-150, 20-100, 20-75, 20-50 100-850 100-800 100-750 100-700 100-650 100-600 100-550 100-500 100-450 100-400 100-350 100- 300, 100-250, 100-200, 100-150, 200-850, 200-800, 200-750, 200-700,

200-650, 200-600, 200-550, 200-500, 200-450, 200-400, 200-350, 200-300, 200-250, 250-900, 250-850,

250-800, 250-750, 250-700, 250-650, 250-600, 250-550, 250-500, 250-450, 250-400, 250-350, 250-300,

300-900, 300-850, 300-800, 300-750, 300-700, 300-650, 300-600, 300-550, 300-500, 300-450, 300-400,

300-350, 350-900, 350-850, 350-800, 350-750, 350-700, 350-650, 350-600, 350-550, 350-500, 350-450,

350-400, 400-900, 400-850, 400-800, 400-750, 400-700, 400-650, 400-600, 400-550, 400-500, 400-450,

450-900, 450-850, 450-800, 450-750, 450-700, 450-650, 450-600, 450-550, 450-500, 500-900, 500-850,

500-800, 500-750, 500-700, 500-650, 500-600, 500-550, 550-900, 550-850, 550-800, 550-750, 550-700,

550-650, 550-600, 600-900, 600-850, 600-800, 600-750, 600-700, 600-650, 650-900, 650-850, 650-800,

650-750, 650-700, 700-900, 700-850, 700-800, 700-750, 750-900, 750-850, 750-800, 800-900, 850-900 or

- 26 042923

850-900 units of ethylene oxide). In some embodiments, one or more cargo molecule(s) consists of a PEG moiety having about 455 units of ethylene oxide (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. PEG fragments described herein may be linear or branched. PEG fragments can be discrete (monodisperse) or non-discrete (polydisperse). Fragments of PEG for use as a PK enhancing cargo molecule may be commercially available. In some embodiments, the one or more cargo molecule(s) comprises a PEG moiety that can act as a modulator or enhancer of PK, as well as another cargo molecule, such as a pharmaceutically active ingredient or compound.

The ανβ6 integrin ligands described contain salts or solvates thereof. Under solvate ligand ανβ6 understand the results of the connection of molecules of an inert solvent with the integrin ligand αγβ6, which are formed due to the force of their mutual attraction. Solvates are, for example, mono- or dihydrates or compounds with alcohols, for example with methanol or ethanol.

Free amino groups or free hydroxyl groups can be provided as substituents of the ανβ6 integrin ligands with appropriate protecting groups.

The αγβ6 integrin ligands also contain, for example, derivatives, i. ανβ6 integrin ligands modified, for example, with alkyl or acyl groups, sugars or oligopeptides, which are cleaved either in vitro or in the body.

In some embodiments, an αγβ6 integrin ligand disclosed herein facilitates delivery of a cargo molecule into the cytosol of a cell presenting ανβ6 integrin on its surface, either through ligand-mediated endocytosis, pinocytosis, or other means. In some embodiments, the ανβ6 integrin ligand disclosed herein facilitates the delivery of a cargo molecule to the cytoplasmic membrane of a cell presenting the ανβ6 integrin.

In some embodiments, the ανβ6 integrin ligand contains the structure represented by

(SEQ ID NO: 109) (Formula IX) wherein Z contains an amino terminal cap (e.g., any of the amino terminal caps described herein or known in the art) and R ⁵ and R ⁶ represent the amino acid side chains of Xaa ¹ and Haa ² respectively.

- 27 042923

In some embodiments, the ανβ6 integrin ligands contain the following structures, wherein Z and R ¹ are as defined for formula III and formula IV herein, and R ⁷ may be OH, J, JR ¹ , J-R'-R ^{2 ,} or Y -R'-R ³ (as each of them is defined for formula III and formula IV in the present description):

(SEQ ID NO: 110) (Formula X)

(SEQ ID NO: 111) (Formula XI)

UN

(SEQ ID NO: 112) (Formula XII)

(SEQ ID NO: 113) (Formula XIII)

- 28 042923

(SEQ ID NO: 116) (Formula XVI)

In some embodiments, the described ανβ6 ligands exhibited increased serum stability compared to the naturally occurring oxb6 integrin binding peptide RGDLATLRQL (SEQ ID NO: 1). As shown in the examples herein, only approximately 5% of the naturally occurring RGDLATLRQL peptide (SEQ ID NO: 1) can be detected after 4 hours incubation at 37° C. in mouse plasma. The RGDLATLRQL peptide (SEQ ID NO: 1) could not be detected after 8 hours at 37° C. in mouse plasma. In some embodiments, the αγβ6 integrin ligand disclosed herein exhibits greater than 20% remaining ligand as determined by HLPC after a 12 hour incubation at 37° C. in mouse plasma. In addition to having increased serum stability from the natural peptide, the described avp6 integrin ligands retained binding to (affinity for) the avp6 integrin.

Pharmaceutical compositions

In some embodiments, provided herein are pharmaceutical compositions that comprise or consist of, or essentially consist of, one or more of the αχβ6 integrin ligands disclosed herein.

Within the scope of the invention, the pharmaceutical composition contains a pharmacologically effective amount of an Active Pharmaceutical Ingredient (API) and optionally one or more pharmaceutically acceptable adjuvants. Pharmaceutically acceptable adjuvant(s) are substances other than the active pharmaceutical ingredient (API, therapeutic product) that are specifically contained in a drug delivery system. Adjuvants do not provide or are not intended to provide a therapeutic effect at the intended dosage. Aids may work to

a) assistance in processing the drug delivery system at the time of receipt;

b) protecting, maintaining or enhancing the stability, bioavailability or patient acceptability of the API;

c) facilitating product identification; and/or

d) enhancing any other feature of the overall safety, efficacy, delivery of the API during storage or use.

The pharmaceutically acceptable adjuvant may or may not be inert.

Adjuvants include, without limitation, absorption enhancers, anti-adhesives,

- 29 042923 blowing agents, antioxidants, binders, buffering agents, carriers, coating agents, colorants, delivery improvers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, dry diluents, fillers, flavors, lubricants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity regulators, carrier media, water repellants and wetting agents.

The pharmaceutical compositions described herein may contain other additional components often found in pharmaceutical compositions. In some embodiments, the additional component is a pharmaceutically active material. Pharmaceutical active materials include, but are not limited to, antipruritics, astringents, local anesthetics or anti-inflammatory agents (eg, antihistamine, diphenhydramine, etc.), a small molecule drug, an antibody, an antibody fragment, aptamers, and/or a vaccine.

The pharmaceutical compositions may also contain preservatives, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, coloring agents, flavoring agents, osmotic salts, buffers, coating agents or antioxidants. They may also contain other therapeutically useful agents.

Pharmaceutical compositions can be administered in many ways depending on whether topical or systemic treatment is required and on the area to be treated. May be administered by any method widely known in the art, such as, but not limited to, local (eg, via a transdermal patch), pulmonary (eg, by inhalation or insufflation of powders or aerosols, including nebulizer, intratracheal, intranasal), epidermal, transdermal , oral or parenteral. Parenteral administration includes, without limitation, intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous (eg, via an implanted device), intracranial, intraparenchymal, intrathecal, and intraventricular administration. In some embodiments, this document describes pharmaceutical compositions administered by subcutaneous injection. Pharmaceutical compositions can be administered orally, for example, in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions. The introduction can also be performed rectally, for example, using suppositories; topically or transdermally, for example using ointments, creams, gels or solutions; or parenterally, for example, using injectable solutions.

Pharmaceutical compositions suitable for injectable use contain sterile aqueous solutions (when soluble in water) or dispersions and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), or phosphate buffered saline. It must be stable under the conditions of preparation and storage and must contain preservatives against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating, such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. In many cases it will be preferable to have isotonic agents, eg sugars, polyalcohols, eg mannitol, sorbitol and sodium chloride in the composition. Prolonged absorption of injectable compositions may be due to the content of an agent in the composition that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be obtained by incorporating in the appropriate solvent, if necessary, the active compound in the required amount with one or a combination of the ingredients listed above, followed by filtered sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile carrier medium which contains the basic dispersion medium and the other necessary ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods include vacuum drying and freeze drying to give a powder of the active ingredient plus any additional desired ingredient from its previously filter-sterilized solution.

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

Active connections can be obtained with media that will protect the connection from

- 30 042923 strictly removed from the body, for example, a controlled release formulation containing implants and microcapsule delivery systems. You can use biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. It will be apparent to those skilled in the art how such formulations can be prepared. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. They can be obtained according to methods known to those skilled in the art, for example, as described in US patent No. 4522811.

The pharmaceutical composition may contain other additional components often found in pharmaceutical compositions. Such additional components include, without limitation, antipruritics, astringents, local anesthetics, or anti-inflammatory agents (eg, antihistamine, diphenhydramine, etc.). As used herein, a pharmacologically effective amount, a therapeutically effective amount or simply an effective amount refers to an effective amount of a pharmaceutically active agent to produce a pharmacological, therapeutic or prophylactic result.

αΎβ6 ligand-containing drugs are also an object of the present invention, as well as methods for preparing such drugs, the methods comprising delivering one or more αΎβ6 ligand-containing compounds, optionally one or more other substances of therapeutic value, in a dosage form suitable for administration to patients -people.

Cells, tissues and non-human organisms.

Cells, tissues, and non-human organisms are provided that contain at least one of the αΎβ6 ligands described herein. A cell, tissue, or non-human organism is obtained by delivering the ave6 ligand to the cell, tissue, or non-human organism by any means available in the art. In some embodiments, the cell is a mammalian cell, including without limitation a human cell.

The αΎβ6 ligands described and the αΎβ6 ligand-containing pharmaceutical compositions disclosed herein may be packaged or contained in a kit, container, pouch, or dispenser. αΎβ6 ligands and pharmaceutical compositions containing αΎβ6 ligands can be packaged in pre-filled syringes or vials.

The embodiments and points provided above are further illustrated with the help of the following non-limiting examples.

Examples

Example 1 Synthesis of αΎβ6 Ligands for Serum Stability Studies.

Rink's Chem-Matrix amide resin was placed in a porous polypropylene syringe and agitated in DCM for 30 minutes prior to use. The following standard conditions for solid phase peptide synthesis were used. Fmoc deprotection was performed by soaking up 40 ml of piperidine:DMF (20:80 v/v) per mmol resin for 20 min. Amide coupling was carried out by soaking the resin with 4 molar equivalents of Fmoc-amino acid, 4 molar equivalents of HBTU, and 10 molar equivalents of diisopropylethylamine in DMF at a concentration of 0.1 M Fmoc-amino acid in DMF for 40 minutes. FmocDap(DNP)-OH was used to couple the DNP chromophore to the resin and a peptide was synthesized from Dap α-amine. Resin digestion was carried out in a trifluoroacetic acid solution for 2 hours. The solvent was reduced to 10% of the original volume with pressurized air and precipitated using Et ₂ O. Microdigestion with TFA and analytical HPLC-MS confirmed the identity of the product. The peptides were then purified to >95% purity on Shimadzu prep HPLC using elution on a wide pore Supelco Discovery BIO C18 column (25 cm x 21 mm, 10 µm particles) with linear gradients of approximately 1 ml/min. Purity was assessed using a Shimadzu analytical HPLC equipped with a Waters XBridge BEH130 C18 column (250 x 6.6 mm, 5 pm particles) using 10-90% solvent B over 50 minutes. Solvent A is H ₂ O:F ₃ CCO ₂ H 100:0.1 v/v, solvent B is CH ₃ CN:F ₃ CCO ₂ H 100:0.1 v/v.

- 31 042923

Fmoc-Dap(DNP)-OH

Example 2 Serum stability of ανβ6 ligands.

Serum stability of ave6 ligands was tested by incubating αγβ6 ligands in mouse serum and analyzing the percentage of uncleaved peptide at different time points. Uncleaved ave6 ligand was determined by analytical HPLC. Separate stock solutions of ave6 ligands were prepared by dissolving the peptides in H ₂ O at >10 mg/mL. The αΎβ6 ligand concentration was estimated using UV/Vis absorbance (DNP: γ=365, ε=17300 M' ¹ cm' ¹ ). The ave6 ligand was diluted to 1 mg/ml ligand in 90% mouse plasma and placed in an incubator at 37°C. At predetermined time points (4, 8, 12, and 24 h), the sample was injected into an analytical HPLC (Shimadzu HPLC) equipped with a Waters XBridge BEH130 C18 column (250 x 6.6 mm, 5 µm particles) using 10-90% solvent B for 50 min. Solvent A is H ₂ O:F ₃ CCO ₂ H 100:0.1 v/v, solvent B is CH ₃ CN:F ₃ CCO ₂ H 100:0.1 v/v.

The percentage of ligand remaining after serum incubation was calculated using the following equation:

Remaining %=[(Area at t=x)+(Area at t=0)] x 100% where Area at t=0 was the area under the ligand peak immediately after plasma dilution of the ligand and peptide peak at time=x.

Each peptide was covalently linked to a PEG8-Dap(DNP) cargo molecule. PEG8-Dap(DNP) was then used to facilitate analysis.

The peptide derivatives were coupled to the PEG8-Dap(DNP) cargo molecule and incubated in mouse serum at 37°C for 4, 8, 12, or 24 hours. The stability of the peptide derivatives was measured by HPLC. The data is shown in the following table. 1 (aminoterminal cap and Xa1Xaa ² are underlined).

- 32 042923

Table 1

Serum stability of ανβ6 ligands

Peptide Derivative SEQ ID NO. Remaining % t=4 h t=8 h t=12 h t=24 h RGDLATLRQL 1 5 <0.1 <0.1 Ac-RGDLATLTQL 2 90 67 34 RGDLAAbuLCitAibL 3 48 16 5 Ac-RGDLAAbuLCitAibL 4 94 82 72 Ac-RGDLAAbuLCitAibL 4 86 75 67 41 Ac-RGDLAAbuLCitAib 5 95 82 73 48 Ac-RGDLAAbuLCit 6 88 77 69 44 Ac-RGDLAAbuL 7 91 85 80 69 Ac-RGDLAAbuLCitAib 5 72 Ac-RGDLAAbuLCitK 8 35 Ac-RGDLAAbuLCitE 9 79 Ac-RGDLAAbuLCitF 10 22 Ac-RGDLAAbuLCitO eleven insoluble Ac-RGDLAAbuLCitG 12 49 Ac-RGDLAAbuLCitAib 5 72 Ac-RGDLAAbuLKAib 13 35 Ac-RGDLAAbuLEAib 14 79 Ac-RGDLAAbuLFAib 15 22 Ac-RGDLAAbuLOAib 16 insoluble Ac-RGDLAAbuLGAib 17 49 Ac-RGDLAAbuLCitAib 5 52 CfRCfECO-RGDLAAbuLCitAib 18 49 CFEtCFbECQ-RGDLAAbuLCitAib 19 49 CFEtCFbECO-RGDLAAbuLCitAib 20 58 CFEFCFbECQ-RGDLAAbuLCitAib 21 51 Ac-RGDLAAbuLCitAib 5 87 Ac-RGDLKAbuLCitAib 22 71 Ac-RGDLEAbuLCitAib 23 98 Ac-RGDLFAbuLCitAib 24 64 Ac-RGDLOAbuLCitAib 25 87 Ac-RGDLAAbuLCitAib 5 85 Ac-RGDLPAbuLCitAib 26 91 Ac-RGDLAKLCitAib 27 72 Ac-RGDLAELCitAib 28 96 Ac-RGDLAELCitAib 29 88 Ac-RGDLAAbuLCitAib 5 72 Ac-RGDLGAbuLCitAib thirty 82 Ac-RGDLAGLCitAib 31 92 Ac-RGDLAPLCitAib 32 84 Ac-RGDLAAbuLCitP 33 68 Ac-RGDLAAbuLCit 6 79

As shown herein, the presence of the amino terminal cap (Z) may provide increased serum stability. In addition, as shown herein, the presence of non-standard amino acids in Xaa ¹ , Xaa ² and/or J (e.g., Xaa ³ and Xaa ⁴ ) in the formulas disclosed herein also provide increased serum stability compared to the natural SEQ peptide. ID NO: 1.

Example 3 Integrin Binding of ανβ6 Ligands.

A. Ligand αvβ6-cargo molecule conjugation.

Each ανβό ligand was coupled to a reversibly modified polymeric cargo molecule 1170-100B. Polymer cargo molecule 1170-100B (56:44 ethoxyethylamine acrylate:propyl acrylate copolymer having MW approximately 45,000) was labeled with Cy5 (NHS linker) and combined with aldehyde-PEG ₂₄ ACit at a 2:1 weight ratio (polymer:aldehyde-PEG ₂₄ -ACit ) in 50 mM HEPES buffer pH 9.0 for 1 hour at RT to form (aldehyde-PEG ₂₄ -ACit) _n -1170-100V, where n is a number greater than 0.

Usually, n was around 10.

The aldehyde-PEG ₂₄ -ACit-modified polymer was then reacted with PEG ₁₂ -ACit at a weight ratio of 1:8 (polymer: PEG ₁₂ -ACit) in 50 mM HEPES buffer, pH 9.0 for 1 h at RT to form (aldehyde-PEG ₂₄ -ACit) _n -1170-100B-(CitA-PEG ₁₂ ) _m , where m is a number greater than 0.

The modified polymer was then purified using a sephadex G-50 centrifuge column and the fluorescence concentration was determined. Su5 after purification mg ..

cone fluorescence. Cy5 was modified with HyNic prior to purification by ^KOHIb purification to facilitate conjugation with the cargo molecule.

polymer^) =

Each ligand αγβ 6

The purified polymer was combined with ave6-(PEG) ₈ -K-HyNic ligand at a weight ratio of 1:1.9 (polymer:ανβ6 ligand) in 50 mM NaOAC-HOAc buffer, pH 5.0 at RT overnight to form a ligand conjugate αvβ6 polymer.

The αγβ 6-polymer ligand conjugate was purified using a sephadex G-50 centrifuge column.

- 34 042923

Ligand ανββ-(PEG) ₈ -K-HyNic

Conjugation efficiency was quantified by measuring the absorptivity of the αγβ6-polymer conjugate at 354 nm using an attenuation factor of 2.9x101 M ^-1 cm ^-1 for the bis-arylhydrazone bond.

Polymer Molar Concentration (mM) _ Polymer Mass Concentration (mg/mL) % Polymer Molecular Weight (Daltons)

Molar concentration ανβό (mM) _ / A ₃₅₄ (con.-au $ 6) - A ₃₅₄ (control con. bezau ^ b)] ^\u003d 29 _TG o molar concentration otvR6 (m ^ A)

Quantity sharp per polymer =--------------------—------ ^! ' polymer molar concentration (mM)

For further analysis, the αγβ6-polymer conjugates were diluted with isotonic glucose solution to the required concentrations.

B. αγβ6 Ligand Binding (Flow Cytometric Analysis).

To evaluate the binding specificity of αγβ6 ligands to αγβ6 integrin, each αγβ6 ligand or negative control peptide was conjugated to a Cy5 labeled polymer (as described above) and evaluated for cell binding. HUH7 (human hepatocellular carcinoma) and SKOV3 (human ovarian carcinoma) cells were determined to show very low cell surface expression of αγβ6 and were used as negative control cell lines. H2009 (human lung epithelial adenocarcinoma) and CAPAN-2 (human pancreatic adenocarcinoma) cells served as αγβ6 positive control cell lines. Cells were separated from the culture flasks with accutase, washed in PBS, and seeded into 5 ml round bottom polystyrene tubes at 200,000 cells in 200 μl of complete medium (culture medium supplemented with fetal calf serum). αγβ 6-polymer ligand conjugates or polymer conjugates without ligand were added at 5 pg/mL (polymer-Cy5 concentration) to cells, mixed and incubated at 37°C for 3 h. Incubation at 37°C facilitated the ligand/receptor interaction, which may result in static binding to the extracellular surface of cells and/or internalized ligand/receptor complexes. The mixture was resuspended at 1 hour intervals. After 3 hours of incubation, cells were washed with 2x4 ml of chilled buffer (PBS-2% FCS) and resuspended in 200 μl of buffer containing 10 μM SYTOX blue dye for live/dead cell selection. Samples were analyzed on a Canto II BD Biosciences cytometer equipped with violet (405 nm), blue (488 nm), and red (633 nm) lasers.

Viable cells were first selected as a blue SYTOX negative population on a C detector with a violet laser. These viable cells were then assessed for red laser conjugate binding/uptake as mean fluorescence intensity (MFI) of the Cy5 fluorophore. Data analysis was performed using FlowJo γ10.1 software. The specific MFI coefficient (sMFIr) for each αγβ6 ligand was determined by the following formula:

MFI value for a specific sample/MFI value without ligand.

As shown in Table. 2 below, only the Ac-RGDLAc-LCitAibL peptide showed moderate binding to SKOV3 cells. This peptide did not show significant binding to HUH7 cells. Thus, none of the tested peptides showed non-specific binding. The negative control peptides AcRGaMeDLAc-LCitAib, AcRGDaMeLAc-LCitAib, RGELATLRQL, AcCitGDLATLCitQL, AcK(Me) ₂ GDLATLRQL, and AcR(Me) ₂ GDLATLRQL, which do not contain RGD, did not show significant binding to cells of the positive control integrin αγβ6, H2009, or CAPAN-2 35 042923 showing no affinity for the integrin ανβ6. Most of the other peptides showed binding to H2009 or CAPAN-2 cells that was comparable to or greater than natural peptides,

RGDLATLRQL and RGDLATL, showing good affinity for the αγβ6 integrin.

table 2

ανβ6 ligand binding to expressing (H2009 and CAPAN-2) and non-expressing (HUH7 and SKOV3) αγβ6 integrin cells

Peptide SEQ ID NO MFI HUH7 SKOV3 H2009 CAPAN-2 Ac-RGDLAc-LCitAibL 4 l,o 3.2 7.7 Ac-RGaMeDLAc-LCitAib 34 0.9 0.9 1.0 Ac-RGDaMeLAc-LCitAib 35 0.9 0.8 0.9 Ac-RGDLAc-L 7 0.9 2.6 7.9 Ac-RGDLAc-LAib 36 0.9 3.2 8.9 RGDLATLRQL 1 1.4 4.7 RGELATLRQL (RGE control) 37 0.99 RGDLATLRQLEEEK-(HyNic) 38 6, 5 meta-guanidine-benzo!c-GDLATLRQL 39 4.5 Ac-RGDLATLRQL 2 4.7 Me-RGDLATLRQL 40 4.8 Guanidinyl-RGDLATLRQL 41 4.3 MeO-PEG8-RGDLATLRQL 42 2.5 Ac-RGDLALLRQL 43 6, 06 Ac-RGDLAc-LRQL 44 6.47 Ac-RGDLAILRQL 45 6.22 Ac-RGDLAVLRQL 46 6.23 Ac-CitGDLATLRQL 47 2.45 Ac-RGDLATLCitQL 48 3.94 Ac-CitGDLATLCitQL 49 0.99 Ac-RGDLATLRAbuL 50 4.26 Ac-RGDLATLRAibL 51 3.9 Ac-RGDLATLRDap(Ac)L 52 4.18 Ac-RGDLATLRCitL 53 4.07 Ac-RGDLATLRNvaL 54 4.26 RGDLATLRQL 55 4.32 Ac-RGDLAc-LCitAibL 4 2.25 4.55 Ac-K(Me) ₂ GDLATLRQL 56 0.9 1.1 Ac-R(Me) ₂ GDLATLRQL 57 0.9 1 Dap(guanidine)-GDLATLRQL 58 1.4 3.4 desamino-RGDLATLRQL 59 0.8 1.1 Ac-RGDL(33-hATLRQL 60 1.2 4.5 Ac-RGDLAibTLRQL 61 1.4 4 Ac-RGDLChaTLRQL 62 1 5.1 RGDLATLRQ 63 1.6 4.7 RGDLATLR 64 1.4 4.6 RGDLATL 65 1.2 2.9 RGDLAT 66 0.9 1.3 Ac-RGDLAibAbuLCitAib 67 Ac-RGDL(33-hAc-LCitAib 68 2.34 Ac-RGDLChAc-LCitAib 69 1.6 Ac-RNMe GDLAT LRQL 70 0.97 4.61 Ac-RGDLAc-LCitAib 5 4.7 Ac-RNMeGDLAc-LCitAib 71 2.6 Ac-RGDNMeLAc-LCitAib 72 1 Ac-RGDLAc-N _Me LCitAib 73 1.3 CH3 CH ₂ -RGDL Ac-LCit Aib 74 4.4

- 36 042923

CH ₃ ( CH ₂ ) 2-RGDLAc-LCitAib 75 3, 9 CH ₃ ( CH ₂ ) s-RGDLAc-LCitAib 76 4.7 CH ₃ ( CH ₂ ) 4-RGDLAc-LCitAib 77 4.1 Ac-RGaMeDLAc-LCitAib 34 1 Ac-RGDaMeLAc-LCitAib 35 1 Ac-RGDLKAbuLCitAib 22 3, 6 Ac-RGDLEAbuLCitAib 23 2.6 Ac-RGDLFAbuLCitAib 24 2.7 Ac-RGDLQAbuLCitAib 25 3.7 Ac-RGDLGAbuLCitAib thirty 3.7 Ac-RGDLPAbuLCitAib 26 1 Ac-RGDLAKLCitAib 27 3, 6 Ac-RGDLAELCitAib 28 2.4 Ac-RGDLAFLCitAib 29 2 Ac-RGDLAQLCitAib 78 3, 6 Ac-RGDLAPLCitAib 32 2.8 Ac-RGDLAc-LCit 6 3.3 Ac-RGDL Ac-LF Aib 15 3, 6 Ac-RGDLAc-LPAib 79 16 Ac-RGDLAc-LCitK 8 5.2 Ac-RGDLAc-LCitE 9 4 Ac-RGDLAc-LCitF 10 3.7 Ac-RGDLAc-LCitQ eleven 4.4 Ac-RGDLAc-LCitG 12 4.8 Ac-RGDLAc-LCitAib 5 3, 8 CH ₃ ( CH ₂ ) iCO-RGDLAc-LCitAib 80 4.2 CH ₃ ( CH ₂ ) ₂ CORGDLAc-LCitAib 19 4.3 CH ₃ (CH ₂ ) sCO-RGDLAc-LCitAib 20 4, 1 CH ₃ ( CH ₂ ) ₄ CO-RGDLAc-LCitAib 21 3, 9 CH ₃ 0 (CH ₂ CH ₂ 0) iCH ₂ CH ₂ C0-RGDLAc-LCitAib 81 3, 8 CH3O (CH ₂ CH ₂ 0 ) ₂ CH ₂ CH ₂ C0-RGDLAc-LCitAib 82 3, 6 CH3O (CH ₂ CH ₂ 0) ₃ CH ₂ GH ₂ G0-RGDLAc-LCitAib 83 3.7 CH3O (CH ₂ CH ₂ 0) ₅ CH ₂ CH ₂ C0-RGDLAc-LCitAib 84 16 Ac-RGDLATLRQL 2 4.7 RGDLAc-LCitAibL 3 4.6 Ac-RGDLAc-LCitAibL 4 4.6 Ac-RGDLAc-L 7 2.6 Ac-RGDLAc-LAib 36 3.2 Ac-RGDLAc-LKAib 13 5.4 Ac-RGDLAc-LEAib 14 19 Ac-RGDLAc-LGAib 17 4 Ac-RGDLAc-LCitP 33 4 Ac-RGDLAc-LQAib 16 4.4 Ac-RGDLAGLCitAib 31 3.8

- 37 042923

Some abbreviations for non-standard amino acids and other chemical groups identified in the previous table have the following chemical structures:

Aib represents α-aminoisobutyric acid

Cit Presents Citrulline

Abu Presents

L-a-aminobutyric acid aMeD is α-methylaspartate

aMeL represents a-methylleucine

Cha presents

Zdiclohexylalanine

Nva introduces Norvaline

K(Me)2 represents

N,N-ε-dimethyllysine

- 38 042923

guanidinylarginine

Example 4 In vivo Intratracheal Administration of Alpha-ENaC Targeting RNAi Agents Conjugated to ανβ6 Integrin Ligands in Rat.

Double-stranded oligonucleotide compositions that contained a sense strand and an antisense strand each of less than 26 nucleotides (i.e., an RNAi agent species) were synthesized according to the solid phase phosphoramidite method according to general techniques known in the art and widely used. in the synthesis of oligonucleotides. Synthesis of the RNAi agents herein was performed on a solid support made from commercially available pre-set pore glass (CPG, 500 A or 600 A obtained from Prime Synthesis, Aston, PA, USA) using either MerMade96E® (Bioautomation) , MerMade12® (Bioautomation) or OP Pilot 100 (GE Healthcare) for synthesis depending on scale. All RNAs and 2'-modified RNAs phos

- 39 042923 foramitides 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 40 wt.% solution of methylamine in water and 28% ammonium hydroxide solution (Aldrich ) with a volume of 1:1 for 1.5 h at 30° C. The solution was evaporated and the dry residue was reconstituted in water.For purification, the crude oligomers were purified by anion exchange HPLC using a 13 μm TSKgel SuperQ-5PW column and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0, and contained 20% acetonitrile, and Buffer B was the same as Buffer A with 1.5 M sodium chloride added.UV traces were recorded at 260 nm. fractions were pooled, then run on size exclusion HPLC using a GE Healthcare XK 26/40 column packed with fine Sephadex G-25 with working buffer of 100 mM ammonium bicarbonate, pH 6.7 and 20% acetonitrile. solutions of RNA (sense and antisense) in 1xPBS (phosphate buffered saline, 1x, Corning, Cellgro) to form the RNAi agent.

Some RNAi agents were lyophilized and stored at -15-25°C. Double concentration was determined by measuring the absorbance of the solution on a UV-Vis spectrometer in 1xPBS. The absorbance of the solution at 260 nm was then multiplied by the conversion factor and the dilution factor to determine the double concentration. Unless otherwise stated, the entire conversion factor was 0.037 mg/(mL-cm). For some experiments, the conversion factor was calculated from the experimentally determined attenuation factor.

The RNAi tools synthesized for Example 4 contained an antisense strand having a nucleobase sequence at least partially complementary to a gene expressing the alpha amiloride-responsive epithelial sodium channel (commonly referred to as alpha-ENaC or SCNN1A) subunit. RNAi agents for alpha-ENaC have been designed to disrupt or inhibit translation of alpha-ENaC messenger RNA (mRNA) transcripts in a sequence-specific manner, thereby inhibiting expression of the alphaENaC gene. The RNAi tools consisted of modified nucleotides and more than one non-phosphodiester bond.

On days 1 and 2 of the study, male Sprague-Dawley rats were dosed intratracheally with a micronebulizer device (Penn Century, Philadelphia, PA) at 200 µl, which included the following drug treatment groups (1) 5% dextrose in aqueous vehicle (D5W);

(2) 1.5 mg/kg anti-alpha-ENaC RNAi agent without ligand (naked RNAi agents) as a formulation with 5% dextrose;

(3) 1.5 mg/kg anti-alpha-ENaC RNAi agent conjugated to the αγβ6 integrin ligand of FIG. 3 (with ανβ6 integrin ligand conjugated at the 5' terminal end of the sense strand) as a formulation with 5% dextrose; or (4) 1.5 mg/kg anti-alpha-ENaC RNAi agent conjugated to an inactive αγβ6 integrin ligand having the structure Ac- ^R GELAc-L-oitAibL (seq id NO: 132) serving as a negative control ligand. Ligand binding to alpha-v integrin receptors is believed to require aspartic acid (D) in the 'RGD' motif, and ligands with glutamic acid (E) substitution have a significantly reduced binding affinity for av integrin. Groups 2, 3 and 4 used the same RNAi anti-alpha-ENaC agent.

The αγβ6 integrin ligand used was synthesized as a TFP ester (as shown in FIG. 1) using general peptide synthesis methods well known in the art and similar to those set forth in Example 1 herein, except for resin cleavage, which was achieved by using 20% HFIP (hexafluoroisopropanol) in DCM (dichloromethane) for 30 minutes to one hour instead of TFA digestion. The 5' terminal end of the sense strand of the RNAi agent was modified with C ₆ amine (-NH2). The αγβ6 TFP-ester integrin ligand was then conjugated to the amino group located at the 5' terminal end of the modified sense strand of the RNAi agent using 3 equivalents of the αγβ6 TFP-ester integrin ligand in 9:1 DMSO:water and an excess of triethylamine as base, with room temperature. Purification was carried out by adding ACN to the solution to precipitate the product and dry under high vacuum.

Four (4) rats per group received treatment. Rats were sacrificed on day 5 of the study and total RNA was isolated from both lungs after collection and homogenization. Alpha-ENaC mRNA representation was quantified by probe-based qPCR, normalized for GAPDH expression, and expressed as a vehicle control group fraction (geometric mean +/- 95% confidence interval).

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Table 3

Relative expression of alpha-ENaC mRNA normalized to the control of example 4

Group Relative expression (Geometric mean) Lower/Upper 95% Confidence Interval (1) carrier medium 5% dextrose 1,000 0.77/1.30 (2) Naked RNAi agent (no ligand) 0.54 0.24/1.22 (3) ανβ6 ligand conjugate FIG. 3-Remedy 0.22 0.11/0.44 (4) conjugate RGE-control ligand- 0.43 0.26/0.73

As shown in Table. 3 above, the ave6 ligand of FIG. 3 conjugated to an RNAi agent to alphaENaC showed an increased relative knockdown of alpha-ENaC mRNA (approximately 78% knockdown) compared to a naked RNAi agent (46% knockdown) and an RNAi agent conjugated to an RGE-control ligand (57% knockdown) alpha -ENaC lung targets in vivo.

Example 5 In vivo Intratracheal Administration of Alpha-ENaC Targeting RNAi Agents Conjugated to ave6 Integrin Ligands in Rat.

Anti-alpha-ENaC RNAi agents similar to those described in Example 4 were synthesized following the same synthetic procedures. On study days 1 and 2, male Sprague-Dawley rats were dosed with a 200 μl micronebulizer device (Penn Century, Philadelphia, PA) which included the following drug groups:

(1) carrier medium D5W;

(2) 1.5 mg/kg ligand-free RNAi agent (naked RNAi agent) as a formulation in D5W;

(3) 1.5 mg/kg anti-alpha-ENaC RNAi agent conjugated to the ave6 integrin ligand of FIG. 3, in the form of a finished form in D5W; or (4) 1.5 mg/kg anti-alpha-ENaC RNAi agent conjugated to an ave6 integrin tridentate ligand having the structure shown in FIG. eleven.

RNAi agents have been developed to inhibit alpha-ENaC gene expression. The same anti-alpha-ENaC RNAi agent was used in groups 2, 3 and 4. The 5' terminal end of the sense strand of the RNAi agents was modified with C ₆ amine (-NH2) as in Example 4. The ave6 integrin ligands of FIG. 3 was synthesized and conjugated to anti-alpha-ENaC RNAi agents following the same procedures of Example 4. When conjugated to the ave6 ligand shown in FIG. 11, the RNAi agent to alpha-ENaC was first functionalized with DBCO-PEG ₅ -ester NHS by conjugation to the 5' amine functionalized terminal end of the sense strand using triethylamine as a base. A tridentate ave6 integrin ligand having a PEG-azide reactive group was synthesized as shown in FIG. 10. After precipitation in a phosphate buffered saline/acetonitrile solvent system, the ave6 integrin tridentate ligand was conjugated to the RNAi agent using copper-free cycloaddition.

Five (5) rats per group received treatment. Rats were sacrificed on day 5 of the study and after collection and homogenization, total RNA was isolated from both lungs. Target mRNA representation was quantified by probe-based qPCR, normalized for GAPDH expression, and expressed as a vehicle control group fraction (geometric mean +/- 95% confidence interval).

Table 4

Relative expression of alpha-ENaC mRNA normalized to the control of example 5

Group Relative expression (Geometric mean) Lower/Upper 95% Confidence Interval (1) carrier medium 5% dextrose 1,000 0.72/1.40 (2) Naked RNAi agent (no ligand) 0.34 0.25/0.45 (3) ανβ6 ligand conjugate FIG. 3-remedy 0.29 0.15/0.55 (4) conjugate [ligand ανβ6 Fig. 11 ((i.e. Ligand ανβ6)h)]-RNAi agent 0.21 0.09/0.51

As shown in Table. 4 above, conjugation of three ave6 ligands (forming a tridentate ligand) as shown in FIG. 11, with the anti-alpha-ENaC RNAi agent, showed increased relative knockdown (approximately 79%) compared to the RNAi agent conjugated to ανβ6 ligand alone (Fig. 3) (71%) and the naked RNAi agent (66 %) in vivo.

Example 6 Intratracheal administration of In vivo RNAi agents targeting alpha-ENaC conjugated to ave6 integrin ligands in rats.

Anti-alpha-ENaC RNAi agents similar to those described in Example 4 were synthesized following the same synthetic procedures. On study days 1 and 2, male Sprague-Dawley rats were dosed with a 200 μl micronebulizer device (Penn Century, Philadelphia, PA) which included the following drug treatment groups:

(1) carrier medium D5W;

(2) 3 mg/kg ligand-free RNAi agent (naked RNAi agent) as a formulation in D5W; or (3) 3.0 mg/kg anti-alpha-ENaC RNAi agent conjugated to the αγβ6 integrin ligand of FIG. 3 as a finished form in D5W.

The anti-alpha-ENaC RNAi agent and av36 integrin ligands were synthesized and conjugated according to the same procedures as described in Example 4.

Five (5) rats per group received treatment. Rats were sacrificed on day 5 of the study and after collection and homogenization, total RNA was isolated from both lungs. Target mRNA representation was quantified by probe-based qPCR, normalized for GAPDH expression, and expressed as a vehicle control group fraction (geometric mean +/- 95% confidence interval).

Table 5

Relative expression of alpha-ENaC mRNA normalized to the control of example 6

Group Relative expression (Geometric mean) Lower/Upper 95% Confidence Interval (1) carrier medium 5% dextrose 1,000 0.76/1.31 (2) Naked RNAi agent (no ligand) 0.49 0.42/0.56 (3) ανβ6 ligand conjugate FIG. 3-Remedy RNAi 0.17 0.12/23

As shown in Table. 5 above, the avp6 ligand of FIG. 3 conjugated to the RNAi agent showed an increased relative knockdown (approximately 83% knockdown) compared to the naked RNAi agent (approximately 51% knockdown) of the lung target in vivo.

Example 7 Oropharyngeal aspiration In vivo RNAi agents targeting a gene expressed in the lung conjugated to ave6 integrin ligands in rats.

Anti-alpha-ENaC RNAi agents similar to those described in Example 4 were synthesized following the same synthetic procedures. On study day 1, male Sprague-Dawley rats were dosed by oropharyngeal aspiration with a dose of 200 µl, which included the following drug groups:

(1) isotonic saline solution;

(2) 0.5 mg/kg RNAi alpha-ENaC targeting agent conjugated to the ave6 integrin ligand of FIG. 3, as a formulation in isotonic saline; or (3) 0.5 mg/kg of an RNAi alpha-ENaC targeting agent conjugated to the ave6 integrin ligand of FIG. 5 as formulated in isotonic saline.

The same RNAi anti-alpha-ENaC agents were used for groups 2 and 3. The 5' terminal end of the sense strand of the RNAi agent was modified with _{C 6} -amine (-NH2) as described in Example 4. RNAi anti-alpha-ENaC agent and avp6 integrin ligands fig. 3 was synthesized and conjugated according to the same procedures as described in Example 4. The avP6 integrin ligands of FIG. 5 was synthesized as a TFP ester, and in addition, the N-terminus of the ave6 integrin ligand was protected with an fmoc group. The RNAi agent was then conjugated in the same manner as described in Example 4, followed by fmoc deprotection using triethylamine as a base.

Five (5) rats per group received treatment. Rats were sacrificed on study day 9 and after collection and homogenization, total RNA was isolated from both lungs. Target mRNA representation was quantified by probe-based qPCR, normalized for GAPDH expression, and expressed as a vehicle control group fraction (geometric mean +/- 95% confidence interval).

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Table 6

Relative expression of alpha-ENaC mRNA normalized to the control of example 7

Group Relative expression (Geometric mean) Lower/Upper 95% Confidence Interval (1) Isotonic Saline Solution 1,000 0.85/1.17 (2) ανβ6 ligand conjugate FIG. 3-Remedy RNAi 0.632 0.49/0.80 (3) ανβ6 ligand conjugate FIG. 5-Remedy RNAi 0.592 0.50/0.70

As shown in Table. 6 above, both ave6 ligands of FIG. 3 and FIG. 5 conjugated with an RNAi agent demonstrated knockdown of lung target alpha-ENaC in vivo.

Example 8 Intratracheal administration of in vivo alpha-ENaC targeting RNAi agents conjugated to ave6 integrin ligands and a poly-L-lysine scaffold in rats.

Anti-alpha-ENaC RNAi agents similar to those described in Example 4 were synthesized following the same synthetic procedures. On days 1 and 2 of the study, male Sprague-Dawley rats were dosed with 200 μl of either (1) D5W (5% dextrose in water) using a microspray device (Penn Century, Philadelphia, RA); or (2) 0.5 mg/kg ligand-free RNAi agent (naked RNAi agent) as a formulation in D5W; or (3) 1.5 mg/kg naked RNAi formulation in D5W; or (4) 5 mg/kg naked RNAi formulation in D5W; or (5) 0.5 mg/kg anti-alpha-ENaC RNAi agent conjugated to the av36 integrin ligand of FIG. 4 by means of a semi-L-linear (PLL) framework, in the form of a finished form in D5W; or (6) 1.5 mg/kg anti-alpha-ENaC RNAi agent conjugated to the av36 integrin ligand of FIG. 4 by means of a PLL framework, as a finished form in D5W; or (7) 5 mg/kg anti-alpha-ENaC RNAi agent conjugated to the ave6 integrin ligand of FIG. 4 by means of a PLL framework as a finished form in D5W.

RNAi agents have been developed to inhibit alpha-ENaC gene expression. The same RNAi anti-alpha-ENaC agent was used in groups 2-7. The αγβ6 integrin ligand was originally synthesized as a TFP ester (shown in FIG. 2). The PLL framework used in groups 5, 6 and 7 of Example 8 was approximately one hundredth (100) L-lysine monomer units (approximately 12 kilodaltons). The poly-L-lysine polymer was modified with 3 equivalents of SMPT (4-succinimidyloxycarbonylalpha-methyl-a(2-pyridyldithio)toluene) and the 5' amine (modified _C6 -amine) of the sense strand of the RNAi agent was modified with SATA (N-succinimidyl S-acetylthioacetate ). Next, the ave6 integrin ligand of FIG. 2 (15 equivalents) was added as a solid and shaken for 1 hour. Protease-cleavable functionalized alanine-citrulline-PEG ₁₂ (10 equivalents; functionalized with p-nitrophenyl carbonate) was then added. After 15 minutes, the SATA-modified RNAi agent (one equivalent) was added dropwise maintaining the pH at 8.6. The remaining lysine groups were functionalized with protease-cleavable functionalized alanine-citrulline PEG12. The product was purified by tangential flow filtration.

Five (5) rats per group were treated. Rats were sacrificed on day 5 of the study and after collection and homogenization, total RNA was isolated from both lungs. Target mRNA representation was quantified by probe-based qPCR, normalized for GAPDH expression, and expressed as a vehicle control group fraction (geometric mean +/- 95% confidence interval). The data is shown in the graph of Fig. 12.

As shown in FIG. 12, the ave6 ligand of FIG. 4 conjugated to a poly-L-lysine scaffold and an RNAi agent showed increased relative knockdown at all three dose levels compared to naked RNAi agent (68% knockdown vs. 47% knockdown at 0.5 mg/kg, 78% knockdown vs. 47% knockdown at 1.5 mg/kg and 86% knockdown versus 75% knockdown at 5 mg/kg).

Example 9 Selective uptake of labeled ave6 ligand conjugates by primary epithelial cells in vitro.

Primary human lung epithelial, endothelial, and smooth muscle cells were cultured and exposed for 24 h to (1) Cy3-labeled (red) polyacrylate polymer scaffold without ligand (no ligand conjugate); or (2) a Cy3 (red) labeled polyacrylate polymer backbone conjugated to the αγβ6 integrin ligand of FIG. 7 (ave6 ligand conjugate).

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Cells were stained with FITC phalloidin (F-actin, green) and Hehest stain (DNA, blue) and visualized by fluorescence microscopy.

Fluorescence microscopy images were obtained using standard methods known in the art. Fluorescent images demonstrated that the Cy3 labeled conjugates without the αvβ6 integrin ligand were not internalized by any cell type. However, Cy3 conjugates with the αvβ6 ligand of FIG. 7 were internalized by primary lung epithelial cells (as indicated by the accumulation of red signal within the endosomal regions of primary lung epithelial cells in the image), but were not internalized by primary endothelial and smooth muscle cells. This shows that the αvβ6 integrin ligands disclosed herein allow for selective internalization by epithelial cells expressing the αvβ6 integrin.

Example 10 Selective uptake of labeled ave6 ligand conjugates by epithelial tissues in vivo.

C57N/6 mice were injected with an intravenous dose of 120 μg of (1) Cy3-labeled (red) polyacrylate polymer scaffold without ligand (no ligand conjugate); or (2) a Cy3-labeled (red) polyacrylate polymer backbone conjugated to the avβ6 integrin ligand of FIG. 7 (avβ6 ligand conjugate); or (3) a Cy3 labeled (red) polyacrylate polymer backbone conjugated to a non-activated avβ6 integrin ligand having the structure Ac-RGELAc-L-CitAibL (SEQ ID NO: 132) which, as previously described in Example 4, is used as the ligand negative control.

Mice were sacrificed 24 hours after injection, and tissues were harvested, fixed, processed, and dissected. Tissue sections were stained with FITC phalloidin (F-actin, green) and Hehest stain (DNA, blue) and visualized by fluorescence microscopy.

A. Bronchiolar epithelial cells of the lung.

Fluorescence microscopy images of lung bronchiolar epithelial cells in mice of Example 10 were obtained using standard methods. From these images, Cy3 labeled conjugates (shown as red labels in the images) with the avβ6 ligand of FIG. 7 were selectively internalized into endosomal compartments by lung bronchiolar epithelial cells in vivo, with essentially no epithelial internalization observed with constructs that did not contain the ligand or control RGE ligand conjugates (i.e., no red was visible in these images).

B. Renal epithelial tissues.

Using standard methods, fluorescence microscopy images of renal tubular epithelial tissues from mice of Example 10 were obtained. From these images, the Cy3-labeled avβ6 ligand conjugates of FIG. 7 were selectively internalized into endosomal compartments by renal tubular epithelial cells in vivo (shown as red labels in the images), while essentially no epithelial internalization was observed with the control conjugates without ligand.

C. Epithelial cells of the gastrointestinal tract.

Using standard methods, fluorescence microscopy images of the murine renal epithelial tissues of Example 10 were obtained. Cy3 labeled avβ6 ligand conjugates FIG. 7 were selectively internalized into endosomal compartments by GI tract epithelial cells in vivo in both the small intestine and gallbladder (shown as red labels in the images), while essentially no epithelial internalization was observed with the control conjugates without ligand.

Other embodiments

It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to explain and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims (18)

1. Ligand of the avβ6 integrin containing RG ¹ DLXaa ¹ Xaa ² L-Xaa ³ Xaa ⁴ LR 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 ¹ is L-alanine; Xaa ² is La-aminobutyric acid (Abu); Xaa ³ represents citrulline; - 44 042923 Xaa ⁴ is α-aminoisobutyric acid (Ab); R ¹ contains PEG and/or a linking group. 2. The αΎβ6 integrin ligand according to claim 1, wherein R ¹ contains a polyethylene glycol having 2-20 ethylene oxide units. 3. An αΎβ6 integrin ligand according to any one of claims 1, 2, wherein said αΎβ6 integrin ligand contains an amino terminal cap. 4. The αΎβ6 integrin ligand of claim 3, wherein the amino terminal cap is selected from the group consisting of CH3CO, CH3CH2CO, CH3(CH3) _2CO , (CH3)3CHCO, CH3( _CH2 )3CO, (CH3^CHNSCHCO, _CH3CH2CH (CH ₃ )CO, (CH3)sCO, CH3(CH2)4CO, CH3SO2, CH3CH2SO2, CH3( _CH2 ) _{2 8O2 ,} (CH3TCHSO2, CH3(CH^O2, ( _{CH3 ) 2CHCH2SO2} , CH ₃ CH ₂ CH(CH ₃ )SO ₂ , (CH ₃ ) ₃ CSO ₂ , PhCO, PhSO ₂ , an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, methyl, ethyl, propyl, butyl, pentyl, NH2NH, PEG, guanidinyl, CH3OCH2CH2OCH2CH2CO, CH3OSHCHSSCHO^SSCHSSCHSO, CH3OSCHSCCHSSCHSSCHSO, CH3OCH2CH2OSH2SO, CH3OSCHSCSCHOSCSCCO, CH3O(CH2CH2O)3CH2CO, CH3O(CH2CH2O)4CH2CO, CH3OSCHSCSCHOSSCHCO, CH3OCH2CH2OCO, CH3O(CH2CH2O)2CO, CH3O(CH2CH2O)3CO, CH3O(CH2CH2O)4CO, CH3O(CH2CH2O) ) ₅ CO, 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, HOCH2CH 2ОСО, NO(CH2CH2O )2CO, HO(CH2CH2O)3CO, HO(CH2CH2O)4CO, HO(CH2CH2O)5CO, CH3CH2OCH2CH2OCH2CH2CO, CH3CH2O(CH2CH2O)2CH2CH2CO, CH3CH2O(CH2CH2O)3CH2CH2CO, CH SHCHSSCHO ^ SSCHSSCHSO, CH3CH2OSH2CH2OSH2CO, CH3SCHOSCHCHSSCHO^SSCHSO, CH3CH2O(CH2CH2O)3CH2CO, CH3SCHOSCHSHCHSSCHO^SSCHSO, CH3CH2OCH2CH2OCO, CH3CH2O(CH2CH2O)2CO, CH3CH2O(CH2CH2O)3CO, CH3CH2O(CH2CH2O)4CO, CH3CH2CH2CH2CO, CH3OCH2CH2CO, HOCH2CH2CO, or CH3CH2OCH2CH2CO. 5. The ave6 integrin ligand of claim 4, wherein the amino terminal cap is _CH3CO . 6. The ave6 integrin ligand of claim 1, wherein said ave6 integrin ligand comprises sequence SEQ ID NO: 4 (Ac-RGDLAAbuLCitAibL). 7. An αΎβ6 integrin ligand according to any one of claims 1 to 6, wherein said αΎβ6 integrin ligand is further conjugated to a cargo molecule. 8. An αΎβ6 integrin ligand according to any one of claims 1 to 7, wherein said αΎβ6 integrin ligand is conjugated to a cargo molecule that contains 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 an acid or polynucleotide, peptide, aptamer, polymer, polyamine, protein, toxin, vitamin, polyethylene glycol, hapten, digoxigenin, biotin, radioactive atom or molecule, or fluorophore. 9. An αΎβ6 integrin ligand according to any one of claims 1 to 8, wherein said αΎβ6 integrin ligand is conjugated to a cargo molecule that contains an oligomeric compound. 10. An αΎβ6 integrin ligand according to claim 9, wherein said oligomeric compound is an RNAi agent. 11. A pharmaceutical composition for delivering a cargo molecule, comprising an ave6 integrin ligand according to any one of claims 7 to 10 and a pharmaceutically acceptable adjuvant. 12. A method for delivering one or more cargo molecules into a cell in vivo, wherein said method comprises administering to a subject a ligand having affinity for an αΎβ6 integrin conjugated to one or more cargo molecules, wherein said αΎβ6 integrin ligand is an ave6 integrin ligand according to any from paragraphs 1-10. 13. The method of claim 12 wherein the cell is selected from the group consisting of type I and II alveolar epithelial cell, goblet cell, secretory epithelial cell, ciliary epithelial cell, corneal and conjunctival epithelial cell, skin epithelial cell, cholangiocyte, enterocyte, ductal epithelial cell, glandular epithelial cell, renal tubule and epithelial tumors (carcinomas). 14. The method according to claim 12 or 13, in which one or more cargo molecules contain an oligomeric compound. 15. The method of claim 14 wherein said oligomeric compound is an RNAi agent. 16. A method for inhibiting expression of a target gene in a cell in vivo, wherein said method comprises administering to a subject an oligomeric compound conjugated to an αΎβ6 integrin ligand according to any one of claims 1 to 10. 17. The method of claim 16 wherein the cell is selected from the group consisting of type I and II alveolar epithelial cell, goblet cell, secretory epithelial cell, ciliary epithelial cell, corneal and conjunctival epithelial cell, skin epithelial cell, cholangiocyte, enterocyte, ductal epithelial cell, glandular epithelial cell, renal tubule and - 45 042923 epithelial tumors (carcinomas). 18. The method according to claim 16 or 17, wherein said oligomeric compound is an RNAi agent.