Bicyclic peptide ligand complex specific for tfr1

Abstract

This document provides a bicyclic peptide ligand complex specifically targeting transferrin receptor 1 (TfR1). Pharmaceutical compositions comprising said bicyclic peptide ligand complex are also provided, as well as the use of said bicyclic peptide ligand complex and pharmaceutical compositions in the prevention, inhibition, or treatment of diseases or conditions via TfR1-mediated therapeutic delivery.

Description

Technical Field

[0001] This invention relates to bicyclic peptide ligand complexes, such as bicyclic peptide ligand complexes specifically targeting transferrin receptor 1 (TfR1). The invention also includes pharmaceutical compositions comprising said bicyclic peptide ligand complexes, and the use of said bicyclic peptide ligand complexes and pharmaceutical compositions in the prevention, inhibition, or treatment of diseases or conditions via TfR1-mediated therapeutic delivery. Background Technology

[0002] Transferrin is a glycoprotein found in vertebrates that binds iron (Fe) and thus mediates the transport of iron (Fe) through the blood plasma. Transferrin is produced in the liver and contains two Fe atoms. 3+ Atom binding sites. Human transferrin consists of... TF It is encoded by genes and produced as a 76 kDa glycoprotein.

[0003] Transferrin glycoprotein binds iron tightly but reversibly. Although less than 0.1% (4 mg) of the total iron in the body is bound to transferrin, it forms the most important iron pool with the highest turnover rate (25 mg / 24 hours). Transferrin has a molecular weight of approximately 80 kDa and contains two specific, high-affinity Fe(III) binding sites. Transferrin has an extremely high affinity for Fe(III) (an association constant of 10 at pH 7.4). 20 M -1 However, its binding capacity gradually decreases as the pH drops below neutral. Transferrin is not limited to binding iron; it also binds to various metal ions. These glycoproteins are found in various body fluids of vertebrates. When not bound to iron or any other metal atom, transferrin is called "deferrotransferrin."

[0004] Transferrin receptor 1 (TfR1), also known as CD71, is a protein synthesized by transferrin receptor 1 in the human body. TFRC TfR1 is a gene-encoded protein. It is essential for the transfer of iron from transferrin into the cell via endocytosis. TfR1 is a transmembrane glycoprotein composed of monomers linked by two disulfide bonds. Each monomer binds to one alloferrin molecule, forming an iron-Tf-TfR complex, which enters the cell via endocytosis.

[0005] TfR1 is a potential novel target in human leukemia and lymphoma cases. TfR1 expressed on endothelial cells of the blood-brain barrier has been used in preclinical studies to allow the delivery of macromolecules, including antibodies, into the brain. Theoretically, this delivery is believed to be achieved through transcellular transport via the endothelial cells of the blood-brain barrier.

[0006] Therefore, there is a need to provide alternative Tfr1-specific complexes to facilitate optimal delivery to the brain. Summary of the Invention

[0007] According to a first aspect of the present invention, a bicyclic peptide ligand complex is provided, comprising:

[0008] (a) One or more bicyclic peptide ligands specifically targeting transferrin receptor 1 (TfR1), the bicyclic peptide ligand comprising a polypeptide and a molecular scaffold, the polypeptide comprising at least three reactive groups separated by at least two ring sequences, the molecular scaffold forming covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular scaffold; and

[0009] (b) Half-life extension (HLE) portion.

[0010] According to another aspect of the invention, a polypeptide or a pharmaceutically acceptable salt thereof is provided, comprising an amino acid sequence as defined herein. According to another aspect of the invention, a peptide ligand comprising a polypeptide or a pharmaceutically acceptable salt thereof as described herein, linked to a molecular scaffold as described herein.

[0011] According to another aspect of the invention, a pharmaceutical composition is provided comprising a combination of a bicyclic peptide ligand or a bicyclic peptide ligand complex as defined herein and one or more pharmaceutically acceptable excipients.

[0012] According to another aspect of the invention, a bicyclic peptide ligand, peptide ligand complex, or pharmaceutical composition as defined herein is provided for the prevention, inhibition, or treatment of a disease or condition via TfR1-mediated therapeutic delivery. Attached Figure Description

[0013] Figure 1 Mean plasma concentration-time curve of B191 after administration of 2.69 mg / kg via intravenous infusion over 15 minutes.

[0014] Figure 2 Individual and mean plasma concentration-time curves of B192 after administration of 3.45 mg / kg via intravenous infusion over 15 minutes.

[0015] Figure 3 Individual and mean plasma concentration-time curves of B193 after administration of 0.28 mg / kg via intravenous infusion over 15 minutes. Detailed Implementation

[0016] definition

[0017] Unless explicitly defined herein, all terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains. For definitions and terms in this field, practitioners may refer specifically to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016).

[0018] All publications, patents and patent applications cited in this article are incorporated herein in their entirety through citation.

[0019] In the context of this disclosure, the term "amino acid" is used in its broadest sense and is intended to include organic compounds containing amine (NH2) and carboxyl (COOH) functional groups, as well as the side chains (e.g., R groups) characteristic of each amino acid. In some embodiments, amino acid refers to naturally occurring L-type α-amino acids or residues. Common single-letter and three-letter abbreviations for naturally occurring amino acids are used herein: A=Ala; C=Cys; D=Asp; E=Glu; F=Phe; G=Gly; H=His; I=Ile; K=Lys; L=Leu; M=Met; N=Asn; P=Pro; Q=Gln; R=Arg; S=Ser; T=Thr; V=Val; W=Trp; and Y=Tyr (Lehninger, AL, (1975) Biochemistry, 2nd edition, pp. 71-92, Worth Publishers, New York). The general term “amino acid” also includes D-amino acids, trans-amino acids, and chemically modified amino acids, such as amino acid analogs, naturally occurring amino acids that are not typically incorporated into proteins (e.g., ortholeucine), and chemically synthesized compounds that have properties known in the art as characteristic of amino acids (e.g., β-amino acids). For example, analogs or mimics of phenylalanine or proline that allow peptide compounds to have the same conformational restrictions as natural Phe or Pro are included within the definition of an amino acid. Such analogs and mimics are referred to herein as “functional equivalents” of the corresponding amino acids. Other examples of amino acids are listed in Roberts and Vellaccio’s *The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer eds.*, Volume 5, page 341, Academic Press, Inc., NY 1983, which is incorporated herein by reference. Table A below provides the chemical properties of 20 major amino acids.

[0020] Table A – Chemical Properties of Amino Acids

[0021]

[0022] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acid residues and their variants and synthetic analogs. Therefore, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids (e.g., chemical analogs of the corresponding naturally occurring amino acids), as well as naturally occurring amino acid polymers. Polypeptides may also undergo maturation or post-translational modification processes, which may include, but are not limited to, glycosylation, proteolytic cleavage, lipolysis, signal peptide cleavage, propeptide cleavage, phosphorylation, etc.

[0023] As used in this article, Abu represents GABA, Aib represents GABA isobutyric acid, Aze represents aziridine, B-MeIle represents β-methyl isoleucine, C5g represents cyclopentylglycine, Cba represents β-cyclobutylalanine, Cbg represents cyclobutylglycine, Chg represents cyclohexylglycine, Cpg represents cyclopropylglycine, EPA represents 2-amino-3-ethyl-valerate, HyP represents trans-4-hydroxy-L-proline, [K(N3)] represents 6-azidolysine, 1Nal represents 1-naphthylalanine, 2Nal represents 2-naphthylalanine, 4Pa... l represents 4-pyridylalanine, tBuAla represents tert-butylalanine, tBuGly represents tert-butylglycine, 3tBuTyr represents 3-tert-butyltyrosine; AzPro represents azidepropyl, Aze represents azacyclobutane, 1Nal represents 1-naphthylalanine, NMeTrp represents N-methyl-tryptophan, K(PYA) represents ε-4-pentynyllysine, Peg represents polyethylene glycol, Pip represents piperidine acid, Sar represents sarcosine, Fl represents fluorescein, and [K(N3)(PYA-maleimide)] represents modified lysine with the following structure:

[0024] .

[0025] peptide ligand complex

[0026] This invention provides a complex comprising a peptide capable of binding to TfR1 and a half-life extension moiety. The half-life extension moiety is described in more detail herein.

[0027] Peptides capable of binding TfR1, as described herein, may be contained, for example, in peptide ligands that contain the polypeptide and are covalently bound to a molecular scaffold (e.g., a molecular scaffold described in more detail herein) such that two or more polypeptide rings are formed on the molecular scaffold. The peptide or peptide ligand may be directly or indirectly (e.g., via a linker) linked to one or more effector groups and / or functional groups, such as one or more cytotoxic agents, radiochelates, or chromophores.

[0028] According to a first aspect of the present invention, a bicyclic peptide ligand complex is provided, comprising:

[0029] (a) One or more bicyclic peptide ligands specifically targeting transferrin receptor 1 (TfR1), the bicyclic peptide ligand comprising a polypeptide and a molecular scaffold, the polypeptide comprising at least three reactive groups separated by at least two ring sequences, the molecular scaffold forming covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular scaffold; and

[0030] (b) Half-life extension (HLE) portion.

[0031] It should be understood that the term "specific targeting of TfR1" refers to the ability of the peptide ligand to bind to transferrin receptor 1 (TfR1). It should also be understood that the effect of the peptide ligand on TfR1 varies depending on the exact epitope it binds to. For example, the effect may be inhibitory (i.e., the peptide ligand blocks / inhibits the binding of transferrin to TfR1) or non-inhibitory (i.e., the peptide ligand does not block / inhibit the binding of transferrin to TfR1).

[0032] Bicyclic peptide ligand

[0033] It should be understood that the present invention relates to "monomer" bicyclic peptides, i.e., those bicyclic peptides containing a single (monomer) bicyclic peptide ligand, and "multimeric" bicyclic peptides, i.e., those bicyclic peptides containing more than one bicyclic peptide ligand (e.g., 2, 3 or 4 bicyclic peptide ligands) conjugated through one or more linkers.

[0034] Monomeric bicyclic peptide ligand

[0035] In one embodiment, the bicyclic peptide ligand complex comprises a single (monomer) bicyclic peptide ligand.

[0036] The bicyclic peptide ligands used in this invention typically comprise a polypeptide having the general formula RG1-X1-RG2-X2-RG3, or a modified derivative thereof, or a pharmaceutically acceptable salt thereof; wherein RG1, RG2, and RG3 represent three reactive groups as defined herein, X1 represents a first chain of a natural or non-natural amino acid, and X2 represents a second chain of a natural or non-natural amino acid. Typically, when the polypeptide binds to a molecular scaffold at the three reactive groups to form a peptide ligand, X1 is linked to the RG1 and RG2 groups to form a first polypeptide ring, and X2 is linked to the RG2 and RG3 groups to form a second polypeptide ring. Therefore, in some embodiments, X1 defines the first ring sequence, and X2 defines the second ring sequence.

[0037] Reactive groups RG1, RG2, and RG3 provide connection points to a molecular scaffold (e.g., a molecular scaffold as defined herein); and are typically contained within the side chains of specific amino acids present in the peptide. Such reactive groups can be, for example, cysteine, homocysteine ​​(hCys, (S)-2-amino-4-thiobutyric acid), βCys ((R)-3-amino-3-mercaptopropionic acid), penicillamine (Pen, (R)-2-amino-3-mercapto-3-methylbutyric acid), Dap ((S)-2,3-diaminopropionic acid), N-alkyl-Dap (e.g., N-methyl-Dap, (S)-2-amino-3-(methylamino)propionic acid), or lysine, or any other suitable reactive group. Reactive groups RG1, RG2, and RG3 can be the same or different. In one embodiment, the reactive group comprises a cysteine ​​residue.

[0038] Inhibitory peptide ligands

[0039] In one embodiment, the peptide ligand specifically targets TfR1 and binds to TfR1 in a manner that blocks / inhibits the binding of transferrin to TfR1.

[0040] In another embodiment, each of the ring sequences comprises 2, 3, 4, 5, 6, 7, 8, or 9 amino acids. In another embodiment, the ring sequence comprises 2, 3, 6, 8, or 9 amino acids. In some embodiments, the bicyclic peptide ligand comprises a first ring sequence comprising 2, 3, 4, 5, 6, 7, 8, or 9 amino acids; and a second ring sequence comprising 2, 3, 4, 5, 6, 7, 8, or 9 amino acids. In some embodiments, the bicyclic peptide ligand comprises a first ring sequence comprising 2, 3, 6, 8, or 9 amino acids; and a second ring sequence comprising 2, 3, 6, 8, or 9 amino acids. In some embodiments, the polypeptide comprising the first and second ring sequences and the reactive group is about 12 to about 16 amino acids long, for example, 13, 14, or 15 amino acids.

[0041] In one embodiment, the polypeptide comprises three reactive groups, such as three cysteine ​​residues, separated by two ring sequences, wherein the first ring sequence consists of two amino acids and the second ring sequence consists of nine amino acids.

[0042] In one embodiment, the polypeptide comprises three reactive groups, such as three cysteine ​​residues, separated by two loop sequences, each consisting of six amino acids.

[0043] In one embodiment, the polypeptide comprises three reactive groups, such as three cysteine ​​residues, separated by two ring sequences, wherein the first ring sequence consists of 3 amino acids and the second ring sequence consists of 8 amino acids.

[0044] In one embodiment of such a peptide ligand, the ring sequence comprises two amino acids. In some embodiments, when the ring sequence comprises two amino acids, the ring sequence comprises a portion of the formula -AA1-AA2-. In some embodiments, AA1 and AA2 are each independently nonpolar amino acids. In some embodiments, AA1 and AA2 are each independently selected from A, V, I, and L. In some embodiments, AA1 and AA2 are each independently selected from A and L. In some embodiments, AA1 is A and AA2 is L. In some embodiments, the portion of the formula -AA1-AA2- is -AL-.

[0045] In some embodiments of such peptide ligands, the ring sequence comprises three amino acids. In some embodiments, when the ring sequence comprises three amino acids, the ring sequence comprises a portion of the formula -AA1-AA2-AA3-. In some embodiments, AA1, AA2, and AA3 are each independently a nonpolar amino acid and / or a charged amino acid. In some embodiments, AA1, AA2, and AA3 are each independently selected from A, V, I and L, D, and E. In some embodiments, AA1 and AA3 are each independently selected from A, V, I, and L. In some embodiments, AA1 and AA3 are each independently selected from A and L. In some embodiments, AA2 is selected from D and E. In some embodiments, AA2 is E. In some embodiments, AA1 is A, AA2 is E, and AA3 is L. In some embodiments, the portion of the formula -AA1-AA2-AA3- is -AEL-.

[0046] In some embodiments of such peptide ligands, the loop sequence comprises 6 amino acids. In some embodiments, when the loop sequence comprises 6 amino acids, the loop sequence comprises a portion of the formula -AA1-AA2-AA3-AA4-AA5-AA6-. In some embodiments, each amino acid in the loop sequence is independently selected from R, E, F, D, T, G, L, A, I, V, S, and K. In some embodiments, each amino acid in the loop sequence is independently selected from R, E, F, D, T, G, L, A, and I. In some embodiments, each amino acid in the loop sequence is independently selected from R, K, E, F, D, T, and S. In some embodiments, each amino acid in the loop sequence is independently selected from R, E, F, D, and T. In some embodiments, each amino acid in the loop sequence is independently selected from G, L, A, V, F, I, E, and D. In some embodiments, each amino acid in the loop sequence is independently selected from G, L, A, F, I, and E. In some implementations, portions of the formula -AA1-AA2-AA3-AA4-AA5-AA6- are selected from -REFFDT- and -GLAFIE-.

[0047] In some embodiments of such peptide ligands, the loop sequence comprises 8 amino acids. In some embodiments, when the loop sequence comprises 8 amino acids, the loop sequence comprises a portion of the formula -AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-. In some embodiments, each amino acid in the loop sequence is independently selected from Y, D, E, G, V, A, I, L, W, S, and T. In some embodiments, each amino acid in the loop sequence is independently selected from Y, D, G, V, W, and S. In some embodiments, the portion of the formula -AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8- is -YDGVYWYS-.

[0048] In some embodiments of such peptide ligands, the loop sequence comprises 9 amino acids. In some embodiments, when the loop sequence comprises 9 amino acids, the loop sequence comprises a portion of the formula -AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-. In some embodiments, each amino acid in the loop sequence is independently selected from N, D, E, W, T, S, L, V, A, P, W, and H. In some embodiments, each amino acid in the loop sequence is independently selected from N, D, W, T, L, P, W, and H. In some embodiments, the portion of the formula -AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9- is -NDWTLPWHH-.

[0049] In some embodiments of such peptide ligands, the loop sequence comprises 4 amino acids. In some embodiments, when the loop sequence comprises 4 amino acids, the loop sequence corresponds to a loop sequence comprising 2 or 3 amino acids, and includes 2 or 1 additional amino acid (respectively), which may be inserted at any position within the amino acid loop sequence. In some embodiments, the loop sequence corresponds to a loop sequence comprising 6, 8, or 9 amino acids, wherein 2, 4, or 5 amino acids are removed (respectively).

[0050] In some embodiments of such peptide ligands, the loop sequence comprises 5 amino acids. In some embodiments, when the loop sequence comprises 5 amino acids, the loop sequence corresponds to a loop sequence comprising 2 or 3 amino acids, and includes 3 or 2 additional amino acids (respectively), which may be inserted at any position within the amino acid loop sequence. In some embodiments, the loop sequence corresponds to a loop sequence comprising 6, 8, or 9 amino acids, wherein 1, 3, or 4 amino acids are removed (respectively).

[0051] In some embodiments of such peptide ligands, the loop sequence comprises 7 amino acids. In some embodiments, when the loop sequence comprises 7 amino acids, the loop sequence corresponds to a loop sequence comprising 2, 3, or 6 amino acids, and includes 5, 4, or 1 additional amino acid (respectively), which may be inserted at any position within the amino acid loop sequence. In some embodiments, the loop sequence corresponds to a loop sequence comprising 8 or 9 amino acids, wherein 1 or 2 amino acids are removed (respectively).

[0052] In one embodiment, the peptide ligand comprises a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide having an amino acid sequence selected from the following:

[0053] RG1-AL-RG2-NDWTLPWHH-RG3;

[0054] RG1-REFFDT-RG2-GLAFIE-RG3; and

[0055] RG1-LEA-RG2-YDGVYWYS-RG3

[0056] Or its variants as described herein; wherein RG1, RG2 and RG3 are as defined herein.

[0057] In one embodiment, the peptide ligand comprises a polypeptide having the following amino acid sequence:

[0058] CALCNDWTLPWHHC (SEQ ID NO: 1);

[0059] CREFFDTCGLAFIEC (SEQ ID NO: 2); and

[0060] CLEACYDGVYWYSC (SEQ ID NO: 3);

[0061] Or its variants as described herein, or its pharmaceutically acceptable salts.

[0062] In one embodiment, the peptide ligand comprises the following amino acid sequence:

[0063] CALCNDWTLPWHHC (SEQ ID NO: 1);

[0064] CREFFDTCGLAFIEC (SEQ ID NO: 2); and

[0065] CLEACYDGVYWYSC (SEQ ID NO: 3);

[0066] Or its pharmaceutically acceptable salt.

[0067] In one embodiment, the peptide ligand comprises a polypeptide as described herein, wherein the polypeptide includes an N-terminal and / or C-terminal addition as described in more detail herein. In some embodiments, the N-terminal addition comprises an N-terminal alanine.

[0068] In some embodiments, the inhibitory peptide ligand comprises a polypeptide as described herein, which is linked (e.g., by covalent bonding) to a molecular scaffold as described herein. In some embodiments, the polypeptide is linked (e.g., covalently bonded) to the molecular scaffold via a bond between the molecular scaffold and each of RG1, RG2, and RG3. When RG1, RG2, and RG3 are cysteine ​​residues, the polypeptide is typically linked to the molecular scaffold via a covalent bond between the thiol group of the cysteine ​​residue and the scaffold. In some embodiments, the scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tripropyl-2-en-1-one (TATA).

[0069] In another embodiment, the molecular scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA), and the peptide ligand comprises an N-terminus and / or a C-terminus and is selected from:

[0070] A-(SEQ ID NO: 1)-A (referred to as B1 in this document);

[0071] A-(SEQ ID NO: 1)-A-[Sar6]-[K-Fl] (referred to as B2 in this paper);

[0072] A-(SEQ ID NO: 2)-A (referred to as B3 in this document);

[0073] A-(SEQ ID NO: 2)-A-[Sar6]-[K-Fl] (referred to as B4 in this paper);

[0074] A-(SEQ ID NO: 3)-A (referred to as B5 in this document); and

[0075] A-(SEQ ID NO: 3)-A-[Sar6]-[K-Fl] (referred to as B6 in this document).

[0076] Sar represents sarcosine, and Fl represents fluorescein; that is, [K-Fl] represents lysine with fluorescein attached at position 6.

[0077] For the purposes of this specification, it is assumed that the inhibitory bicyclic peptide is cyclized with TATA to produce a trisubstituted structure. However, as will be clearly seen from the description of the invention presented herein, cyclization can be performed using any suitable molecular scaffold that forms a covalent bond with a reactive group of the polypeptide, resulting in at least two polypeptide rings. Cyclization occurs on the first, second, and third reactive groups (RG1, RG2, and RG3), respectively. When the reactive group is cysteine, cyclization occurs on the first, second, and third cysteine ​​residues, respectively.

[0078] Non-inhibitory peptide ligands

[0079] In one embodiment, the peptide ligand specifically targets TfR1 and binds to TfR1 in a manner that does not inhibit / block the binding of transferrin to TfR1.

[0080] In one embodiment, the peptide ligand comprises a polypeptide comprising two ring sequences, each ring sequence comprising 3, 4, 5, 6, or 7 amino acids. In another embodiment, the peptide ligand comprises a polypeptide comprising two ring sequences, each ring sequence comprising 3, 4, 6, or 7 amino acids. In one embodiment, the bicyclic peptide ligand comprises a polypeptide comprising a ring sequence containing about 7 amino acids and a ring sequence containing about 3 amino acids. In another embodiment, the bicyclic peptide ligand comprises a polypeptide comprising a ring sequence containing about 6 amino acids and a ring sequence containing about 4 amino acids. In some embodiments, the length of the polypeptide comprising the first and second ring sequences and the reactive group is about 12 to about 16 amino acids, for example, 13, 14, or 15 amino acids.

[0081] In another embodiment, the ring sequence contains 3 or 7 amino acids.

[0082] In one embodiment, the polypeptide comprises three reactive groups, such as three cysteine ​​residues separated by two ring sequences, wherein the first ring sequence consists of 7 amino acids and the second ring sequence consists of 3 amino acids. In another embodiment, the polypeptide comprises three reactive groups, such as three cysteine ​​residues separated by two ring sequences, wherein the first ring sequence consists of 6 amino acids and the second ring sequence consists of 4 amino acids.

[0083] In some embodiments, the ring sequence comprises 3 amino acids. In some embodiments, the ring sequence comprising 3 amino acids has the amino acid sequence -AA1-AA2-AA3-. In some embodiments, each of AA1 to AA3 is any amino acid, such as any natural or non-natural amino acid. In some embodiments, AA1 is selected from [tBuGly], [EPA], [Chg], [tBuAla], [C5g], [Cbg], [Cpg], [B-MeIle], I, A, Y, and [3HyV]. In some embodiments, AA1 is selected from [tBuGly], [EPA], [Chg], [tBuAla], [C5g], [Cbg], [Cpg], [B-MeIle], and [3HyV]. In some embodiments, AA1 is selected from I, A, and Y. In some embodiments, AA1 is I or [tBuGly]. In some embodiments, AA1 is I. In some embodiments, AA1 is [tBuGly]. In some embodiments, AA2 is S, A, [HSer], T, D, E, N, or Q. In some embodiments, AA2 is S. In some embodiments, AA3 is any amino acid. In some embodiments, AA3 is a natural amino acid. In some embodiments, AA3 is a non-natural amino acid. In some embodiments, AA3 is W, Y, [2Nal], [3tBuTyr], [1Nal], [4Pal], A, [DOPA], [pCaPhe], [pCoPhe], or [hTyr]. In some embodiments, AA3 is W, Y, or A. In some embodiments, AA3 is Y. In some embodiments, a portion of -AA1-AA2-AA3- is -I / A / D / [tBuGly]–S / A / E–W / Y / A-. In some embodiments, a portion of -AA1-AA2-AA3- is -I / [tBuGly]–S–Y-. In some implementations, the portion -AA1-AA2-AA3- is –I–S–Y-. In some implementations, the portion -AA1-AA2-AA3- is –[tBuGly]–S–Y-.

[0084] In some embodiments of such peptide ligands, the loop sequence comprises 4 amino acids. In some embodiments, when the loop sequence comprises 4 amino acids, the loop sequence corresponds to a loop sequence comprising 3 amino acids and includes one additional amino acid, which can be inserted at any position within the amino acid loop sequence. In some embodiments, the loop sequence comprising 4 amino acids has the amino acid sequence -AA1-AA2-AA3-AA4-. In some embodiments, each of AA1 to AA4 is any amino acid, such as any natural or non-natural amino acid. In some embodiments, each of AA1 to AA4 is independently selected from G, D, E, W, Y, [2Nal], [3tBuTyr], [1Nal], [4Pal], and A. In some embodiments, each of AA1 to AA4 is independently selected from G, D, E, W, Y, and A. In some embodiments, each of AA1 to AA4 is independently selected from G, D, E, and Y. In some embodiments, AA1 is G. In some embodiments, AA2 is D. In some embodiments, AA3 is E. In some embodiments, AA4 is any amino acid. In some embodiments, AA4 is W, Y, [2Nal], [3tBuTyr], [1Nal], [4Pal], or A. In some embodiments, AA4 is W, Y, or A. In some embodiments, AA4 is Y. In some embodiments, a portion of -AA1-AA2-AA3-AA4- is -GDEW / Y / A-. In some embodiments, a portion of -AA1-AA2-AA3-AA4- is -GDEY-.

[0085] In some embodiments, the ring sequence comprises 7 amino acids. In some embodiments, the ring sequence comprising 7 amino acids has the amino acid sequence -AA1-AA2-AA3-AA4-AA5-AA6-AA7-. In some embodiments, each of AA1 to AA7 is independently any natural or non-natural amino acid. In some embodiments, each of AA1 to AA7 is independently selected from S, A, D, W, L, G, P, Y, Q, [dA], H, [tBuAla], [K(N3)], E, [Aib], [Cba], [Gla], [Abu], [dS], [HyP], [DOPA], [dT], [Oxa], [hTyr], [dD], [Cis-HyP], N, [pCaPhe], [dE], I, [pCoPhe], S, [dN], [Aze], T, [dQ], and [dY]. In some embodiments, A1 is selected from S, P, A, [K(N3)], [HyP], [Oxa], and [Cis-HyP]. Typically, A1 is selected from S and P. In one embodiment, A1 is S. In one embodiment, A1 is P. In some embodiments, A2 is selected from A, S, P, Q, G, [HyP], [Aib], N, I, [Aze], [dA], [K(N3)], [Oxa], and [Cis-HyP]. Typically, A2 is selected from P and [HyP]. In one embodiment, A2 is P. In one embodiment, A2 is [HyP]. In some embodiments, A3 is selected from D, A, E, and [Gla]. Typically, A3 is D. In some embodiments, A4 is selected from D, A, S, [Aib], [Abu], and [K(N3)]. Typically, A4 is A. In some embodiments, A5 is selected from W, Y, H, A, [DOPA], [hTyr], [pCaPhe], and [pCoPhe]. Typically, A5 is H or Y. In one embodiment, A5 is H. In one embodiment, A5 is Y. In some embodiments, A6 is selected from L, Q, [tBuAla], [Cba], [Abu], [Aib], A, S, T, D, E, and N. Typically, A6 is L. In some embodiments, A7 is selected from G, [dA], A, [dS], [dT], [dD], [dE], [dN], [dQ], [dY], S, D, Y, and N. Typically, A7 is G. In some embodiments, the portion -AA1-AA2-AA3-AA4-AA5-AA6-AA7- has the sequence -AA1-AA2-DAH / YLG-. In some implementations, the portion -AA1-AA2-AA3-AA4-AA5-AA6-AA7- has the sequence –S / P / A / [K(N3)] / [HyP] / [Oxa] / [Cis-HyP]-AA2-DAH / YLG-.In some implementations, the portion -AA1-AA2-AA3-AA4-AA5-AA6-AA7- has the sequence -AA1-A / S / P / Q / G / [HyP] / [Aib] / N / I / [Aze] / [dA] / [K(N3)] / [Oxa] / [Cis-HyP]-DAH / YLG-.

[0086] In some embodiments, the portion -AA1-AA2-AA3-AA4-AA5-AA6-AA7- has the sequence -S / PP / [HyP]-DAH / YLG-. In some embodiments, the portion -AA1-AA2-AA3-AA4-AA5-AA6-AA7- has the sequence -SPDAHLG-. In some embodiments, the portion -AA1-AA2-AA3-AA4-AA5-AA6-AA7- has the sequence -P[HyP]DAHLG-. In some embodiments, the portion -AA1-AA2-AA3-AA4-AA5-AA6-AA7- has the sequence -SPDAYLG-. In some embodiments, the portion -AA1-AA2-AA3-AA4-AA5-AA6-AA7- has the sequence -P[HyP]DAYLG-.

[0087] In some embodiments of such peptide ligands, the loop sequence comprises 6 amino acids. In some embodiments, when the loop sequence comprises 6 amino acids, the loop sequence corresponds to a loop sequence comprising 7 amino acids, wherein one amino acid is deleted from said sequence. In some embodiments, the loop sequence comprising 6 amino acids has the amino acid sequence -AA1-AA2-AA3-AA4-AA5-AA6-.

[0088] In some embodiments, each of AA1 to AA6 is any amino acid, such as any natural or non-natural amino acid. In some embodiments, each of AA1 to AA6 is independently selected from Y, L, P, D, W, [tBuAla], A, S, Q, G, [HyP], [Aib], N, I, [Aze], [dA], and [K(N3)]. In some embodiments, each of AA1 to AA6 is independently selected from Y, L, P, D, W, and [tBuAla].

[0089] In some embodiments, AA1 is Y. In some embodiments, AA2 is L. In some embodiments, AA3 is selected from A, S, P, Q, G, [HyP], [Aib], N, I, [Aze], [dA], [K(N3)], L, [Oxa], and [Cis-Hyp]. Typically, A3 is selected from P and [HyP], most commonly P. In some embodiments, A4 is selected from D, A, and P. Typically, A4 is D. In some embodiments, AA5 is W. In some embodiments, AA6 is [tBuAla]. In some embodiments, part of -AA1-AA2-AA3-AA4-AA5-AA6- is -YLPDW-[tBuAla]-.

[0090] In some embodiments, the first loop sequence contains 6 or 7 amino acids and is selected from:

[0091] SADDWLG;

[0092] SSDAYLG;

[0093] PPDAHLG;

[0094] PQDAYLG;

[0095] PPDSWQG;

[0096] SPDAHLG;

[0097] PGDAHLG;

[0098] PPDSHLG;

[0099] P[HyP]DAYLG;

[0100] S[HyP]DAHLG;

[0101] P[Aib]DAHLG;

[0102] P[Aib]DAYLG;

[0103] SADAHLG;

[0104] S[Aib]DAHLG;

[0105] PPDAYLG;

[0106] S[Aib]DAYLG;

[0107] APDAHLG;

[0108] SPDAYLG;

[0109] PNDAHLG;

[0110] PIDAHLG;

[0111] SPD[Aib]HLG;

[0112] SPDAH[tBuAla]G;

[0113] SPDAH[Cba]G;

[0114] SPD[Abu]HLG;

[0115] S[Aze]DAHLG;

[0116] SPDDHLG;

[0117] SPDSHLG;

[0118] SPDAH[Abu]G;

[0119] P[dA]DAHLG;

[0120] SPDAHL[dA];

[0121] SPDAH[Aib]G;

[0122] SPAAHLG;

[0123] SPDAALG;

[0124] SPDAHAG;

[0125] SPDAHLA;

[0126] [K(N3)]PDAHLG;

[0127] S[K(N3)]DAHLG;

[0128] SPD[K(N3)]HLG;

[0129] P[HyP]DAHLG;

[0130] [HyP][HyP]DAYLG

[0131] [Oxa][HyP]DAYLG

[0132] [Cis-HyP][HyP]DAYLG

[0133] P[Oxa]DAYLG

[0134] P[Cis-HyP]DAYLG

[0135] P[HyP]DA[DOPA]LG

[0136] P[HyP]DA[hTyr]LG

[0137] P[HyP]DA[pCaPhe]LG

[0138] P[HyP]DA[pCoPhe]LG

[0139] P[HyP]DAYL[dS]

[0140] P[HyP]DAYL[dT]

[0141] P[HyP]DAYL[dD]

[0142] P[HyP]DAYL[dE]

[0143] P[HyP]DAYL[dN]

[0144] P[HyP]DAYL[dQ]

[0145] P[HyP]DAYL[dY]

[0146] P[HyP]DAYLS

[0147] P[HyP]DAYLD

[0148] P[HyP]DAYLY;

[0149] P[HyP]DAYLN;

[0150] P[HyP]EAYLG;

[0151] P[HyP][Gla]AYLG;

[0152] P[HyP]DAYSG;

[0153] P[HyP]DAYTG;

[0154] P[HyP]DAYDG;

[0155] P[HyP]DAYEG;

[0156] P[HyP]DAYNG;

[0157] P[HyP]DAYQG; and

[0158] YLPDW[tBuAla];

[0159] and its variants as described herein;

[0160] And its pharmaceutically acceptable salts.

[0161] In some embodiments, the second ring sequence contains 3 or 4 amino acids and is selected from...

[0162] ISW;

[0163] ISY;

[0164] [tBuGly]SY;

[0165] [EPA]SY;

[0166] [Chg]SY;

[0167] IS[2Nal];

[0168] IS[3tBuTyr];

[0169] IS[1Nal];

[0170] IS[4Pal];

[0171] [tBuAla]SY;

[0172] [C5g]SY;

[0173] [Cbg]SY;

[0174] [Cpg]SY;

[0175] [B-MeIle]SY;

[0176] ASY;

[0177] IAY;

[0178] ISA;

[0179] [tBuGly]S[DOPA];

[0180] [tBuGly]S[pCaPhe];

[0181] [tBuGly]S[pCoPhe];

[0182] [tBuGly]S[hTyr];

[0183] [tBuGly][HSer]Y;

[0184] [tBuGly]TY;

[0185] [tBuGly]DY;

[0186] [tBuGly]EY;

[0187] [tBuGly]NY;

[0188] [tBuGly]QY;

[0189] YSY;

[0190] [3HyV]SY; and

[0191] GDEY;

[0192] and its variants as described herein;

[0193] And its pharmaceutically acceptable salts.

[0194] In some embodiments, the peptide ligand comprises a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide having an amino acid sequence selected from:

[0195] RG1-SADDWLG-RG2-ISW-RG3;

[0196] RG1-SSDAYLG-RG2-ISW-RG3;

[0197] RG1-PPDAHLG-RG2-ISW-RG3;

[0198] RG1-PQDAYLG-RG2-ISW-RG3;

[0199] RG1-PPDSWQG-RG2-ISY-RG3;

[0200] RG1-SPDAHLG-RG2-ISY-RG3;

[0201] RG1-PGDAHLG-RG2-ISY-RG3;

[0202] RG1-PPDSHLG-RG2-ISY-RG3;

[0203] RG1-SADDWLG-RG2-ISY-RG3;

[0204] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3;

[0205] RG1-P[HyP]DAYLG-RG2-ISY-RG3;

[0206] RG1-S[HyP]DAHLG-RG2-ISY-RG3;

[0207] RG1-P[Aib]DAHLG-RG2-[tBuGly]SY-RG3;

[0208] RG1-PPDAHLG-RG2-ISY-RG3;

[0209] RG1-P[Aib]DAYLG-RG2-[tBuGly]SY-RG3;

[0210] RG1-SADAHLG-RG2-ISY-RG3;

[0211] RG1-S[Aib]DAHLG-RG2-[tBuGly]SY-RG3;

[0212] RG1-SPDAHLG-RG2-[EPA]SY-RG3;

[0213] RG1-PPDAYLG-RG2-[tBuGly]SY-RG3;

[0214] RG1-S[Aib]DAYLG-RG2-[tBuGly]SY-RG3;

[0215] RG1-APDAHLG-RG2-ISY-RG3;

[0216] RG1-P[Aib]DAHLG-RG2-ISY-RG3;

[0217] RG1-SPDAYLG-RG2-[tBuGly]SY-RG3;

[0218] RG1-SPDAHLG-RG2-[tBuGly]SY-RG3;

[0219] RG1-PNDAHLG-RG2-ISY-RG3;

[0220] RG1-PIDAHLG-RG2-ISY-RG3;

[0221] RG1-SPDAYLG-RG2-ISY-RG3;

[0222] RG1-PPDAYLG-RG2-ISY-RG3;

[0223] RG1-S[Aib]DAHLG-RG2-ISY-RG3;

[0224] RG1-SPDAHLG-RG2-[Chg]SY-RG3;

[0225] RG1-APDAHLG-RG2-ISY-RG3;

[0226] RG1-SPDAHLG-RG2-IS[2Nal]-RG3;

[0227] RG1-SPDAHLG-RG2-IS[3tBuTyr]-RG3;

[0228] RG1-SPD[Aib]HLG-RG2-ISY-RG3;

[0229] RG1-SPDAHLG-RG2-IS[1Nal]-RG3;

[0230] RG1-SPDAH[tBuAla]G-RG2-ISY-RG3;

[0231] RG1-SPDAH[Cba]G-RG2-ISY-RG3;

[0232] RG1-SPDAHLG-RG2-ISW-RG3;

[0233] RG1-SPD[Abu]HLG-RG2-ISY-RG3;

[0234] RG1-S[Aze]DAHLG-RG2-ISY-RG3;

[0235] RG1-SPDDHLG-RG2-ISY-RG3;

[0236] RG1-SPDSHLG-RG2-ISY-RG3;

[0237] RG1-SPDAH[Abu]G-RG2-ISY-RG3;

[0238] RG1-SPDAHLG-RG2-IS[4Pal]-RG3;

[0239] RG1-P[dA]DAHLG-RG2-ISY-RG3;

[0240] RG1-SPDAYLG-RG2-[tBuAla]SY-RG3;

[0241] RG1-SPDAHLG-RG2-[C5g]SY-RG3;

[0242] RG1-SPDAHLG-RG2-[Cbg]SY-RG3;

[0243] RG1-SPDAHL[dA]-RG2-ISY-RG3;

[0244] RG1-SPDAH[Aib]G-RG2-ISY-RG3;

[0245] RG1-SPDAHLG-RG2-[Cpg]SY-RG3;

[0246] RG1-SPDAHLG-RG2-[B-MeIle]SY-RG3;

[0247] RG1-SADAHLG-RG2-ISY-RG3;

[0248] RG1-SPAAHLG-RG2-ISY-RG3;

[0249] RG1-SPDAALG-RG2-ISY-RG3;

[0250] RG1-SPDAHAG-RG2-ISY-RG3;

[0251] RG1-SPDAHLA-RG2-ISY-RG3;

[0252] RG1-SPDAHLG-RG2-ASY-RG3;

[0253] RG1-SPDAHLG-RG2-IAY-RG3;

[0254] RG1-SPDAHLG-RG2-ISA-RG3;

[0255] RG1-[K(N3)]PDAHLG-RG2-ISY-RG3;

[0256] RG1-S[K(N3)]DAHLG-RG2-ISY-RG3;

[0257] RG1-SPD[K(N3)]HLG-RG2-ISY-RG3;

[0258] RG1-P[HyP]DAHLG-RG2-[tBuGly]SY-RG3;

[0259] RG1-[HyP][HyP]DAYLG-RG2-[tBuGly]SY-RG3-EPW;

[0260] RG1-[Oxa][HyP]DAYLG-RG2-[tBuGly]SY-RG3-EPW;

[0261] RG1-[Cis-HyP][HyP]DAYLG-RG2-[tBuGly]SY-RG3-EPW;

[0262] RG1-P[Oxa]DAYLG-RG2-[tBuGly]SY-RG3-EPW;

[0263] RG1-P[Cis-HyP]DAYLG-RG2-[tBuGly]SY-RG3-EPW;

[0264] RG1-P[HyP]DA[DOPA]LG-RG2-[tBuGly]SY-RG3-EPW;

[0265] RG1-P[HyP]DA[hTyr]LG-RG2-[tBuGly]SY-RG3-EPW;

[0266] RG1-P[HyP]DA[pCaPhe]LG-RG2-[tBuGly]SY-RG3-EPW;

[0267] RG1-P[HyP]DA[pCoPhe]LG-RG2-[tBuGly]SY-RG3-EPW;

[0268] RG1-P[HyP]DAYL[dS]-RG2-[tBuGly]SY-RG3-EPW;

[0269] RG1-P[HyP]DAYL[dT]-RG2-[tBuGly]SY-RG3-EPW;

[0270] RG1-P[HyP]DAYL[dD]-RG2-[tBuGly]SY-RG3-EPW;

[0271] RG1-P[HyP]DAYL[dE]-RG2-[tBuGly]SY-RG3-EPW;

[0272] RG1-P[HyP]DAYL[dN]-RG2-[tBuGly]SY-RG3-EPW;

[0273] RG1-P[HyP]DAYL[dQ]-RG2-[tBuGly]SY-RG3-EPW;

[0274] RG1-P[HyP]DAYL[dY]-RG2-[tBuGly]SY-RG3-EPW;

[0275] RG1-P[HyP]DAYLS-RG2-[tBuGly]SY-RG3-EPW;

[0276] RG1-P[HyP]DAYLD-RG2-[tBuGly]SY-RG3-EPW;

[0277] RG1-P[HyP]DAYLY-RG2-[tBuGly]SY-RG3-EPW;

[0278] RG1-P[HyP]DAYLN-RG2-[tBuGly]SY-RG3-EPW;

[0279] RG1-P[HyP]DAYLG-RG2-[tBuGly]S[DOPA]-RG3-EPW;

[0280] RG1-P[HyP]DAYLG-RG2-[tBuGly]S[pCaPhe]-RG3-EPW;

[0281] RG1-P[HyP]DAYLG-RG2-[tBuGly]S[pCoPhe]-RG3-EPW;

[0282] RG1-P[HyP]DAYLG-RG2-[tBuGly]S[hTyr]-RG3-EPW;

[0283] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-E[HyP]W;

[0284] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-E[Oxa]W;

[0285] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-E[Cis-HyP]W;

[0286] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-EPY;

[0287] RG1-P[HyP]EAYLG-RG2-[tBuGly]SY-RG3-EPW;

[0288] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-EP[DOPA];

[0289] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-EP[pCaPhe];

[0290] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-EP[pCoPhe];

[0291] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-EP[hTyr];

[0292] RG1-P[HyP][Gla]AYLG-RG2-[tBuGly]SY-RG3-EPW;

[0293] RG1-P[HyP]DAYSG-RG2-[tBuGly]SY-RG3-EPW;

[0294] RG1-P[HyP]DAYTG-RG2-[tBuGly]SY-RG3-EPW;

[0295] RG1-P[HyP]DAYDG-RG2-[tBuGly]SY-RG3-EPW;

[0296] RG1-P[HyP]DAYEG-RG2-[tBuGly]SY-RG3-EPW;

[0297] RG1-P[HyP]DAYNG-RG2-[tBuGly]SY-RG3-EPW;

[0298] RG1-P[HyP]DAYQG-RG2-[tBuGly]SY-RG3-EPW;

[0299] RG1-P[HyP]DAYLG-RG2-[tBuGly][HSer]Y-RG3-EPW;

[0300] RG1-P[HyP]DAYLG-RG2-[tBuGly]TY-RG3-EPW;

[0301] RG1-P[HyP]DAYLG-RG2-[tBuGly]DY-RG3-EPW;

[0302] RG1-P[HyP]DAYLG-RG2-[tBuGly]EY-RG3-EPW;

[0303] RG1-P[HyP]DAYLG-RG2-[tBuGly]NY-RG3-EPW;

[0304] RG1-P[HyP]DAYLG-RG2-[tBuGly]QY-RG3-EPW;

[0305] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-DPW;

[0306] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-[Gla]PW;

[0307] RG1-P[HyP]DAYLG-RG2-YSY-RG3-EPW;

[0308] RG1-P[HyP]DAYLG-RG2-[3HyV]SY-RG3-EPW; and

[0309] RG1-YLPDW[tBuAla]-RG2-GDEY-RG3;

[0310] Or its variants as described herein; wherein RG1, RG2 and RG3 are as defined herein.

[0311] In one embodiment, the peptide ligand comprises a polypeptide or a pharmaceutically acceptable salt thereof, the polypeptide having the following amino acid sequence:

[0312] CSADDWLGCISWC (SEQ ID NO: 4);

[0313] CSSDAYLGCISWC (SEQ ID NO: 5);

[0314] CPPDAHLGCISWC (SEQ ID NO: 6);

[0315] CPQDAYLGCISWC (SEQ ID NO: 7);

[0316] CPPDSWQGCISYC (SEQ ID NO: 8);

[0317] CSPDAHLGCISYC (SEQ ID NO: 9) (referred to as B7 in this document);

[0318] CPGDAHLGCISYC (SEQ ID NO: 10);

[0319] CPPDSHLGCISYC (SEQ ID NO: 11);

[0320] CSADDWLGCISYC (SEQ ID NO: 12);

[0321] CP[HyP]DAYLGC[tBuGly]SYC (SEQ ID NO: 13);

[0322] CP[HyP]DAYLGCISYC (SEQ ID NO: 14);

[0323] CS[HyP]DAHLGCISYC (SEQ ID NO: 15);

[0324] CP[Aib]DAHLGC[tBuGly]SYC (SEQ ID NO: 16);

[0325] CPPDAHLGCISYC (SEQ ID NO: 17);

[0326] CP[Aib]DAYLGC[tBuGly]SYC (SEQ ID NO: 18);

[0327] CSADAHLGCISYC (SEQ ID NO: 19);

[0328] CS[Aib]DAHLGC[tBuGly]SYC (SEQ ID NO: 20);

[0329] CSPDAHLGC[EPA]SYC (SEQ ID NO: 21);

[0330] CPPDAYLGC[tBuGly]SYC (SEQ ID NO: 22);

[0331] CS[Aib]DAYLGC[tBuGly]SYC (SEQ ID NO: 23);

[0332] CAPDAHLGCISYC (SEQ ID NO: 24);

[0333] CP[Defect]DAHLGCISYC (SEQ ID NO: 25);

[0334] CSPDAYLGC[tBuGly]SYC (SEQ ID NO: 26);

[0335] CSPDAHLGC[tBuGly]SYC (SEQ ID NO: 27);

[0336] CPNDAHLGCISYC (SEQ ID NO: 28);

[0337] CPIDAHLGCISYC (SEQ ID NO: 29);

[0338] CSPDAYLGCISYC (SEQ ID NO: 30);

[0339] CPPDAYLGCISYC (SEQ ID NO: 31);

[0340] CS[Aib]DAHLGCISYC (SEQ ID NO: 32);

[0341] CSPDAHLGC[Chg]SYC (SEQ ID NO: 33);

[0342] CAPDAHLGCISYC (SEQ ID NO: 34);

[0343] CYLPDW[tBuAla]CGDEYC (SEQ ID NO: 35);

[0344] CSPDAHLGCIS[2Nal]C (SEQ ID NO: 36);

[0345] CSPDAHLGCIS[3tBuTyr]C (SEQ ID NO: 37);

[0346] CSPD[Aib]HLGCISYC (SEQ ID NO: 38);

[0347] CSPDAHLGCIS[1Nal]C (SEQ ID NO: 39);

[0348] CSPDAH[tBuAla]GCISYC (SEQ ID NO: 40);

[0349] CSPDAH[Cba]GCISYC (SEQ ID NO: 41);

[0350] CSPDAHLGCISWC (SEQ ID NO: 42);

[0351] CSPD[Abu]HLGCISYC (SEQ ID NO: 43);

[0352] CS[Aze]DAHLGCISYC (SEQ ID NO: 44);

[0353] CSPDDHLGCISYC (SEQ ID NO: 45);

[0354] CSPDSHLGCISYC (SEQ ID NO: 46);

[0355] CSPDAH[Abu]GCISYC (SEQ ID NO: 47);

[0356] CSPDAHLGCIS[4Pal]C (SEQ ID NO: 48);

[0357] CP[dA]DAHLGCISYC (SEQ ID NO: 49);

[0358] CSPDAYLGC[tBuAla]SYC (SEQ ID NO: 50);

[0359] CSPDAHLGC[C5g]SYC (SEQ ID NO: 51);

[0360] CSPDAHLGC[Cbg]SYC (SEQ ID NO: 52);

[0361] CSPDAHL[dA]CISYC (SEQ ID NO: 53);

[0362] CSPDAH[Aib]GCISYC (SEQ ID NO: 54);

[0363] CSPDAHLGC[Cpg]SYC (SEQ ID NO: 55);

[0364] CSPDAHLGC[B-MeIle]SYC (SEQ ID NO: 56);

[0365] CSADAHLGCISYC (SEQ ID NO: 57);

[0366] CSPAAHLGCISYC (SEQ ID NO: 58);

[0367] CSPDAALGCISYC (SEQ ID NO: 59);

[0368] CSPDAHAGCISYC (SEQ ID NO: 60);

[0369] CSPDAHLACISYC (SEQ ID NO: 61);

[0370] CSPDAHLGCASYC (SEQ ID NO: 62);

[0371] CSPDAHLGCIAYC (SEQ ID NO: 63);

[0372] CSPDAHLGCISAC (SEQ ID NO: 64);

[0373] C[K(N3)]PDAHLGCISYC (SEQ ID NO: 65);

[0374] CS[K(N3)]DAHLGCISYC (SEQ ID NO: 66);

[0375] CSPD[K(N3)]HLGCISYC (SEQ ID NO: 67);

[0376] CP[HyP]DAHLGC[tBuGly]SYC (SEQ ID NO: 68);

[0377] Or its variants as described herein.

[0378] In one embodiment, the peptide ligand comprises the following amino acid sequence:

[0379] CSADDWLGCISWC (SEQ ID NO: 4);

[0380] CSSDAYLGCISWC (SEQ ID NO: 5);

[0381] CPPDAHLGCISWC (SEQ ID NO: 6);

[0382] CPQDAYLGCISWC (SEQ ID NO: 7);

[0383] CPPDSWQGCISYC (SEQ ID NO: 8);

[0384] CSPDAHLGCISYC (SEQ ID NO: 9) (referred to as B7 in this document);

[0385] CPGDAHLGCISYC (SEQ ID NO: 10);

[0386] CPPDSHLGCISYC (SEQ ID NO: 11);

[0387] CSADDWLGCISYC (SEQ ID NO: 12);

[0388] CP[HyP]DAYLGC[tBuGly]SYC (SEQ ID NO: 13);

[0389] CP[HyP]DAYLGCISYC (SEQ ID NO: 14);

[0390] CS[HyP]DAHLGCISYC (SEQ ID NO: 15);

[0391] CP[Aib]DAHLGC[tBuGly]SYC (SEQ ID NO: 16);

[0392] CPPDAHLGCISYC (SEQ ID NO: 17);

[0393] CP[Aib]DAYLGC[tBuGly]SYC (SEQ ID NO: 18);

[0394] CSADAHLGCISYC (SEQ ID NO: 19);

[0395] CS[Aib]DAHLGC[tBuGly]SYC (SEQ ID NO: 20);

[0396] CSPDAHLGC[EPA]SYC (SEQ ID NO: 21);

[0397] CPPDAYLGC[tBuGly]SYC (SEQ ID NO: 22);

[0398] CS[Aib]DAYLGC[tBuGly]SYC (SEQ ID NO: 23);

[0399] CAPDAHLGCISYC (SEQ ID NO: 24);

[0400] CP[Defect]DAHLGCISYC (SEQ ID NO: 25);

[0401] CSPDAYLGC[tBuGly]SYC (SEQ ID NO: 26);

[0402] CSPDAHLGC[tBuGly]SYC (SEQ ID NO: 27);

[0403] CPNDAHLGCISYC (SEQ ID NO: 28);

[0404] CPIDAHLGCISYC (SEQ ID NO: 29);

[0405] CSPDAYLGCISYC (SEQ ID NO: 30);

[0406] CPPDAYLGCISYC (SEQ ID NO: 31);

[0407] CS[Aib]DAHLGCISYC (SEQ ID NO: 32);

[0408] CSPDAHLGC[Chg]SYC (SEQ ID NO: 33);

[0409] CAPDAHLGCISYC (SEQ ID NO: 34);

[0410] CYLPDW[tBuAla]CGDEYC (SEQ ID NO: 35);

[0411] CSPDAHLGCIS[2Nal]C (SEQ ID NO: 36);

[0412] CSPDAHLGCIS[3tBuTyr]C (SEQ ID NO: 37);

[0413] CSPD[Aib]HLGCISYC (SEQ ID NO: 38);

[0414] CSPDAHLGCIS[1Nal]C (SEQ ID NO: 39);

[0415] CSPDAH[tBuAla]GCISYC (SEQ ID NO: 40);

[0416] CSPDAH[Cba]GCISYC (SEQ ID NO: 41);

[0417] CSPDAHLGCISWC (SEQ ID NO: 42);

[0418] CSPD[Abu]HLGCISYC (SEQ ID NO: 43);

[0419] CS[Aze]DAHLGCISYC (SEQ ID NO: 44);

[0420] CSPDDHLGCISYC (SEQ ID NO: 45);

[0421] CSPDSHLGCISYC (SEQ ID NO: 46);

[0422] CSPDAH[Abu]GCISYC (SEQ ID NO: 47);

[0423] CSPDAHLGCIS[4Pal]C (SEQ ID NO: 48);

[0424] CP[dA]DAHLGCISYC (SEQ ID NO: 49);

[0425] CSPDAYLGC[tBuAla]SYC (SEQ ID NO: 50);

[0426] CSPDAHLGC[C5g]SYC (SEQ ID NO: 51);

[0427] CSPDAHLGC[Cbg]SYC (SEQ ID NO: 52);

[0428] CSPDAHL[dA]CISYC (SEQ ID NO: 53);

[0429] CSPDAH[Aib]GCISYC (SEQ ID NO: 54);

[0430] CSPDAHLGC[Cpg]SYC (SEQ ID NO: 55);

[0431] CSPDAHLGC[B-MeIle]SYC (SEQ ID NO: 56);

[0432] CSADAHLGCISYC (SEQ ID NO: 57);

[0433] CSPAAHLGCISYC (SEQ ID NO: 58);

[0434] CSPDAALGCISYC (SEQ ID NO: 59);

[0435] CSPDAHAGCISYC (SEQ ID NO: 60);

[0436] CSPDAHLACISYC (SEQ ID NO: 61);

[0437] CSPDAHLGCASYC (SEQ ID NO: 62);

[0438] CSPDAHLGCIAYC (SEQ ID NO: 63);

[0439] CSPDAHLGCISAC (SEQ ID NO: 64);

[0440] C[K(N3)]PDAHLGCISYC (SEQ ID NO: 65);

[0441] CS[K(N3)]DAHLGCISYC (SEQ ID NO: 66);

[0442] CSPD[K(N3)]HLGCISYC (SEQ ID NO: 67);

[0443] CP[HyP]DAHLGC[tBuGly]SYC (SEQ ID NO: 68);

[0444] Wherein Abu represents aminobutyric acid, Aib represents aminoisobutyric acid, Aze represents azacyclobutane, B-MeIle represents β-methylisoleucine, C5g represents cyclopentylglycine, Cba represents β-cyclobutylalanine, Cbg represents cyclobutylglycine, Chg represents cyclohexylglycine, Cpg represents cyclopropylglycine, EPA represents 2-amino-3-ethyl-valerate, HyP represents trans-4-hydroxy-L-proline, [K(N3)] represents 6-azidolysine, 1Nal represents 1-naphthylalanine, 2Nal represents 2-naphthylalanine, 4Pal represents 4-pyridylalanine, tBuAla represents tert-butylalanine, tBuGly represents tert-butylglycine, 3tBuTyr represents 3-tert-butyltyrosine, or pharmaceutically acceptable salts thereof.

[0445] In some implementations, the first loop sequence contains 7 amino acids and is selected from:

[0446] SADDWLG;

[0447] SSDAYLG;

[0448] PPDAHLG;

[0449] PQDAYLG;

[0450] PPDSWQG;

[0451] SPDAHLG;

[0452] PGDAHLG; and

[0453] PPDSHLG;

[0454] and its variants as described herein;

[0455] And its pharmaceutically acceptable salts.

[0456] In some embodiments, the second ring sequence contains three amino acids and is selected from ISW and ISY;

[0457] and its variants as described herein;

[0458] And its pharmaceutically acceptable salts.

[0459] In some embodiments, the peptide ligand comprises a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide having an amino acid sequence selected from:

[0460] RG1-SADDWLG-RG2-ISW-RG3;

[0461] RG1-SSDAYLG-RG2-ISW-RG3;

[0462] RG1-PPDAHLG-RG2-ISW-RG3;

[0463] RG1-PQDAYLG-RG2-ISW-RG3;

[0464] RG1-PPDSWQG-RG2-ISY-RG3;

[0465] RG1-SPDAHLG-RG2-ISY-RG3;

[0466] RG1-PGDAHLG-RG2-ISY-RG3;

[0467] RG1-PPDSHLG-RG2-ISY-RG3;

[0468] RG1-SADDWLG-RG2-ISY-RG3; and

[0469] RG1-P[HyP]DAHLG-RG2-[tBuGly]SY-RG3

[0470] Or its variants as described herein; wherein RG1, RG2 and RG3 are as defined herein.

[0471] In another embodiment, the peptide ligand comprises the following amino acid sequence:

[0472] CSADDWLGCISWC (SEQ ID NO: 4);

[0473] CSSDAYLGCISWC (SEQ ID NO: 5);

[0474] CPPDAHLGCISWC (SEQ ID NO: 6);

[0475] CPQDAYLGCISWC (SEQ ID NO: 7);

[0476] CPPDSWQGCISYC (SEQ ID NO: 8);

[0477] CSPDAHLGCISYC (SEQ ID NO: 9);

[0478] CPGDAHLGCISYC (SEQ ID NO: 10);

[0479] CPPDSHLGCISYC (SEQ ID NO: 11);

[0480] CSADDWLGCISYC (SEQ ID NO: 12); and

[0481] CP[HyP]DAHLGC[tBuGly]SYC

[0482] Or its variants as described herein;

[0483] Its pharmaceutically acceptable salt.

[0484] In another embodiment, the peptide ligand comprises the following amino acid sequence:

[0485] CSADDWLGCISWC (SEQ ID NO: 4);

[0486] CSSDAYLGCISWC (SEQ ID NO: 5);

[0487] CPPDAHLGCISWC (SEQ ID NO: 6);

[0488] CPQDAYLGCISWC (SEQ ID NO: 7);

[0489] CPPDSWQGCISYC (SEQ ID NO: 8);

[0490] CSPDAHLGCISYC (SEQ ID NO: 9);

[0491] CPGDAHLGCISYC (SEQ ID NO: 10);

[0492] CPPDSHLGCISYC (SEQ ID NO: 11); and

[0493] CSADDWLGCISYC (SEQ ID NO: 12);

[0494] Or its pharmaceutically acceptable salt.

[0495] In another embodiment, the peptide ligand comprises a polypeptide having the amino acid sequence RG1-P[HyP]DAHLG-RG2-[tBuGly]SY-RG3, or a variant thereof as described herein, or a pharmaceutically acceptable salt thereof, wherein RG1, RG2, and RG3 are as defined herein. In yet another embodiment, the peptide ligand comprises the amino acid sequence CP[HyP]DAHLGC[tBuGly]SYC (SEQ ID NO: 68), wherein HyP represents trans-4-hydroxy-L-proline and tBuGly represents tert-butylglycine.

[0496] This document also provides a peptide ligand specifically targeting transferrin receptor 1 (TfR1), wherein the peptide ligand comprises a polypeptide having the amino acid sequence as described herein. For the avoidance of doubt, in some embodiments, the peptide ligand further comprises a half-life extension portion, such as the half-life extension portion described herein. In some embodiments, the peptide ligand does not further comprise a half-life extension portion, such as the half-life extension portion described herein. It should be understood that the peptide ligand of SEQ ID NO: 68 constitutes a novel peptide ligand specific to Tfr1. Therefore, this document provides a peptide ligand specifically targeting transferrin receptor 1 (TfR1), wherein the peptide ligand comprises a polypeptide having the amino acid sequence RG1-P[HyP]DAHLG-RG2-[tBuGly]SY-RG3, or a variant thereof as described herein, or a pharmaceutically acceptable salt thereof, wherein RG1, RG2, and RG3 are as defined herein. Therefore, according to another aspect of the invention, a peptide ligand specifically targeting transferrin receptor 1 (TfR1) is provided, wherein the peptide ligand comprises the amino acid sequence CP[HyP]DAHLGC[tBuGly]SYC (SEQ ID NO: 68), wherein HyP represents trans-4-hydroxy-L-proline and tBuGly represents tert-butylglycine.

[0497] In some embodiments, the peptide comprises an N-terminal and / or C-terminal addition. In some embodiments, the N-terminal and / or C-terminal addition comprises one or more natural or non-natural amino acids. In some embodiments, the N-terminal and / or C-terminal addition comprises one or more groups selected from the following: A, G, [Sar], N, [1Nal], W, [dW], H, M, E, V, T, S, [NMeTrp], F, [AzPro], [dY], [Pip], P, [Aze], [dP], I, Q, [K(N3)], [K(N3)(PYA–maleimide], PEG (e.g., PEG5 to PEG30, such as PEG10, PEG12, PEG14, PEG16, PEG18, PEG20, PEG22, PEG24, PEG26, PEG28, PEG30). In some embodiments, one or more N-terminal and / or C-terminal additions comprise about 1 to about 10, for example about 2 to about 5, such as 2, 3, or 4 such groups. Examples of N-terminal modification include elongating the peptide by one or more amino acids or amino acid analogs (e.g., alanine (A) or variants thereof), or N-terminal acetylation (represented by "Ac"). Examples of C-terminal modification include elongating the peptide by one or more amino acids or amino acid analogs (e.g., alanine (A)), or C-terminal amidation, i.e., converting the C-terminal carboxylic acid group (-CO(O)H or -CO(O)-) to an amide -C(O)NH2. In some embodiments, the N-terminal and / or C-terminal additions comprise one or more N-terminal additions selected from the following:

[0498] A-;

[0499] Ac-;

[0500] [Fl]G[Sar5]-A-;

[0501] N[1Nal]N-;

[0502] NWN-;

[0503] Ac-NWN-;

[0504] Ac-N[1Nal]N-;

[0505] N[dW]N-;

[0506] Ac-N[dW]N-;

[0507] HWM-;

[0508] NEV-;

[0509] HTS-;

[0510] Ac-N[NMeTrp]N-;

[0511] N[NMeTrp]N-;

[0512] Ac-A-;

[0513] ESF-;

[0514] [AzPro]-NWN-;

[0515] [AzPro]-; and

[0516] Ac-N[dY]N-;

[0517] And / or one or more C-terminals selected from the following:

[0518] -A;

[0519] -A-[Sar6]-[K-Fl];

[0520] -[Sar6]-[K-Fl];

[0521] -E[Pip]W;

[0522] -EPW;

[0523] -E[Aze]W;

[0524] -E[dP]W;

[0525] -PHP;

[0526] -PIVH;

[0527] -EHQE;

[0528] -[K(N3)];

[0529] -EPW-[K(N3)];

[0530] -E-[dP]-W-[K(N3)];

[0531] -E-[Aze]-W-[K(N3)];

[0532] -E-[Pip]-W-[K(N3)];

[0533] -[K(N3)(PYA-maleimide];

[0534] -EPW-[Peg10]-[K(N3)];

[0535] -EPW-[Peg24]-[K(N3)]; and

[0536] -EPWGGSGGS-[K(N3)].

[0537] In some embodiments, the peptide ligand comprises the polypeptide of SEQ ID NO: 68 and includes one or more N-terminal and / or C-terminal additions as described herein. Exemplary peptides include: Ac-(RG1-P[HyP]DAHLG-RG2-[tBuGly]SY-RG3); for example, Ac-(SEQ ID NO: 68) (referred to herein as B191). In some embodiments, the peptide ligand comprises the polypeptide of SEQ ID NO: 13 and includes one or more N-terminal and / or C-terminal additions as described herein.

[0538] In some embodiments, the non-inhibitory peptide ligand comprises a polypeptide as described herein, which is linked (e.g., by covalent bonding) to a molecular scaffold as described herein. In some embodiments, the polypeptide is linked (e.g., covalently bonded) to the molecular scaffold via a bond between the molecular scaffold and each of RG1, RG2, and RG3. When RG1, RG2, and RG3 are cysteine ​​residues, the polypeptide is typically linked to the molecular scaffold via a covalent bond between the thiol group of the cysteine ​​residue and the scaffold. In some embodiments, the scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethylone) (TATB).

[0539] In another embodiment, the molecular scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethylone) (TATB), and the peptide ligand comprises an N-terminal and / or C-terminal addition selected from:

[0540] A-(SEQ ID NO: 4)-A (referred to as B8 in this document);

[0541] A-(SEQ ID NO: 4)-A-[Sar6]-[K-Fl] (referred to as B9 in this paper);

[0542] A-(SEQ ID NO: 5)-A (referred to as B10 in this document);

[0543] A-(SEQ ID NO: 5)-A-[Sar6]-[K-Fl] (referred to as B11 in this paper);

[0544] A-(SEQ ID NO: 6)-A (referred to as B12 in this document);

[0545] Ac-(SEQ ID NO: 6) (referred to as B13 in this article);

[0546] A-(SEQ ID NO: 7)-A (referred to as B14 in this document);

[0547] Ac-(SEQ ID NO: 7) (referred to as B15 in this document);

[0548] A-(SEQ ID NO: 8)-A (referred to as B16 in this document);

[0549] A-(SEQ ID NO: 8)-A-[Sar6]-[K-Fl] (referred to as B17 in this paper);

[0550] A-(SEQ ID NO: 9)-A (referred to as B18 in this document);

[0551] A-(SEQ ID NO: 9)-A-[Sar6]-[K-Fl] (referred to as B19 in this paper);

[0552] (SEQ ID NO: 9)-[Sar6]-[K-Fl] (referred to as B20 in this paper);

[0553] Ac-(SEQ ID NO: 9)-A-[Sar6]-[K-Fl] (referred to as B21 in this paper);

[0554] Ac-(SEQ ID NO: 9)-[Sar6]-[K-Fl] (referred to as B22 in this paper);

[0555] [Fl]G[Sar5]-A-(SEQ ID NO: 9)-A (referred to as B23 in this paper);

[0556] N[1Nal]N-(SEQ ID NO: 9) (referred to as B24 in this paper);

[0557] Ac-(SEQ ID NO: 9)-E[Pip]W (referred to as B25 in this paper);

[0558] Ac-(SEQ ID NO: 9)-EPW (referred to as B26 in this paper);

[0559] NWN-(SEQ ID NO: 9) (referred to as B27 in this document);

[0560] NWN-(SEQ ID NO: 9)-A (referred to as B28 in this article);

[0561] Ac-(SEQ ID NO: 9)-E[Aze]W (referred to as B29 in this paper);

[0562] Ac-NWN-(SEQ ID NO: 9) (referred to as B30 in this document);

[0563] Ac-(SEQ ID NO: 9)-E[dP]W (referred to as B31 in this paper);

[0564] Ac-N[1Nal]N-(SEQ ID NO: 9) (referred to as B32 in this paper);

[0565] N[dW]N-(SEQ ID NO: 9) (referred to as B33 in this article);

[0566] Ac-N[dW]N-(SEQ ID NO: 9) (referred to as B34 in this article);

[0567] HWM-(SEQ ID NO: 9)-A (referred to as B35 in this article);

[0568] A-(SEQ ID NO: 9)-PHP (referred to as B36 in this article);

[0569] A-(SEQ ID NO: 9)-EPW (referred to as B37 in this document);

[0570] NEV-(SEQ ID NO: 9)-A (referred to as B38 in this document);

[0571] A-(SEQ ID NO: 9)-PIVH (referred to as B39 in this article);

[0572] Ac-(SEQ ID NO: 9) (referred to as B40 in this article);

[0573] HTS-(SEQ ID NO: 9)-A (referred to as B41 in this document);

[0574] Ac-N[NMeTrp]N-(SEQ ID NO: 9) (referred to as B42 in this paper);

[0575] N[NMeTrp]N-(SEQ ID NO: 9) (referred to as B43 in this paper);

[0576] Ac-A-(SEQ ID NO: 9)-A (referred to as B44 in this document);

[0577] A-(SEQ ID NO: 9)-EHQE (referred to as B45 in this document);

[0578] ESF-(SEQ ID NO: 9)-A (referred to as B46 in this document);

[0579] NWN-(SEQ ID NO: 9)-[K(N3)] (referred to as B47 in this paper);

[0580] Ac-NWN-(SEQ ID NO: 9)-[K(N3)] (referred to as B48 in this paper);

[0581] [AzPro]-NWN-(SEQ ID NO: 9) (referred to as B49 in this document);

[0582] Ac-(SEQ ID NO: 9)-EPW-[K(N3)] (referred to as B50 in this paper);

[0583] [AzPro]-(SEQ ID NO: 9)-EPW (referred to as B51 in this document);

[0584] Ac-(SEQ ID NO: 9)-[K(N3)] (referred to as B52 in this paper);

[0585] [AzPro]-(SEQ ID NO: 9) (referred to as B53 in this document);

[0586] Ac-N[dY]N-(SEQ ID NO: 9)-[K(N3)] (referred to as B54 in this paper);

[0587] Ac-(SEQ ID NO: 9)-E-[dP]-W-[K(N3)] (referred to as B55 in this paper);

[0588] Ac-(SEQ ID NO: 9)-E-[Aze]-W-[K(N3)] (referred to as B56 in this paper);

[0589] Ac-(SEQ ID NO: 9)-E-[Pip]-W-[K(N3)] (referred to as B57 in this paper);

[0590] Ac-(SEQ ID NO: 9)-[K(N3)(PYA-maleimide] (referred to as B58 in this article);

[0591] Ac-(SEQ ID NO: 9)-EPW-[Peg 10 ]-[K(N3)] (referred to as B59 in this paper);

[0592] Ac-(SEQ ID NO: 9)-EPW-[Peg 24 ]-[K(N3)] (referred to as B60 in this paper);

[0593] Ac-(SEQ ID NO: 9)-EPWGGSGGS-[K(N3)] (referred to as B61 in this paper);

[0594] A-(SEQ ID NO: 10)-A (referred to as B62 in this document);

[0595] Ac-(SEQ ID NO: 10) (referred to as B63 in this article);

[0596] A-(SEQ ID NO: 11)-A (referred to as B64 in this document);

[0597] Ac-(SEQ ID NO: 11) (referred to as B65 in this document);

[0598] A-(SEQ ID NO: 12)-A (referred to as B66 in this document);

[0599] Ac-(SEQ ID NO: 12) (referred to as B67 in this document);

[0600] Ac-(SEQ ID NO: 13) (referred to as B68 in this article);

[0601] Ac-(SEQ ID NO: 13)-EPW (referred to as B69 in this paper);

[0602] Ac-NWN-(SEQ ID NO: 13) (referred to as B70 in this document);

[0603] NWN-(SEQ ID NO: 13) (referred to as B71 in this document);

[0604] A-(SEQ ID NO: 13)-A (referred to as B72 in this document);

[0605] Ac-(SEQ ID NO: 13)-[K(N3)] (referred to as B73 in this paper);

[0606] Ac-NWN-(SEQ ID NO: 13)-[K(N3)] (referred to as B74 in this paper);

[0607] Ac-(SEQ ID NO: 13)-EPW-[K(N3)] (referred to as B75 in this paper);

[0608] Ac-(SEQ ID NO: 14) (referred to as B76 in this article);

[0609] Ac-(SEQ ID NO: 14)-[K(N3)] (referred to as B77 in this paper);

[0610] [AzPro]-(SEQ ID NO: 14) (referred to as B78 in this document);

[0611] Ac-(SEQ ID NO: 15) (referred to as B79 in this document);

[0612] A-(SEQ ID NO: 15)-A (referred to as B80 in this document);

[0613] Ac-(SEQ ID NO: 15)-[K(N3)] (referred to as B81 in this paper);

[0614] [AzPro]-(SEQ ID NO: 15) (referred to as B82 in this document);

[0615] A-(SEQ ID NO: 16)-A (referred to as B83 in this document);

[0616] TYMN-(SEQ ID NO: 17)-A (referred to as B84 in this article);

[0617] A-(SEQ ID NO: 17)-A (referred to as B85 in this document);

[0618] A-(SEQ ID NO: 18)-A (referred to as B86 in this document);

[0619] Ac-(SEQ ID NO: 19) (referred to as B87 in this document);

[0620] A-(SEQ ID NO: 20)-A (referred to as B88 in this document);

[0621] A-(SEQ ID NO: 21)-A (referred to as B89 in this document);

[0622] A-(SEQ ID NO: 22)-A (referred to as B90 in this document);

[0623] A-(SEQ ID NO: 23)-A (referred to as B91 in this document);

[0624] Ac-(SEQ ID NO: 24) (referred to as B92 in this article);

[0625] A-(SEQ ID NO: 25)-A (referred to as B93 in this document);

[0626] A-(SEQ ID NO: 26)-A (referred to as B94 in this document);

[0627] A-(SEQ ID NO: 27)-A (referred to as B95 in this document);

[0628] IDSN-(SEQ ID NO: 28)-A (referred to as B96 in this document);

[0629] WGKS-(SEQ ID NO: 29)-A (referred to as B97 in this document);

[0630] A-(SEQ ID NO: 30)-A (referred to as B98 in this document);

[0631] A-(SEQ ID NO: 31)-A (referred to as B99 in this document);

[0632] A-(SEQ ID NO: 32)-A (referred to as B100 in this document);

[0633] A-(SEQ ID NO: 33)-A (referred to as B101 in this document);

[0634] A-(SEQ ID NO: 34)-A-[Sar6]-[K-Fl] (referred to as B102 in this paper);

[0635] A-(SEQ ID NO: 35)-A (referred to as B103 in this document);

[0636] A-(SEQ ID NO: 36)-A (referred to as B104 in this document);

[0637] A-(SEQ ID NO: 37)-A (referred to as B105 in this document);

[0638] A-(SEQ ID NO: 38)-A (referred to as B106 in this document);

[0639] A-(SEQ ID NO: 39)-A (referred to as B107 in this document);

[0640] A-(SEQ ID NO: 40)-A (referred to as B108 in this document);

[0641] A-(SEQ ID NO: 41)-A (referred to as B109 in this document);

[0642] A-(SEQ ID NO: 42)-A (referred to as B110 in this document);

[0643] A-(SEQ ID NO: 43)-A (referred to as B111 in this document);

[0644] A-(SEQ ID NO: 44)-A (referred to as B112 in this document);

[0645] Ac-(SEQ ID NO: 44) (referred to as B113 in this article);

[0646] Ac-(SEQ ID NO: 44)-[K(N3)] (referred to as B114 in this paper);

[0647] [AzPro]-(SEQ ID NO: 44) (referred to as B115 in this document);

[0648] A-(SEQ ID NO: 45)-A (referred to as B116 in this document);

[0649] A-(SEQ ID NO: 46)-A (referred to as B117 in this document);

[0650] A-(SEQ ID NO: 47)-A (referred to as B118 in this document);

[0651] A-(SEQ ID NO: 48)-A (referred to as B119 in this document);

[0652] A-(SEQ ID NO: 49)-A (referred to as B120 in this document);

[0653] A-(SEQ ID NO: 50)-A (referred to as B121 in this document);

[0654] A-(SEQ ID NO: 51)-A (referred to as B122 in this document);

[0655] A-(SEQ ID NO: 52)-A (referred to as B123 in this document);

[0656] A-(SEQ ID NO: 53)-A (referred to as B124 in this document);

[0657] A-(SEQ ID NO: 54)-A (referred to as B125 in this document);

[0658] A-(SEQ ID NO: 55)-A (referred to as B126 in this document);

[0659] A-(SEQ ID NO: 56)-A (referred to as B127 in this document);

[0660] A-(SEQ ID NO: 57)-A-[Sar6]-[K-Fl] (referred to as B128 in this paper);

[0661] A-(SEQ ID NO: 58)-A-[Sar6]-[K-Fl] (referred to as B129 in this paper);

[0662] A-(SEQ ID NO: 59)-A-[Sar6]-[K-Fl] (referred to as B130 in this paper);

[0663] A-(SEQ ID NO: 60)-A-[Sar6]-[K-Fl] (referred to as B131 in this paper);

[0664] A-(SEQ ID NO: 61)-A-[Sar6]-[K-Fl] (referred to as B132 in this paper);

[0665] A-(SEQ ID NO: 62)-A-[Sar6]-[K-Fl] (referred to as B133 in this paper);

[0666] A-(SEQ ID NO: 63)-A-[Sar6]-[K-Fl] (referred to as B134 in this paper);

[0667] A-(SEQ ID NO: 64)-A-[Sar6]-[K-Fl] (referred to as B135 in this paper);

[0668] Ac-(SEQ ID NO: 65) (referred to as B136 in this article);

[0669] Ac-(SEQ ID NO: 66) (referred to as B137 in this document);

[0670] Ac-(SEQ ID NO: 67) (referred to as B138 in this document);

[0671] Ac-(SEQ ID NO: 68) (referred to as B191 in this article);

[0672] Ac-(SEQ ID NO: 68)-EPW (referred to as B196 in this paper);

[0673] Ac-(SEQ ID NO: 68)-EPW-[K(N3)] (referred to herein as B197); and

[0674] Ac-(SEQ ID NO: 68)-EPW-[K(PYA)] (referred to as B198 in this paper);

[0675] Where AzPro represents azidepropyl, Aze represents azacyclobutane, 1Nal represents 1-naphthylalanine, NMeTrp represents N-methyl-tryptophan, K(PYA) represents ε-4-pentynyllysine, [K(N3)] represents 6-azidolysine, Peg represents polyethylene glycol, Pip represents piperidine acid, Sar represents sarcosine, Fl represents fluorescein, and [K(N3)(PYA-maleimide)] represents a modified lysine with the following structure:

[0676] .

[0677] In another embodiment, the molecular scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethylone) (TATB), and the peptide ligand comprises an N-terminal and / or C-terminal addition, and is selected from:

[0678] Ac-(SEQ ID NO: 68) (referred to as B191 in this article);

[0679] Ac-(SEQ ID NO: 68)-EPW (referred to as B196 in this paper);

[0680] Ac-(SEQ ID NO: 68)-EPW-[K(N3)] (referred to herein as B197); and

[0681] Ac-(SEQ ID NO: 68)-EPW-[K(PYA)] (referred to as B198 in this paper);

[0682] Where [K(N3)] represents 6-azidolysine, and K(PYA) represents ε-4-pentyneyllysine.

[0683] It is understood that the bicyclic peptide ligands B191 and B196 to B198 constitute novel bicyclic peptide ligands specific for TfR1. Therefore, according to another aspect of the invention, a bicyclic peptide ligand specifically targeting transferrin receptor 1 (TfR1) is provided, wherein the peptide ligand is B191 or B196 to B198.

[0684] Specifically, B191 was tested in the SPR assay described in WO 2022 / 101633 and demonstrated to have a Kd of 20 nM with human TfR1 and a Kd of 379 nM with cynomolgus monkey TfR1. Therefore, according to another aspect of the invention, a bicyclic peptide ligand specifically targeting transferrin receptor 1 (TfR1) is provided, wherein the peptide ligand is B191.

[0685] In an alternative embodiment, the molecular scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA), and the peptide ligand comprises an N-terminal and / or C-terminal addition, and is as follows:

[0686] Ac-(SEQ ID NO: 13) (referred to as B139 in this document).

[0687] Other examples of bicyclic peptides that specifically target TfR1 include:

[0688] C[HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 69, referred to as B140 in this document);

[0689] C[Oxa][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 70, referred to as B141 in this document);

[0690] C[Cis-HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 71, referred to as B142 in this document);

[0691] CP[Oxa]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 72, referred to as B143 in this document);

[0692] CP[Cis-HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 73, referred to as B144 in this document);

[0693] CP[HyP]DA[DOPA]LGC[tBuGly]SYCEPW (SEQ ID NO: 74, referred to as B145 in this document);

[0694] CP[HyP]DA[pCaPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 75, referred to as B146 in this document);

[0695] CP[HyP]DA[pCoPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 76, referred to as B147 in this document);

[0696] CP[HyP]DA[hTyr]LGC[tBuGly]SYCEPW (SEQ ID NO: 77, referred to as B148 in this document);

[0697] CP[HyP]DAYL[dS]C[tBuGly]SYCEPW (SEQ ID NO: 78, referred to as B149 in this document);

[0698] CP[HyP]DAYL[dT]C[tBuGly]SYCEPW (SEQ ID NO: 79, referred to as B150 in this document);

[0699] CP[HyP]DAYL[dD]C[tBuGly]SYCEPW (SEQ ID NO: 80, referred to as B151 in this document);

[0700] CP[HyP]DAYL[dE]C[tBuGly]SYCEPW (SEQ ID NO: 81, referred to as B152 in this document);

[0701] CP[HyP]DAYL[dN]C[tBuGly]SYCEPW (SEQ ID NO: 82, referred to as B153 in this document);

[0702] CP[HyP]DAYL[dQ]C[tBuGly]SYCEPW (SEQ ID NO: 83, referred to as B154 in this document);

[0703] CP[HyP]DAYL[dY]C[tBuGly]SYCEPW (SEQ ID NO: 84, referred to as B155 in this document);

[0704] CP[HyP]DAYLSC[tBuGly]SYCEPW (SEQ ID NO: 85, referred to as B156 in this document);

[0705] CP[HyP]DAYLDC[tBuGly]SYCEPW (SEQ ID NO: 86, referred to as B157 in this document);

[0706] CP[HyP]DAYLYC[tBuGly]SYCEPW (SEQ ID NO: 87, referred to as B158 in this document);

[0707] CP[HyP]DAYLNC[tBuGly]SYCEPW (SEQ ID NO: 88, referred to as B159 in this document);

[0708] CP[HyP]DAYLGC[tBuGly]S[DOPA]CEPW (SEQ ID NO: 89, referred to herein as B160);

[0709] CP[HyP]DAYLGC[tBuGly]S[pCaPhe]CEPW (SEQ ID NO: 90, referred to as B161 in this document);

[0710] CP[HyP]DAYLGC[tBuGly]S[pCoPhe]CEPW (SEQ ID NO: 91, referred to herein as B162);

[0711] CP[HyP]DAYLGC[tBuGly]S[hTyr]CEPW (SEQ ID NO: 92, referred to as B163 in this document);

[0712] CP[HyP]DAYLGC[tBuGly]SYCE[HyP]W (SEQ ID NO: 93, referred to as B164 in this document);

[0713] CP[HyP]DAYLGC[tBuGly]SYCE[Oxa]W (SEQ ID NO: 94, referred to as B165 in this document);

[0714] CP[HyP]DAYLGC[tBuGly]SYCE[Cis-HyP]W (SEQ ID NO: 95, referred to as B166 in this document);

[0715] CP[HyP]DAYLGC[tBuGly]SYCEPY (SEQ ID NO: 96, referred to as B167 in this document);

[0716] CP[HyP]DAYLGC[tBuGly]SYCEP[DOPA] (SEQ ID NO: 97, referred to as B168 in this document);

[0717] CP[HyP]DAYLGC[tBuGly]SYCEP[pCaPhe] (SEQ ID NO: 98, referred to as B169 in this document);

[0718] CP[HyP]DAYLGC[tBuGly]SYCEP[pCoPhe] (SEQ ID NO: 99, referred to as B170 in this document);

[0719] CP[HyP]DAYLGC[tBuGly]SYCEP[hTyr] (SEQ ID NO: 100, referred to as B171 in this document);

[0720] CP[HyP]EAYLGC[tBuGly]SYCEPW (SEQ ID NO: 101, referred to as B172 in this document);

[0721] CP[HyP][Gla]AYLGC[tBuGly]SYCEPW (SEQ ID NO: 102, referred to as B173 in this document);

[0722] CP[HyP]DAYSGC[tBuGly]SYCEPW (SEQ ID NO: 103, referred to as B174 in this document);

[0723] CP[HyP]DAYTGC[tBuGly]SYCEPW (SEQ ID NO: 104, referred to as B175 in this document);

[0724] CP[HyP]DAYDGC[tBuGly]SYCEPW (SEQ ID NO: 105, referred to as B176 in this document);

[0725] CP[HyP]DAYEGC[tBuGly]SYCEPW (SEQ ID NO: 106, referred to as B177 in this document);

[0726] CP[HyP]DAYNGC[tBuGly]SYCEPW (SEQ ID NO: 107, referred to as B178 in this document);

[0727] CP[HyP]DAYQGC[tBuGly]SYCEPW (SEQ ID NO: 108, referred to as B179 in this document);

[0728] CP[HyP]DAYLGC[tBuGly][HSer]YCEPW (SEQ ID NO: 109, referred to as B180 in this document);

[0729] CP[HyP]DAYLGC[tBuGly]TYCEPW (SEQ ID NO: 110, referred to as B181 in this document);

[0730] CP[HyP]DAYLGC[tBuGly]DYCEPW (SEQ ID NO: 111, referred to as B182 in this document);

[0731] CP[HyP]DAYLGC[tBuGly]EYCEPW (SEQ ID NO: 112, referred to as B183 in this document);

[0732] CP[HyP]DAYLGC[tBuGly]NYCEPW (SEQ ID NO: 113, referred to as B184 in this document);

[0733] CP[HyP]DAYLGC[tBuGly]QYCEPW (SEQ ID NO: 114, referred to as B185 in this document);

[0734] CP[HyP]DAYLGC[tBuGly]SYCDPW (SEQ ID NO: 115, referred to as B186 in this document);

[0735] CP[HyP]DAYLGC[tBuGly]SYC[Gla]PW (SEQ ID NO: 116, referred to as B187 in this document);

[0736] CP[HyP]DAYLGCYSYCEPW (SEQ ID NO: 117, referred to herein as B188); and

[0737] CP[HyP]DAYLGC[3HyV]SYCEPW (SEQ ID NO: 118, referred to herein as B189),

[0738] Cis-HyP represents cis-L-4-hydroxyproline, DOPA represents 3,4-dihydroxyphenylalanine, Gla represents L-γ-carboxyglutamic acid, HyP represents hydroxyproline, HSerr represents homoserine, hTyr represents homotyrosine, 3HyV represents 3-hydroxy-L-valine, Oxa represents oxazolidin-4-carboxylic acid, pCaPhe represents L-4-carbamoylphenylalanine, pCoPhe represents 4-carboxy-L-phenylalanine, and tBuGly represents tert-butylglycine.

[0739] The polypeptide may have an amino acid sequence as defined herein (e.g., a polypeptide according to any one of SEQ ID NO: 1 to 118), wherein one, two, three, four, or five amino acids, typically one, two, or three, are modified as described herein. In some embodiments, the polypeptide may have at least 70% sequence identity with any of the peptides described herein (e.g., any one of SEQ ID NO: 1-118), such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. It should be understood that the term "sequence identity" as used herein is understood to mean the percentage identity between two protein sequences (e.g., SEQ ID NO: X and SEQ ID NO: Y), as measured, for example, using the methods described herein.

[0740] In some embodiments, the inhibitory peptide ligand comprises a polypeptide as described herein, which is linked (e.g., by covalent bonding) to a molecular scaffold as described herein. In some embodiments, the polypeptide is linked (e.g., covalently bonded) to the molecular scaffold via a bond between the molecular scaffold and each of RG1, RG2, and RG3. When RG1, RG2, and RG3 are cysteine ​​residues, the polypeptide is typically linked to the molecular scaffold via a covalent bond between the thiol group of the cysteine ​​residue and the scaffold. In some embodiments, the scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tripropyl-2-en-1-one (TATA) or 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethylone) (TATB).

[0741] For the purposes of this specification, it is assumed that the non-inhibitory bicyclic peptide is cyclized with TATA or TATB to produce a trisubstituted structure. However, as can be clearly seen from the description of the invention presented herein, cyclization can be performed using any suitable molecular scaffold that forms a covalent bond with the reactive group of the peptide, such that at least two peptide rings are formed. In some embodiments, the peptide is attached to the molecular scaffold via a bond (e.g., covalent bonding) between the molecular scaffold and each of RG1, RG2, and RG3. When RG1, RG2, and RG3 are cysteine ​​residues, cyclization occurs on the first, second, and third cysteine ​​residues, respectively.

[0742] In another embodiment, the pharmaceutically acceptable salt is selected from free acids or sodium, potassium, calcium, or ammonium salts.

[0743] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry). Molecular biological, genetic, and biochemical methods employ standard techniques (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999), 4th ed., John Wiley & Sons, Inc.), which are incorporated herein by reference.

[0744] Multimeric bicyclic peptide ligand

[0745] In one embodiment, the bicyclic peptide ligand complex comprises more than one bicyclic peptide (e.g., 2, 3, or 4) conjugated via one or more linkers. The bicyclic peptides may be the same or different. In some embodiments, at least one bicyclic peptide in the complex is a peptide ligand as described in more detail herein (e.g., a monomeric bicyclic peptide ligand). In some embodiments, the bicyclic peptide ligand complex comprises at least two (i.e., 2, 3, or 4) monomeric bicyclic peptide ligands as defined herein.

[0746] This aspect of the invention describes a series of polymerized bicyclic peptides that utilize various chemical connectors and hinges of different lengths and rigidities at different linkage sites within the bicyclic peptides to bind and activate TfR1 with broad potency and efficacy.

[0747] Those skilled in the art will understand that this aspect of the invention provides multiple arrangements of (multimeric) bicyclic peptides, which offer synergistic benefits due to the properties produced by the multimeric binding complex compared to the corresponding monomeric binding complexes comprising a single bicyclic peptide. For example, the multimeric binding complexes of this aspect of the invention typically have a higher level of binding potency or affinity (as measured herein by Kd values) than their monomeric counterparts. Furthermore, the multimeric binding complexes of the invention are designed to be small enough for renal clearance.

[0748] Beyond theory, it is believed that polymerized bicyclic peptides can activate receptors through homologous crosslinking with more than one identical receptor. Therefore, in one embodiment, the bicyclic peptide ligand specifically targets the same target within TfR1. In another embodiment, the multimeric binding complex comprises at least two identical bicyclic peptide ligands. "Identical" means bicyclic peptides having the same amino acid sequence; crucially, the same amino acid sequence refers to the binding portion of the bicyclic peptide (e.g., the sequence may differ at the linker position). In this embodiment, each bicyclic peptide within the multimeric binding complex will bind to the exact same epitope on the same target of TfR1—thus, the resulting target-bound complex will produce a homodimer (if the multimeric complex comprises two identical bicyclic peptides), a homotrimer (if the multimeric complex comprises three identical bicyclic peptides), or a homotetramer (if the multimeric complex comprises four identical bicyclic peptides), etc.

[0749] In an alternative embodiment, the multimeric binding complex comprises at least two distinct bicyclic peptide ligands. "Distinct" means bicyclic peptides with different amino acid sequences. In this embodiment, the different bicyclic peptide ligands within the multimeric binding complex will bind to different epitopes on TfR1—therefore, the resulting target-bound complex will produce biepisodes (if the multimeric complex comprises two distinct bicyclic peptides), triepisodes (if the multimeric complex comprises three distinct bicyclic peptides), or tetraepisodes (if the multimeric complex comprises four distinct bicyclic peptides), etc.

[0750] Beyond theory, it is believed that polymerized bicyclic peptides can activate receptors by heterologously crosslinking different targets (e.g., different target sites on TfR1). Therefore, in one embodiment, the bicyclic peptide ligand is specifically targeted at different targets on TfR1. It should be understood that in this embodiment, the multimeric binding complex comprises at least two different bicyclic peptide ligands (i.e., bicyclic peptide ligands with different amino acid sequences). In this embodiment, each bicyclic peptide within the multimeric binding complex will bind to a different epitope on TfR1—thus, the resulting target-bound complex will produce a bispecific multimeric binding complex (if the multimeric complex comprises two different bicyclic peptides), a trispecific multimeric binding complex (if the multimeric complex comprises three different bicyclic peptides), a tetraspecific multimeric binding complex (if the multimeric complex comprises four different bicyclic peptides), and so on.

[0751] It should be understood that the multipolymer binding complex of the present invention can be designed to bind to a series of different targets on TfR1.

[0752] The bicyclic peptides within the multimeric binding complex of the present invention can be assembled using a variety of different schemes. For example, there can be a central hinge or branch portion, from which spacer or arm elements extend, each element containing a bicyclic peptide. Alternatively, it is conceivable that a ring-shaped support element can support multiple inwardly or outwardly projecting bicyclic peptides.

[0753] In one embodiment, each bicyclic peptide ligand is connected to the central hinge portion via a spacer group.

[0754] It should be understood that the spacer group can be linear and connects a single bicyclic peptide to the central hinge portion. Therefore, in one embodiment, the multimer-binding complex comprises a compound of formula (I):

[0755]

[0756] (I)

[0757] CHM represents the central hinge section;

[0758] Bicyclic represents a bicyclic peptide ligand as defined herein; and

[0759] m represents an integer selected from 2 to 10.

[0760] In one implementation, m represents an integer selected from 2, 3, or 4.

[0761] In another implementation, m represents 2.

[0762] When m represents 2, it should be understood that the central hinge section will require 2 connection points. Therefore, in one implementation, m represents 2, and CHM is the base order of equation (A):

[0763]

[0764] (A)

[0765] This group, along with other groups that can be used to link bicyclic peptide ligands together in multimeric binding complexes, will be described in more detail herein.

[0766] Dimer

[0767] In one embodiment, the multimeric binding complex comprises two identical bicyclic peptides and includes the dimer binding complex described in Table A below:

[0768] Table A: Exemplary Dimeric Binding Complexes of the Present Invention

[0769]

[0770] In one embodiment, the bicyclic peptide ligand specifically targeting transferrin receptor 1 (TfR1) is selected from any one of B1 to B191 or B196 to B198.

[0771] In one embodiment, the multimeric binding complex further comprises one or more effector groups and / or functional groups, such as one or more cytotoxic agents, radiochelates, or chromophores. In one embodiment, the multimeric binding complex further comprises a fluorophore.

[0772] connector

[0773] In embodiments providing a multimeric binding complex comprising at least one bicyclic peptide ligand as described herein (e.g., at least two, three, or four bicyclic peptide ligands as described herein), the peptide ligands may be conjugated together by any suitable linker. Such linkers can also be used to attach bicyclic peptide ligands (or bicyclic peptide ligand complexes as described herein) to a payload, such as an effector group as described in more detail herein. Linkers as described herein can also be used to attach bicyclic peptide ligands to HLE portions as described in more detail herein.

[0774] In one embodiment, the linker is a linear linker or a branched linker. In some embodiments, the linker is a branched linker and includes three or four branches. In some embodiments, the linker is capable of binding three or four bicyclic peptide ligands. In some embodiments, the linker includes three branches and is capable of binding three bicyclic peptide ligands. In some embodiments, the linker includes four branches and is capable of binding four bicyclic peptide ligands. In some embodiments, the linker includes one or more repeating monomeric groups.

[0775] In some embodiments, the linker is a bidentate or polydentate group with a length of about 0.3 nm to about 300 nm. In some embodiments, the linker length is about 0.5 nm to about 200 nm, for example about 1 nm to about 100 nm, for example about 1.5 nm to about 50 nm, for example about 2 nm to about 20 nm, for example about 3 nm to about 10 nm. In some embodiments, the linker length is a continuous length. In some embodiments, the length is determined when the linker is in an aqueous solution under physiological conditions (e.g., phosphate-buffered saline, pH 7.4, 37°C), and in some embodiments, it can be determined using atomic force microscopy.

[0776] In some embodiments, the connector comprises an alkyl, alkenyl, or ynyl group, such as C 1-30 Alkyl (e.g., C10) 2-12 For example, C 2-6 Alkyl), C 2-30 alkenyl (e.g., C) 2-12 For example, C 2-6 alkenyl) or C 2-30 alkynyl groups (e.g., C) 2-12 For example, C 2-6 (Alkynyl) group. When the connector contains an alkyl, alkenyl, or alkynyl group, the group can be straight-chain or branched, and can be optionally substituted.

[0777] In some embodiments, the connector includes one or more connecting portions, such as one or more poly(alkylene glycol) groups, such as poly(ethylene glycol) or poly(propylene glycol). In some embodiments, the connector may include one or more groups, such as amine groups, amide groups; alkylene groups; urethane groups; ether groups; ester groups; disulfide bonds; hydrazone groups; sulfonamide groups; thioether groups; or cyclic groups, preferably 4-12 membered carbocyclic or heterocyclic groups, 5-12 membered heteroaryl or C6 groups. 6-12 Aryl; wherein the alkylene, alkenylene, ynylene, poly(alkylene glycol), amine, and cyclic groups are each optionally substituted independently.

[0778] In some embodiments, the linker comprises one or more amino acids or amino acid analogs. In some embodiments, the linker comprises about 1 to about 5 amino acids or amino acid analogs. In some embodiments, the side chains of two amino acids or amino acid analogs in the linker are linked together. In some embodiments, the linker comprises a portion of the following form:

[0779]

[0780] Each R 1 For H or C 1-4 Alkyl; each R 2 Selected from the side chain of amino acids (e.g., typical amino acids) or C 1-4 Alkyl, the C 1-4 Alkyl groups can be, for example, OH, SH, SC. 1-4 Alkyl, aryl (which can be substituted with OH), heteroaryl, C(O)OH, C(O)NH2, N + H3, NH (C=N) + H2)NH2 substitution, for example, R 2 It may contain arginine or high-arginine side chains; and LINK is a linker, wherein LINK is optionally C. 2-8 An alkylene group (e.g., an alkylene group) optionally capped or substituted by one or more groups, such as amine groups, amide groups; alkylene groups; urethane groups; ether groups; ester groups; disulfide bonds; hydrazone groups; sulfonamide groups; thioether groups; or cyclic groups as defined herein; for example, LINK may contain C 3-6 Alkylene groups, which are terminated by or interrupted by amide groups; for example, LINK can contain the form -C 1-4 Alkylene-NHC(O)-C 1-4The alkylene moiety (e.g., -C4 alkylene-NHC(O)-C1 alkylene-). In some embodiments, the moiety is attached to a polypeptide contained in a peptide ligand or bicyclic peptide ligand as defined herein, for example at the N-terminus or C-terminus of the polypeptide contained in a peptide ligand or bicyclic peptide ligand as defined herein.

[0781] In some embodiments, the linker includes one or more reactive groups for reacting with bicyclic peptide ligands as described herein. Exemplary reactive groups include azide groups (e.g., which, in the presence of suitable conditions, such as in the presence of an azide-alkynyl cycloaddition catalyst, can react with an alkynyl group on a bicyclic peptide ligand as described herein, for example, to form a 1,2,3-triazole group); carboxylic acids and their activated derivatives (e.g., NHS esters) (e.g., which, in the presence of suitable conditions, can react with an amine group on a bicyclic peptide ligand as described herein, for example, to form an amide bond), etc.

[0782] In some embodiments, when the connector contains a poly(alkylene glycol), the poly(alkylene glycol) is either poly(ethylene glycol)(PEG) or poly(propylene glycol)(PPG). In some embodiments, the connector contains one or more PEGs. n The PEG group, wherein n represents the number of consecutive ethylene glycol units in each PEG group. In some embodiments, n is an integer from about 2 to about 25, for example from about 3 to about 10. In some embodiments, the connector comprises one or more branches, such as three branches, and each branch contains PEG. n Group.

[0783] In some embodiments, when the linker comprises an amide group, the amide group has the formula -NHC(O)- or -C(O)NH-. In some embodiments, when the linker comprises an amine group, the amine group has the formula N(R)3, wherein each R may be the same or different. In some embodiments, each R comprises a bicyclic peptide ligand as described herein, which, for example, comprises one or more PEGs as described herein. n The linker group is attached to the amine nitrogen. In some embodiments, when the linker contains a cyclic group, the cyclic group is a C6 aryl group. In some embodiments, when the linker contains an alkylene group, the alkylene group is a C6 aryl group. 1-3 Alkylene.

[0784] In one implementation, the linker is a linear linker. Without being bound by theory, it is believed that the advantage of a linear linker is that it allows for the presence of a first peptide ligand at one end and a second peptide ligand at the other end, and / or, for example, an HLE portion as described herein.

[0785] In some implementations, the linear connector comprises groups of the following formula:

[0786] (Alk) m —PEG n —(Alk) m

[0787] Each Alk is independently C 1-4 Alkylene; each m is independently 0 or 1; n is an integer from about 2 to about 25. In some embodiments, the linear linker may contain an amide group (e.g., which can be interrupted). In some embodiments, the linear linker may contain a group of the following formula.

[0788] PEG n -(Alk) m -amide-(Alk) m -PEG n

[0789] Each Alk is independently C 1-4 Alkylene; each m is independently 0 or 1; each n is independently an integer from about 2 to about 25; amide is an amide group (-NHC(O)- or -C(O)NH-). In some embodiments, the linear linker may also comprise an amino acid or an artificial amino acid, such as gGlu. In some embodiments, the linker may comprise groups for reacting with a first bicyclic peptide ligand as described herein, and groups for reacting with a second peptide ligand and / or, for example, the HLE moiety as described herein.

[0790] In some implementations, the connector comprises groups of the following formula

[0791] (Alk) m -amide-(Alk) m

[0792] Each Alk is independently C 1-4 Alkylene; each m is independently 0 or 1; and the amide is an amide group (-NHC(O)- or -C(O)NH-). In some embodiments, the linker also includes a capping group, such as an azide (N3) group (which can be used to react with an alkyne group, for example, on a bicyclic peptide ligand or HLE moiety (e.g., contained in a PYA group (e.g., K(PYA)))) or an alkyne group (which can be used to react with an azide group, for example, on a bicyclic peptide ligand or HLE moiety (e.g., contained in a K(N3) group)).

[0793] In some implementations, the linear connector is a group of the following formula.

[0794] N3—(Alk) m —PEG n —(Alk) m —Q

[0795] Each Alk is independently C 1-3 Alkylene; each m is independently 0 or 1; n is an integer from about 2 to about 25; and Q is a carboxylic acid (C(O)OH) or its activated derivative (e.g., an NHS-ester group).

[0796] Those skilled in the art will understand that, when conjugated to bicyclic peptide ligands as described herein, the azide group typically reacts with an alkyne group on one bicyclic peptide ligand (e.g., with an alkyne group contained in the K(PYA) moiety as described herein), while a carboxylic acid or NHS-ester typically reacts with an amine group on another bicyclic peptide ligand (e.g., with the N-terminal amino group of the polypeptide as described herein). Similarly, when conjugated to a payload (e.g., an effector group), the azide or carboxylic acid group can react with a complementary group (e.g., an alkyne or amide group on the payload).

[0797] In one implementation, the linker is a branched linker. Without being bound by theory, it is believed that the advantage of a branched linker is that it allows for the presence of a first peptide ligand at one end and two or more second peptide ligands and / or HLE moieties at the other end.

[0798] In some implementations, the branched connector comprises groups of the following formula

[0799] [(Alk) m —(PEG n ) v —(Alk) m ] x -W-[(Alk) m —(PEG n ) v —(Alk) m ] y

[0800] Each Alk is independently C 1-4 Alkylene, which is independently and optionally terminated with an amide group, an -O- group or a C(O) group; each m is independently 0, 1 or 2; n is an integer from about 2 to about 25; each v is independently 0 or 1; W is an N or benzene ring; and x and y are each independently 0 or 1 to about 4 integers; where x + y equals the number of branches in the branched linker; for example, a linker containing three branches may have x = 1 and y = 2 or x = 2 and y = 1.

[0801] In some embodiments, the alkylene group in the branched linker may be interrupted or capped by an amide group (-NHC(O)- or -C(O)NH-). In some embodiments, the linker may contain one or more groups for reaction with one or more bicyclic peptide ligands (e.g., as described herein) and / or one or more HLE moieties (e.g., as described herein). In some embodiments, an azide (N3) group may be used to react with, for example, an alkyne group on a bicyclic peptide ligand or HLE moieties (e.g., an alkyne group contained in a PYA group (e.g., K(PYA))); and / or an alkyne group may be used to react with, for example, an azide group on a bicyclic peptide ligand or HLE moieties (e.g., an azide group contained in a K(N3) group). In some embodiments, the branched linker is a group of the following formula.

[0802] [N3—(Alk) m —PEG n —(Alk) m -] x -W-[-(Alk) m —PEG n —(Alk) m —Q] y

[0803] Each Alk is independently C 1-3 Alkylene, optionally and independently terminated with an amide group, an -O- group, or a C(O) group; each m is independently 0, 1, or 2; n is an integer from about 2 to about 25; Q is a carboxylic acid or an activated derivative thereof (e.g., an N-ester group); W is an N or benzene ring; and x and y are each independently an integer from 0 or 1 to about 4; wherein x + y equals the number of branches in the branched linker; for example, a linker containing three branches may have x = 1 and y = 2 or x = 2 and y = 1. For the avoidance of ambiguity, when the linker contains multiple [N3—(Alk)]... m —PEG n —(Alk) m -] and / or [-(Alk)] m —PEG n —(Alk) m When the [Q] group is used, the groups can be the same or different. In some embodiments, the branched connector is...

[0804]

[0805] serial number

[0806] When referring to the positions of amino acid residues within the peptides of the present invention, cysteine ​​residues are omitted from the numbering because they are constant. Therefore, the numbering of amino acid residues within the peptides of the present invention is as follows:

[0807] -C-A1-L2-C-N3-D4-W5-T6-L7-P8-W9-H 10 -H 11 -C- (SEQ ID NO: 1).

[0808] Molecular format

[0809] The N-terminus or C-terminus extension of the double-loop core sequence is added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal biotin-G-Sar5 tail would be represented as:

[0810] [Biot]-G-[Sar5]-A-(SEQ ID NO: X).

[0811] [Biot] represents biotin, and Sar represents creatine.

[0812] Reverse peptide sequence

[0813] Given the publications of Nair et al. (2003) J. Immunol. 170(3), 1362-1373, it is expected that the peptide sequences disclosed herein will also function in their reverse form. For example, the sequence is reversed (i.e., the N-terminus becomes the C-terminus, and vice versa), and its stereochemistry is also reversed (i.e., D-amino acids become L-amino acids, and vice versa).

[0814] peptide ligand definition

[0815] As mentioned herein, peptide ligands refer to peptides, peptide compounds, or peptide mimics that are covalently bound to a molecular scaffold. Typically, such peptides, peptide compounds, or peptide mimics comprise a peptide having a natural or non-natural amino acid, two or more reactive groups (i.e., cysteine, homocysteine ​​(hCys, (S)-2-amino-4-thiobutyric acid), βCys ((R)-3-amino-3-mercaptopropionic acid), or penicillamine (Pen, (R)-2-amino-3-mercapto-3-methylbutyric acid), Dap ((S)-2,3-diaminopropionic acid), or N-alkyl-Dap (e.g., N-methyl-Dap, (S)-2-amino-3-(methylamino)propionic acid)) that are capable of forming covalent bonds with the scaffold, and a sequence between the reactive groups that is referred to as a ring sequence because it forms a ring when the peptide, peptide compound, or peptide mimic is bound to the scaffold. In this invention, peptides, peptide compounds, or peptide mimics typically contain at least three cysteine ​​residues (referred to herein as Ci, Cii, and Ciiii) and form at least two loops on the scaffold.

[0816] Therefore, in some embodiments, this disclosure provides peptides, peptide ligands, or peptide ligand complexes as provided herein, wherein one or more cysteine ​​residues of one or more polypeptides comprising said peptide, peptide ligand, or peptide ligand complex are replaced with homocysteine ​​(hCys), βCys, penicillamine (Pen), Dap, or N-methyl-Dap. In some embodiments, one or more (e.g., 1, 2, or 3) cysteine ​​residues of any polypeptide described herein are replaced with homocysteine ​​(hCys), βCys, penicillamine (Pen), Dap, or N-methyl-Dap.

[0817] peptide specificity

[0818] As described above, in some embodiments, the provided peptide and ligands containing such peptides (described in more detail herein) are specifically targeted at TfR1.

[0819] As used herein, the term "specificity" ("specific binding," etc.) in its broadest sense refers to a peptide or peptide ligand that binds to its biological target. In some embodiments, the peptide or peptide ligand binds to its biological target in a specific manner; that is, the binding to the biological target is not nonspecific. In some embodiments, peptides exhibiting nonspecific binding are heterogeneous; that is, the peptide is capable of binding to a variety of different biological species, typically including both the target target and off-target binding sites, such as binding sites on cell types other than the target cell type. Therefore, in some embodiments, peptides or peptide ligands selected or designed to specifically bind to their intended targets do not exhibit heterogeneous binding to off-target binding sites.

[0820] In some embodiments, binding to a target is binding to a specific epitope on the target. Peptides or peptide ligands can be engineered to specifically target a particular epitope, or can be identified by suitable screening methods (e.g., display techniques, such as phage display) that can be used to develop high-affinity binders to a given target (e.g., an epitope). Alternatively, peptides or peptide ligands that specifically bind to a biological target (e.g., a cellular target) can be identified without knowing the specific epitope they bind to. In some embodiments, peptides or peptide ligands that specifically bind to a target or epitope have a high affinity for that target or epitope. In some embodiments, the binding affinity of a peptide or peptide ligand to its epitope can be expressed as its dissociation constant (KD, also written as Kd). Typically, the KD of a peptide or peptide ligand that specifically binds to a biological target will be less than 10 µM, for example, less than 1 µM. Typically, peptides or peptide ligands that specifically bind to biological targets have a nanomolar affinity (KD) for that target, such as less than 100 nM, less than 20 nM, less than 10 nM, less than 5 nM, or even less than 1 nM. Binding affinity can be determined by methods known in the art, such as SPR and competitive assays. Some suitable assays are described in the examples.

[0821] In some embodiments, the peptide or peptide ligand that specifically binds to a biological target will have a higher (lower KD) affinity for that specific binding site than for other binding sites. For example, a peptide or peptide ligand that specifically binds to TfR1 typically binds to TfR1 with a higher affinity than to other binding sites. In some embodiments, the binding affinity of the peptide or peptide ligand that specifically binds to the biological target to the desired binding site is at least twice, for example, at least five times, for example, at least ten times, for example, at least twenty times, for example, at least fifty times, for example, at least one hundred times, for example, at least one thousand times or more than that of the peptide to any other off-target binding site.

[0822] Advantages of peptide ligands

[0823] Certain bicyclic peptides of the present invention possess numerous advantageous properties, making them suitable as pharmaceutical molecules for injection, inhalation, nasal administration, ophthalmic application, oral administration, or topical application. These advantageous properties include:

[0824] - Species cross-reactivity. This is a typical requirement for preclinical pharmacodynamic and pharmacokinetic assessments;

[0825] - Protease stability. Bicyclic peptide ligands should, in most cases, exhibit stability against plasma proteases, epithelial (“membrane-anchored”) proteases, gastrointestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across different species to allow for the development of bicyclic peptide lead candidates in animal models and their safe administration to humans.

[0826] - Ideal solubility characteristics. This is a function of the ratio of charged and hydrophilic residues to hydrophobic residues and intramolecular / intermolecular hydrogen bonds, which are important for formulation and absorption purposes; and

[0827] - It has an optimal plasma half-life in circulation. Depending on the clinical indication and treatment regimen, it may be necessary to develop bicyclic peptides with short or long in vivo exposure times to manage chronic or acute disease states. The optimal exposure time will depend on the need for sustained exposure (to obtain maximum therapeutic efficacy) versus the need for short exposure times (to minimize the toxicological effects of sustained exposure to the agent).

[0828] Pharmaceutically acceptable salts

[0829] It should be understood that, within the scope of this invention, references to peptide ligands include the salt form of said ligands.

[0830] The salts of the present invention can be synthesized by conventional chemical methods from a parent compound containing a basic or acidic moiety, such as... Pharmaceutical Salts: Properties, Selection, and Use The method described in (P. Heinrich Stahl (ed.), Camille G. Wermuth (ed.), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002). Typically, such salts can be prepared by reacting the free acidic or basic form of these compounds with a suitable base or acid in water or an organic solvent, or in a mixture of both.

[0831] Acid addition salts (monosal or diosal) can be formed from a variety of acids (inorganic and organic). Examples of acid addition salts include monosal or diosal salts formed with acids selected from: acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetaminobenzoic acid, butyric acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, decanoic acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclohexane, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactopyric acid, gentian acid, glucoheponic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-ketoglutarate, ethanol. Acids, hippuric acid, hydrohalic acids (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), hydroxyethanesulfonic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanate, p-toluenesulfonic acid, undecenoic acid and valeric acid, as well as acylated amino acids and cation exchange resins.

[0832] A specific group of salts consists of salts formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, ethanesulfonic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (methanesulfonates), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid, and lacturonic acid. One specific salt is hydrochloride. Another specific salt is acetate.

[0833] If the compound is anionic, or has functional groups that may be anionic (e.g., -COOH may be -COO), - Then, it can form salts with organic or inorganic bases to generate suitable cations. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions, such as Li₂. + Na + and K + Alkaline earth metal cations, such as Ca²⁺ + and Mg² + ; and other cations, such as Al³ + or Zn² + Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH4+). + ) and substituted ammonium ions (e.g., NH3R) +NH2R2 + NHR3 + NR4 + Examples of suitable substituted ammonium ions are derived from those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. A common example of a quaternary ammonium ion is N(CH3)4. + .

[0834] In one implementation, the pharmaceutically acceptable salt is selected from sodium, potassium, calcium, or ammonium salts.

[0835] When the peptides of the present invention contain amine functional groups, they can, for example, form quaternary ammonium salts by reacting with an alkylating agent according to methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the peptides of the present invention.

[0836] The peptides (including peptide ligands comprising said peptides) of the present invention can exist in zwitterionic form. Such compounds can also be provided in the form of pharmaceutically acceptable salts. Suitable salts include those formed with pharmaceutically acceptable acids that donate protons to negatively charged groups (e.g., COO- groups) and counterions to positively charged groups (e.g., quaternary nitrogen atoms) to balance the positive charge. Suitable pharmaceutically acceptable acids include hydrochloric acid, sulfonic acids (including methanesulfonic acid and toluenesulfonic acid), ascorbic acid, and citric acid. Hydrochloric acid and sulfonic acids are preferred, especially hydrochloric acid. Alternatively, the zwitterion can be combined with a pharmaceutically acceptable base, such as hydroxides of alkali metals (e.g., sodium or potassium) and alkaline earth metals (e.g., calcium or magnesium).

[0837] Modified derivatives

[0838] It should be understood that modified derivatives of peptide ligands as defined herein are within the scope of this invention.

[0839] In some implementations, the modified derivatives include functional fragments, derivatives, and variants of the sequences provided herein.

[0840] As those skilled in the art will understand, fragments of amino acid sequences include deletion variants of such sequences, wherein one or more, for example, at least 1, 2, 3, 4, or 5 amino acids are deleted. Deletions may occur at the C-terminus or N-terminus of the reference sequence, or within the reference sequence.

[0841] Derivatives of amino acid sequences include modified sequences, including sequences modified in vivo or in vitro. Many different protein modifications are known to those skilled in the art, including modifications that introduce novel functional groups into amino acid residues, modifications that protect reactive amino acid residues, or modifications that couple amino acid residues to chemical moieties (e.g., reactive functional groups for attachment to linkers of such amino acid residues). Exemplary modifications that can be made to the provided peptides and ligands are described in more detail herein.

[0842] Derivatives of amino acid sequences include addition variants of such sequences in which one or more, for example, at least 1, 2, 3, 4, or 5 amino acids are added to or introduced into a reference sequence. Addition can occur at the C-terminus or N-terminus of the reference sequence, or within the reference sequence. Variations of amino acid sequences include sequences in which one or more amino acid residues (e.g., at least 1, 2, 3, 4, or 5 amino acids) in the reference sequence are exchanged by one or more substitution residues. Variations of amino acid sequences include sequences carrying naturally occurring amino acids and / or non-natural amino acids.

[0843] Variants, derivatives, and fragments of the above-described amino acid sequence generally retain at least some of the activity / functionality of the reference sequence. In a preferred embodiment, the variants, derivatives, and fragments substantially retain their biological functions as described herein. Thus, in one embodiment, the variants, derivatives, and fragments retain the binding specificity of the reference sequence, i.e., the ability to specifically bind NPR3. In one such embodiment, the variants, derivatives, and fragments bind the same epitopes as the reference sequence. In another embodiment, the variants, derivatives, and fragments retain the binding affinity of the reference sequence. Generally, variants, derivatives, and fragments of the reference sequence have increased / improved activity / functionality compared to the reference sequence.

[0844] In some implementations, variants, derivatives, or fragments of the amino acid sequence are represented by their percentage identity with a reference sequence. Determining percentage identity is a routine procedure within the capabilities of those skilled in the art. Suitable methods include CLUSTAL W (Thompson et al., Nucleic Acids Research, 22(22) 4673-4680(1994)) and iterative optimization (Gotoh, J. Mol. Biol. 264(4) 823-838(1996)); and the methods described in Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. In one exemplary method, two amino acid sequences are compared to optimize the alignment score using a vacancy opening penalty of 10, a vacancy extension penalty of 1, and a "blosum 62" scoring matrix of Henikoff and Henikoff (as above). The percentage identity is then calculated using the following formula: [100 × (T / L)]; where T = the total number of perfect matches and L = the length of the longer sequence plus the number of vacancy introduced into the longer sequence to align the two sequences.

[0845] In some implementations, the variants, derivatives, or fragments of the reference sequence have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher identity with the reference sequence.

[0846] Examples of such suitable modified derivatives include one or more modifications selected from the following: N-terminal and / or C-terminal modification; replacement of one or more amino acid residues with one or more non-natural amino acid residues, or vice versa; replacement of one or more amino acids (e.g., one or more natural amino acids) with one or more isosteric and / or isoelectronic amino acids; replacement of one or more natural amino acids with one or more isosteric and / or isoelectronic non-natural amino acids, or vice versa (e.g., replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of spacer groups; replacement of one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues; The following modifications are made: replacing one or more amino acid residues with one or more alternative amino acids, such as alanine; replacing one or more L-amino acid residues with one or more D-amino acid residues; N-alkylating one or more amide bonds in a bicyclic peptide ligand; replacing one or more peptide bonds with alternative bonds; modifying the peptide backbone length; replacing the hydrogen on the α-carbon of one or more amino acid residues with other chemical groups; modifying amino acids (e.g., cysteine, lysine, glutamic acid / aspartic acid, and tyrosine) with suitable amine, thiol, carboxylic acid, and phenolic reactive agents to functionalize the amino acids; and introducing or replacing amino acids with orthogonal reactive amino acids suitable for functionalization, such as introducing amino acids with azido or alkynyl groups, which respectively allow functionalization with the alkynyl or azido groups.

[0847] Amino acid residues can typically be replaced with other amino acid residues having similar chemical structures, similar chemical properties, or similar side chain volumes (“conservative substitution”). The introduced amino acids can have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acids they replace. Alternatively, a conservative substitution can introduce another aromatic or aliphatic amino acid at a previously present position. Conservative amino acid variations are well known in the art and can be selected based on the properties of the 20 major amino acids defined in Table A above. In cases where amino acids have similar polarity, this can also be determined by referring to the hydropathological scale of the amino acid side chain, as is well known to those skilled in the art.

[0848] In one embodiment, the modified derivative comprises N-terminal and / or C-terminal modifications. In a further embodiment, the modified derivative comprises N-terminal modification using suitable amino-reactive chemistry and / or C-terminal modification using suitable carboxyl-reactive chemistry. In a further embodiment, the N-terminal or C-terminal modification comprises the addition of an effector group, including but not limited to cytotoxic agents, radiochelates, or chromophores.

[0849] In another embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, during peptide synthesis, the N-terminal residue is capped with acetic anhydride or other suitable reagent to obtain an N-terminal acetylated molecule. This embodiment offers the advantage of removing potential recognition sites for aminopeptidases and avoids the possibility of bicyclic peptide degradation.

[0850] In an alternative embodiment, N-terminal modification includes the addition of a spacer group that facilitates the conjugation of effector groups and maintains the efficacy of the bicyclic peptide on its target.

[0851] In another embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal residue is synthesized as an amide during peptide synthesis, thereby yielding a C-terminal amidated molecule. This embodiment offers the advantage of removing a potential recognition site for carboxypeptidase and reduces the likelihood of the bicyclic peptide being degraded by proteolytic hydrolysis.

[0852] In one embodiment, the modified derivative comprises replacing one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids having isosteric / isoelectron side chains can be selected, which are neither recognized by degrading proteases nor have any adverse effect on target efficacy.

[0853] Alternatively, non-natural amino acids with restricted amino acid side chains can be used, thereby hindering the conformational and spatial hydrolysis of adjacent peptide bonds. Specifically, these involve proline analogs, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and cyclic amino acids, one simple derivative being aminocyclopropionic acid.

[0854] In one embodiment, the modified derivative includes the addition of a spacer group. In a further embodiment, the modified derivative comprises N-terminal cysteine ​​(C... i ) and / or C-terminal cysteine ​​(C iii Add spacer groups.

[0855] In one embodiment, the modified derivative includes replacing one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues. In a further embodiment, the modified derivative includes replacing tryptophan residues with naphthylalanine or alanine residues. This embodiment provides the advantage of improving the pharmaceutical stability characteristics of the resulting bicyclic peptide ligand.

[0856] In one embodiment, the modified derivative comprises replacing one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises replacing one or more hydrophobic amino acid residues with one or more charged amino acid residues. The proper balance between charged and hydrophobic amino acid residues is an important characteristic of bicyclic peptide ligands. For example, hydrophobic amino acid residues affect the degree of plasma protein binding and thus the concentration of the free effective fraction in plasma, while charged amino acid residues (especially arginine) may affect the interaction of the peptide with phospholipid membranes on cell surfaces. This combination can affect the half-life, volume of distribution, and exposure of the peptide drug and can be adjusted according to clinical endpoints. Furthermore, the correct combination and amount of charged and hydrophobic amino acid residues can reduce irritation at the injection site (if the peptide drug is administered subcutaneously).

[0857] In one embodiment, the modified derivative comprises replacing one or more L-amino acid residues with one or more D-amino acid residues. It is believed that this embodiment increases protein hydrolytic stability through steric hindrance and the tendency of D-amino acids to stabilize β-turn conformation (Tugyi et al. (2005) PNAS, 102(2), 413-418).

[0858] In one embodiment, the modified derivative comprises the removal of any amino acid residues and their replacement with alanine (e.g., D-alanine). This embodiment offers the advantage of identifying key binding residues and removing potential proteolytic attack sites.

[0859] It should be noted that each of the above modifications is specifically designed to improve the potency or stability of the peptide. Further potency improvements based on modifications can be achieved through the following mechanisms:

[0860] - The incorporation of hydrophobic components that utilize hydrophobic effects and result in a lower dissociation rate enables higher affinity;

[0861] - The incorporation of charged groups that utilize long-range ion interactions leads to faster association rates and higher affinities (see, for example, Schreiber et al.). Rapid, electrostatically assisted association of proteins(1996), Nature Struct. Biol. 3, 427-31); and

[0862] - Incorporate additional constraints into the peptide, such as by properly constraining the side chains of amino acids to minimize entropy loss during target binding; constraining the torsion angle of the main chain to minimize entropy loss during target binding; and introducing additional cyclization into the molecule for the same reasons.

[0863] (For reviews, see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[0864] Isotope changes

[0865] This invention includes all pharmaceutically acceptable (radioactive) isotope-labeled peptide ligands of this invention, wherein one or more atoms are replaced by atoms having the same atomic number but with a different atomic mass or mass number than those commonly found in nature; and peptide ligands of this invention wherein are attached with a metal chelating group (referred to as an "effecton") capable of accommodating the relevant (radioactive) isotope; and peptide ligands of this invention wherein certain functional groups are covalently replaced with the relevant (radioactive) isotope or isotope-labeled functional groups.

[0866] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention include isotopes of hydrogen, such as... 2 H(D) and 3 H(T); isotopes of carbon, for example 11 C 13 C and 14 C; isotopes of chlorine, for example 36 Cl; isotopes of fluorine, for example 18 F; Isotopes of iodine, such as 123 I, 125 I and 131 I; Isotopes of nitrogen, such as 13 N and 15 N; isotopes of oxygen, such as 15 O、 17 O and 18 O; isotopes of phosphorus, such as 32 P; isotopes of sulfur, such as S; isotopes of copper, such as 64 Cu; isotopes of gallium, such as 67 Ga or 68 Ga; isotopes of yttrium, for example 90Y; isotopes of lutetium, for example 177 Lu; and bismuth isotopes, such as 213 Bi.

[0867] Certain isotope-labeled peptide ligands of the present invention, such as those incorporating radioactive isotopes, can be used for drug and / or substrate tissue distribution studies, and for clinical assessment of the presence or absence of targets on diseased tissues. The peptide ligands of the present invention can also possess valuable diagnostic properties because they can be used to detect or identify the formation of complexes between labeled compounds and other molecules, peptides, proteins, enzymes, or receptors. Detection or identification methods can use compounds labeled with labeling agents (e.g., radioactive isotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, amylopectin, and luciferase)). Radioactive isotope tritium (i.e....) 3 H(T)) and carbon-14 (i.e. 14 C) It is particularly suitable for this purpose due to its ease of incorporation and convenient detection methods.

[0868] Use heavier isotopes (e.g., deuterium, i.e.) 2 Replacing H(D) may provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dose requirement, and may therefore be preferred in some cases.

[0869] Using isotopes that emit positrons (e.g.) 11 C 18 F, 15 O and 13 The replacement of N) can be used in positron emission tomography (PET) studies to examine target occupancy.

[0870] The isotope-labeled compounds of the peptide ligands of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the accompanying examples, using a suitable isotope-labeling reagent to replace the previously used unlabeled reagent.

[0871] Molecular scaffold

[0872] In one embodiment, the molecular scaffold comprises a non-aromatic molecular scaffold. The term "non-aromatic molecular scaffold" as used herein refers to any molecular scaffold that does not contain an aromatic (i.e., unsaturated) carbocyclic or heterocyclic ring system as defined herein.

[0873] Suitable examples of non-aromatic molecular scaffolds are described in Heinis et al. (2014) Angewandte Chemie, International Edition 53(6) 1602-1606.

[0874] As mentioned in the aforementioned literature, molecular scaffolds can be small molecules, such as small organic molecules.

[0875] In one embodiment, the molecular scaffold can be a macromolecule. In one embodiment, the molecular scaffold is a macromolecule composed of amino acids, nucleotides, or carbohydrates.

[0876] In one embodiment, the molecular scaffold includes reactive groups capable of reacting with functional groups of a polypeptide to form covalent bonds.

[0877] Molecular scaffolds may contain chemical groups that are linked to peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, acid anhydrides, succinimides, maleimides, alkyl halides, and acyl halides.

[0878] In one embodiment, the molecular scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tripropyl-2-en-1-one (also known as triacryloylhexahydro-s-triazine (TATA)).

[0879]

[0880] TATA.

[0881] Therefore, the bicyclic peptide of the present invention is reacted with the peptide's reactive groups (e.g., on RG1, RG2, and RG3; e.g., on C). i C ii and C iii Cyclication on a cysteine ​​residue leads to the formation of a trisubstituted 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tripropyl-1-one derivative of TATA with the following structure:

[0882]

[0883] (also described as)

[0884] ,

[0885] in This indicates the junction of three cysteine ​​residues.

[0886] In an alternative embodiment, the molecular scaffold is 1,1',1''-(1,3,5-triazine-1,3,5-triyl)tris(2-bromoethylone) (TATB).

[0887] Therefore, in relation to the bicyclic peptide of the present invention at C i C ii and C iii Following cyclization of cysteine ​​residues, the molecular scaffold forms a trisubstituted derivative of TATB with the following structure:

[0888]

[0889] TATB.

[0890] synthesis

[0891] The peptides of this invention can be synthesized using standard techniques and then reacted in vitro with a molecular scaffold. Standard chemical methods can be used when performing this operation. This enables the rapid, large-scale preparation of soluble materials for further downstream experiments or validation. Such methods can be accomplished using conventional chemical methods, such as those disclosed in Timmerman et al. (see above).

[0892] Therefore, the present invention also relates to the preparation of peptides or conjugates selected as described herein, wherein the preparation includes optional additional steps as described below. In one embodiment, these steps are performed on a final product peptide / conjugate prepared by chemical synthesis.

[0893] Optionally, when preparing the conjugate or complex, amino acid residues in the target polypeptide can be substituted.

[0894] Peptides can also be extended to incorporate, for example, another ring, and thus introduce multiple specificities.

[0895] To extend the peptide, chemical extension can be performed at its N-terminus, C-terminus, or within the ring using orthogonally protected lysine (and its analogues) using standard solid-phase or liquid-phase chemistry. An activated or activatable N-terminus or C-terminus can be introduced using standard (biological) conjugation techniques. Alternatively, it can be added by fragment condensation or native chemical linking (e.g., as described in Dawson et al., 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779) or by enzymes (e.g., using subtiligases, as described in Chang et al., Proc Natl Acad Sci US A., Dec 20, 1994; 91(26):12544-8 or Hikari et al., Bioorganic & Medicinal Chemistry Letters, Vol. 18, No. 22, Nov 15, 2008, pp. 6000-6003).

[0896] Alternatively, the peptide can be further extended or modified by disulfide conjugation. This has the added advantage that the first and second peptides can dissociate from each other once in the reducing environment of the cell. In this case, a molecular scaffold (e.g., TATA or TATB) can be added during the chemical synthesis of the first peptide to react with three cysteine ​​groups; then another cysteine ​​or thiol can be attached to the N-terminus or C-terminus of the first peptide, such that the cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide, forming a disulfide-linked bicyclic peptide-peptide conjugate.

[0897] Furthermore, the addition of other functional groups or effector groups can be achieved in the same manner, using appropriate chemical methods, by coupling at the N-terminus or C-terminus or via side chains. In one embodiment, the coupling is carried out in a manner that does not inhibit the activity of either entity.

[0898] In some embodiments, the peptides provided herein can be synthesized via solid-phase synthesis. In some embodiments, the synthesis of peptide ligands provided herein may include solid-phase synthesis of the peptides as described herein. In some embodiments, solid-phase synthesis includes Fmoc solid-phase peptide synthesis (e.g., as described in more detail herein). In some embodiments, the synthesized peptide is cyclized with a molecular scaffold as described herein. In some embodiments, the cyclized scaffold is purified, for example, by lyophilization.

[0899] Extended half-life (HLE) portion

[0900] It should be understood that the HLE portion may contain any suitable agent capable of prolonging the half-life of the resulting complex. The term "prolonged half-life" includes any agent capable of increasing the duration of the resulting complex in the host body before it is metabolized, compared to the duration of the complex without the HLE portion. The half-life can be, for example, the plasma half-life after administration of the complex to a subject.

[0901] It will be apparent to those skilled in the art that the HLE portion is well known in the art. Any suitable half-life extender can be used. As will be understood by those skilled in the art, the HLE portion may also be referred to as an "exposure modifier" or "exposure-modifying portion" (EM portion).

[0902] In one embodiment, the HLE portion is the part capable of binding albumin (e.g., serum albumin, particularly human serum albumin (HSA)). HSA is the most abundant protein in plasma (approximately 40 g / L). It exhibits remarkable solubility and stability, and crucially, has a half-life of approximately 19 days in the human body. HSA has the ability to bind a variety of hydrophobic small molecules, such as up to seven long-chain fatty acids simultaneously, and has two known binding sites for medium-chain fatty acids.

[0903] Examples of albumin binders include:

[0904] (i) Short-chain fatty acids (i.e. fatty acids with 5 or fewer carbon atoms), such as butyric acid (C4);

[0905] (ii) Medium-chain fatty acids (i.e. fatty acids with 6 to 12 carbon atoms), such as hexanoic acid (C6), octanoic acid (C8), decanoic acid (C10), or lauric acid (C12);

[0906] (iii) Long-chain fatty acids (i.e. fatty acids with 13 to 21 carbon atoms), such as myristic (C14) acid, palmitic (C16) acid, stearic (C18) acid, and arachidic (C20) acid;

[0907] (iv) Very long-chain fatty acids (i.e. fatty acids with 22 or more carbon atoms), such as behenic acid (C22), ceramide (C24) and ceramide (C26);

[0908] (v) Genentech’s small molecule FVIIa peptide-albumin binding inhibitor (Bioorg Med Chem Lett, 2002, 12(20), 2883-6 and Bioorg Med Chem Lett, 2003, 13(9), 1513-5), which greatly increased plasma half-life in rabbits;

[0909] (vi) The Fmoc protecting group has been shown to significantly reduce the hydrolysis of compounds in the presence of HSA (Int J PepRes Ther, 2007, 13(1-2), 12);

[0910] (vii) Coumarin derivatives, such as warfarin-type coumarin derivatives, have been shown to improve in vivo half-life in rats;

[0911] (viii) Evans blue, which binds to the Sudlow site I of HSA with low molar affinity (2.5 µM) and has a long half-life in blood (Theranostics, 2016, 6(2), 243-53);

[0912] (ix) Diflunisal, which binds to sites I and II of HSA with an affinity of about 3 µM (J Med Chem 2017, 60(17), 7434-46);

[0913] (x) Indomethacin, which binds primarily to site I of HSA with an affinity of 0.7 µM (J Med Chem 2017, 60(17), 7434-46);

[0914] (xi) Lithocholic acid (bile acid), an example of an endogenous molecule, can control blood glucose levels for more than 24 hours when conjugated with insulin (Pharm Res 2006, 23(1), 49-55);

[0915] (xii)4-(p-iodophenyl)butyric acid (Albutag) and related phenylbutyric acid examples have been found to be stable in mouse and human serum albumin (Angew Chem Int Ed, 2008, 47(17), 3196-3201);

[0916] (xiii) PSMA derivatives, such as RPS-068 (Eur J Nucl Med Mol Imaging 2018, 45(11), 1841-51), RPS-072 (J Nucl Med 2019, 60(5), 656-63), PSMA-ALB-56 (MolPharmaceutics 2018, 15, 2297-306), NODAGA analogs (Cu) (Mol Pharmaceutics 2018, 15, 5556-64), or those described in EP 3 778 592;

[0917] (xiv) peptides, such as F-Tag (Nat Commun 2017, 8, 16092), 89D03 (WO 2011 / 095545), [Ac][K(N3)]GRLIEDICLPRWGCLWEDD (SEQ ID NO: 119; SA21; J Biol Chem2002, 277(38), 35035-43);

[0918] (xv) Liraglutide (Diabetes Care 2002; 25:1398-1404);

[0919] (xvi) Smegglutinin (Med Chem; 2015, 58, 7370-7380);

[0920] (xvii)Knob domain antibody fragment;

[0921] (xviii) Polyethylene glycolated compounds, such as those described in WO 2020 / 205948;

[0922] (xix) Compounds containing small molecule aromatic compounds, such as those described in WO 2008 / 053360; and

[0923] (xx)PPB03 (Bioconjugate Chem 2017, 28(9), 2372-2383).

[0924] In one specific implementation, HLE is selected from:

[0925] (i) Short-chain fatty acids (i.e. fatty acids with 5 or fewer carbon atoms), such as butyric acid (C4);

[0926] (ii) Medium-chain fatty acids (i.e. fatty acids with 6 to 12 carbon atoms), such as hexanoic acid (C6), octanoic acid (C8), decanoic acid (C10), or lauric acid (C12);

[0927] (iii) Long-chain fatty acids (i.e., fatty acids with 13 to 21 carbon atoms), such as myristic (C14) acid, palmitic (C16) acid, stearic (C18) acid, and arachidic (C20) acid; and

[0928] (iv) Very long-chain fatty acids (i.e. fatty acids with 22 or more carbon atoms), such as behenic acid (C22), ceramide (C24) and ceramide (C26).

[0929] In another specific implementation, HLE is selected from:

[0930] (ii) Medium-chain fatty acids (i.e. fatty acids with 6 to 12 carbon atoms), such as hexanoic acid (C6), octanoic acid (C8), decanoic acid (C10), or lauric acid (C12);

[0931] (iii) Long-chain fatty acids (i.e. fatty acids with 13 to 21 carbon atoms), such as myristic (C14) acid, palmitic (C16) acid, stearic (C18) acid, and arachidic (C20) acid.

[0932] In yet another specific implementation scheme, HLE is selected from:

[0933] (iii) Long-chain fatty acids (i.e. fatty acids with 13 to 21 carbon atoms), such as myristic (C14) acid, palmitic (C16) acid, stearic (C18) acid, and arachidic (C20) acid.

[0934] In one specific implementation, HLE is selected from peptides, cyclic peptides, bicyclic peptides, fatty acids, or lipids.

[0935] In one specific embodiment, the HLE moiety is a fatty acid, such as a long-chain fatty acid, particularly a C18 diacid, especially octadecanoic acid. In some embodiments, the HLE moiety is a saturated fatty acid. In some embodiments, the HLE moiety is an unsaturated fatty acid. In some embodiments, the HLE moiety is a straight-chain or branched-chain fatty acid. Data are presented herein as in Example 3. Figure 2 As shown in Table 2, the HLE-containing bicyclic peptide ligand complex (B192) containing octadecanoic acid exhibits an increased half-life of 7.8 hours (compared to a half-life of 0.28 hours for the HLE-deficient equivalent complex B191).

[0936] In some embodiments, the HLE moiety is a C4-C30 fatty acid. In some embodiments, the HLE moiety is a C4-C26 fatty acid. In some embodiments, the HLE moiety is a C6-C20 fatty acid. In some embodiments, the HLE moiety is a C4, C6, C8, C10, C12, C14, C16, C18, or C20 fatty acid. In some embodiments, the HLE moiety is a peptide. In some embodiments, the HLE moiety is a protein. In some embodiments, the HLE moiety is an antibody or a fragment thereof. In some embodiments, the HLE moiety is an albumin-binding antibody or a fragment thereof. In some embodiments, the HLE moiety is an albumin-binding ligand. In some embodiments, the HLE moiety is an organic molecule. In some embodiments, the HLE moiety is a dye. In some embodiments, the HLE moiety is a polymer. In some embodiments, the HLE moiety is or contains PEG.

[0937] In some embodiments, the HLE moiety comprises one, two, three, or more fatty acid groups. For example, in some embodiments, the HLE moiety comprises a dilipid group. In some embodiments, the dilipid comprises two fatty acid groups conjugated to a bicyclic peptide ligand. In some embodiments, the dilipid group has the following form

[0938]

[0939] Wherein the wavy line indicates the connection point with the compound, n is an integer from about 1 to about 10, and each m is independently an integer from about 6 to about 20, for example, an integer from about 10 to about 15. Examples of suitable fatty acid groups include C16 fatty acids. In some embodiments, the dilipid group has the following form, where the wavy line indicates the connection point with the compound, and n is an integer from about 1 to about 10.

[0940]

[0941] In some embodiments, the HLE moiety comprises myristic acid, hexadecenoic acid, octadecanoic acid, octadecanoic acid, palmitic acid, palmitic acid, 16-tetraazole-hexadecanoic acid, 16-sulfohexadecanoic acid, docosanoic acid, 1,2-distearate-sn-glycerol-3-phosphoethanolamine, lauric acid, dimerlauric acid, tetramerlauric acid, or α,ω-octadecanoic acid. In some embodiments, the HLE moiety comprises a lipid conjugate or a substituent as described in any of WO2016161374, WO2019217459, WO2020257194, WO2021092371, WO2023064530, or WO2024129748.

[0942] In some embodiments, the HLE portion comprises a fatty acid and PEG. In some embodiments, the HLE portion comprises a fatty acid conjugate with PEG. In some embodiments, the HLE portion comprises a C4-C26 fatty acid, such as a C4, C6, C8, C10, C12, C14, C16, C18, or C20 fatty acid conjugate with PEG. The PEG portion comprises about 2 to about 50, for example, about 3 to about 20, such as about 4 to about 10 PEG monomer units. In some embodiments, the PEG portion is interrupted by a linking portion such as an amide group. In some embodiments, the PEG portion comprises a group of the formula (PEG)nL-(PEG)m, wherein n and m are each independently an integer from about 1 to about 10, for example, an integer from about 2 to about 5, such as 2, 3, 4, or 5; and wherein L is a linking group. In some embodiments, L is a linking portion comprising an amide group, a carbamate group, or a carbonyl group. In some embodiments, the PEG portion comprises (PEG)2-CH2-C(O)NH-(PEG)2.

[0943] In some implementations, the HLE portion includes a part of the following formula:

[0944] [Fatty acid]-[Connector 1]-[PEG]-[Terminal];

[0945] in

[0946] [Fatty acids] are C4-C26 fatty acids;

[0947] [Connector 1] is a connector; for example, [Connector 1] may be composed of or contain amino acids or artificial amino acids (e.g., gGlu);

[0948] [PEG] is the PEG portion, which contains 2 to about 50 PEG units and is optionally interrupted by one or more linking portions; for example, [PEG] contains (PEG)2-CH2-C(O)NH-(PEG)2;

[0949] [Terminal] is an optional capping group that can be used to link the HLE moiety to a bicyclic peptide ligand as described herein, such as an amino acid or an artificial amino acid; for example, lysine.

[0950] For example, in some embodiments, the HLE portion comprises a C4, C6, C8, C10, C12, C14, C16, C18, C20, C22, C24, C26, C28, or C30 fatty acid (e.g., C12, C14, C16, C18, C20, C22, or C24 fatty acid, e.g., C16, C18, or C20 fatty acid, e.g., C18 fatty acid), which is linked to the PEG portion of the formula (PEG)nL-(PEG)m via an amino acid (e.g., gGlu), wherein n and m are each independently an integer from about 1 to about 5, e.g., 2, 3, or 4; e.g., n and m are each 2; and L is a group of the formula -CH2-C(O)NH-; and wherein the PEG portion is linked to the side chain of a lysine amino acid via an amide bond, the lysine amino acid being either linked to or part of the N-terminus or C-terminus of a polypeptide contained in a bicyclic peptide ligand as described herein. An example is the HLE moiety used in compound B192 described in Example 3:

[0951]

[0952] Wherein the [terminus] is an optional capping group that can be used to link the HLE moiety to a bicyclic peptide ligand as described herein, such as an amino acid or an artificial amino acid; for example, lysine. When the [terminus] is a lysine residue contained in or linked to a bicyclic peptide ligand, the HLE moiety can have the following forms:

[0953]

[0954] In one specific embodiment, the HLE moiety is an albumin-binding peptide, such as SA21. SA21 has the amino acid sequence [Ac]RLIEDICLPRWGCLWEDD[CONH2], wherein the cysteine ​​residue is cyclized via a disulfide bond; an N-terminal acetyl group and / or a C-terminal amide group may or may not be present. In some embodiments (where the HLE moiety is an albumin-binding peptide), the albumin-binding peptide is a derivative of SA21 containing a reactive group for conjugation with a bicyclic peptide ligand as described herein. Any suitable reactive group can be used. In some embodiments, the reactive group is present on a reactive amino acid that may be inserted into the sequence of SA21, for example at the N-terminus or C-terminus, typically at the N-terminus. A suitable reactive group is an azide group, which can be used, for example, for a click reaction with a corresponding alkyne group attached to the bicyclic moiety (e.g., an alkyne group contained in the [K(PYA)] (N-pentyneyl-L-lysine) group). In some embodiments, the azide group may be attached to an amino acid such as lysine (e.g., in the [K(N3)] group). The [K(N3)] group can be present at the N-terminus or C-terminus of the SA21 peptide, for example, at the N-terminus. In some embodiments, the amino acid containing the reactive group (e.g., the [K(N3)] group) is linked to the N-terminus of the SA-21 peptide via a spacer region (e.g., another amino acid, such as glycine or lysine). Therefore, in some embodiments, the HLE portion is a derivative of SA-21 (i.e., an albumin-binding derivative), such as ([Ac][K(N3)]GRLIEDICLPRWGCLWEDD (SEQ ID NO: 119). Data herein are as shown in Example 3, Figure 3 As shown in Table 3, the HLE-containing bicyclic peptide ligand complex (B193) containing SA21 exhibits an increased half-life of 22.6 hours (compared to a half-life of 0.28 hours for the equivalent complex B191 lacking HLE). In some embodiments, the HLE portion is a variant of SA-21 containing one or more modifications as described herein in the context of peptide variants contained in bicyclic peptide ligands; for example, one or more amino acid modifications may be made to the SA-21 sequence.

[0955] In some embodiments, the HLE moiety is ABDcon or a variant thereof. ABDcon has the amino acid sequence LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA. In some embodiments, the HLE moiety is ABDcon-azide. ABDcon-azide has the amino acid sequence [K(N3)]LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA. In some embodiments, the HLE moiety is ABDcon or a variant of ABDcon-azide containing one or more modifications as described herein in the context of polypeptide variants contained in bicyclic peptide ligands; for example, one or more amino acid modifications may be made to the ABDcon-azide sequence.

[0956] In some embodiments, the HLE portion is ABM00001 or a variant thereof. ABM00001 has the amino acid sequence GVSDFYKNLINRAKTVEGVHALIGHI. In some embodiments, the HLE portion is PP001 or a variant thereof. PP001 has the amino acid sequence LASAKEAANAELDAYGVSDFYKRLIDKAKTVEGVEALKDAILAALP. In some embodiments, the HLE portion is PEP or a variant thereof. PEP has the amino acid sequence DITGAALLEAKEAAINELKQYGISDYYVTLINKAKTVEGVNALKAEILSALP. In some embodiments, the HLE is PEP-X or a variant thereof. PEP-X has the amino acid sequence DITGAALLEAKDAAINELKQYGISDYYVHLINKADTVEGVNALKDEILSALP. In some embodiments, the variant may contain one or more modifications, such as one or more amino acid modifications, as described herein in the context of peptide variants contained in bicyclic peptide ligands.

[0957] In some embodiments, the HLE portion is an FN3 domain, such as those disclosed in WO 11,203,630 or WO 2017 / 210425 (each incorporated herein by reference). In some embodiments, the HLE portion is an albumin-binding peptide, such as those disclosed in WO 2009 / 016043, WO 2012 / 004384, WO 2013 / 177398, WO 2014 / 048977 or WO 2015 / 091957 (each incorporated herein by reference). In some embodiments, the HLE portion is an ankyrin repeat domain, such as those disclosed in WO 2016 / 156596 (which is incorporated herein by reference). In some embodiments, the HLE portion is the VHH domain of an albumin-binding antibody, as disclosed in WO 2017 / 201488 or WO2018 / 104444 (each incorporated herein by reference). In some embodiments, the HLE portion is the VHH domain of an albumin-binding antibody and has the amino acid sequence EVQLVESGGGLVQPGNSLRLSCAASGFTFSRFGMSWVRQAPGKGLEWV SSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIgGSLSRSSQGTLVTVSS or a variant thereof. In some embodiments, the HLE is ScFv, such as disclosed in WO2012 / 162068 (incorporated herein by reference). In some embodiments, the HLE portion is nanofibrin, such as disclosed in WO 2022 / 171852 (incorporated herein by reference). In some embodiments, the HLE is an IgG binding moiety. In some embodiments, the IgG binding moiety is a protein G analog (e.g., TTYKLVINGKTLKGETTTKAVDAETAAAAFAQYANDNGVDGVWTYDDATKTFTVTE) or a variant thereof; or FcIII (DC‡AWHLGELVWC‡T) or a variant thereof; or PAM (GK(K(YTR)YTR)K(YTR)YTR) or a variant thereof; or 15-IGP (Ac-DC‡AYHKGELVWC‡TFH-NH2) or a variant thereof. In some embodiments, the HLE moiety is an FcRn binding moiety. In some embodiments, the FcRn moiety is Mezo_1 (QRFC‡TGHFGGLYPC‡NGP) or a variant thereof; Mezo_283 (C2H4-[(C(O)NH-RF[Pen])) ‡ -TGHFT-[Sar]-[NMeL]-YPC ‡]2) or its variants; BM07918(EQDAAAHEIRWLPNLTFDQRVAFIHKLAD) or its variants; ZFcRn_2(VDAKYAKEQDAAAHEIRWLPNLTFDQRVAFIHKLADDPSQSSELLSEAKKLNDSQAPK) or its variants; FcBP(Y286H)(QRFC‡TGHFGGLHPC‡NG) or its variants; or SYN1327(Ac-RF-[Pen]‡-TGHFG-[Sar]-[NMeL]-YPC‡) or its variants; wherein ‡ This indicates that two amino acids are linked by a disulfide bond.

[0958] In some embodiments, the FcRn binding moiety comprises a peptide (e.g., a cyclic peptide), such as those disclosed in WO 2007 / 098420, WO2014 / 205072, or WO 2014 / 140366 (each incorporated herein by reference). In some embodiments, the variant may comprise one or more modifications, such as one or more amino acid modifications, as described herein in the context of peptide variants contained in bicyclic peptide ligands.

[0959] In some embodiments, the HLE moiety increases the half-life (e.g., plasma half-life) of the bicyclic peptide ligand by at least 2, 3, 4, 5, 10, 20, 50, 100, 200, or 500 times or more compared to the half-life of the bicyclic peptide ligand without the HLE moiety.

[0960] It should be understood that the HLE moiety can be directly bonded to the bicyclic peptide ligand, i.e., via a covalent bond, or a spacer or linker can exist between the bicyclic peptide ligand and the HLE moiety. The design and construction of such spacer or linker will be obvious to those skilled in the art.

[0961] Pharmaceutical Composition

[0962] According to another aspect of the invention, a pharmaceutical composition is provided comprising a bicyclic peptide ligand or a bicyclic peptide ligand complex as defined herein and one or more pharmaceutically acceptable excipients.

[0963] Typically, the peptide ligands and complexes of the present invention are used in purified form with pharmacologically suitable excipients or carriers. These excipients or carriers typically comprise aqueous or alcohol / aqueous solutions, emulsions, or suspensions, including saline and / or buffer media. Parenteral media include sodium chloride solution, Ringer's dextran, dextran and sodium chloride, and lactated Ringer's solution. If it is desired to keep the peptide complex in suspension, suitable physiologically acceptable adjuvants may be selected from thickeners such as carboxymethyl cellulose, polyvinylpyrrolidone, gelatin, and alginate.

[0964] Intravenous mediators include fluids and nutritional supplements, as well as electrolyte supplements, such as those based on Ringer's dextran. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases (Mack (1982) Remington's Pharmaceutical Sciences, 16th edition).

[0965] The peptide ligands and complexes of the present invention can be used as a composition for single administration or in combination with other pharmaceutical agents. These other pharmaceutical agents may include antibodies, antibody fragments, and various immunotherapeutic agents such as cyclosporine, methotrexate, doxorubicin, or cisplatin, as well as immunotoxins. Other examples of other pharmaceutical agents that can be administered alone or in combination with the peptide ligands of the present invention include cytokines, lymphokines, other hematopoietic factors, thrombolytic and antithrombotic factors. Pharmaceutical compositions may include a “mixture” of various cytotoxic or other pharmaceutical agents with the protein ligands of the present invention, or even combinations of selected polypeptides with different specificities according to the present invention, such as polypeptides using different target ligand selections, whether or not they are mixed prior to administration.

[0966] The administration route of the pharmaceutical composition according to the invention can be any route commonly known to those skilled in the art. For treatment, the peptide ligand of the invention can be administered to any patient according to standard techniques. Administration can be performed by any suitable modality, including parenteral, intravenous, intramuscular, intraperitoneal, percutaneous, pulmonary, or, suitably, direct infusion via catheter. Preferably, the pharmaceutical composition according to the invention is administered intravenously. The dosage and frequency of administration will depend on the patient's age, sex, and condition, concurrent administration of other medications, contraindications, and other parameters that the clinician must consider.

[0967] The peptide ligands and complexes of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has proven effective and can be employed using lyophilization and reconstitution techniques known in the art. Those skilled in the art will understand that lyophilization and reconstitution may result in varying degrees of activity loss, and may require upward adjustment to compensate for this.

[0968] Compositions containing the peptide ligands and complexes or mixtures thereof of the present invention can be administered for prophylactic and / or therapeutic treatment. In some therapeutic applications, an amount sufficient to achieve at least partial blockage, inhibition, modulation, killing, or some other measurable parameter of a selected cell population is defined as a “therapeutic effective dose.” The amount required to achieve this dose will depend on the severity of the disease, but typically ranges from 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, more commonly from 0.05 to 2.0 mg / kg / dose. For prophylactic applications, compositions containing the peptide ligands or mixtures thereof of the present invention may also be administered at similar or slightly lower doses.

[0969] Compositions containing peptide ligands or complexes according to the invention can be used in preventative and therapeutic settings to help alter, inactivate, kill, or remove selected target cell populations in mammals. Furthermore, the peptide ligands described herein can be used in vitro or selectively in vitro to kill, deplete, or otherwise effectively remove target cell populations from heterogeneous cell collections. Blood from mammals can be combined in vitro with selected peptide ligands according to standard techniques to kill unwanted cells or otherwise remove them from the blood for return to the mammal.

[0970] Payload

[0971] In one embodiment, the bicyclic peptide ligand complex also includes a payload in addition to an antisense oligonucleotide (ASO).

[0972] Therefore, according to another aspect of the present invention, a bicyclic peptide ligand complex is provided, comprising:

[0973] (a) One or more bicyclic peptide ligands specifically targeting transferrin receptor 1 (TfR1), the bicyclic peptide ligand comprising a polypeptide and a molecular scaffold, the polypeptide comprising at least three reactive groups separated by at least two ring sequences, the molecular scaffold forming covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular scaffold.

[0974] (b) The extended half-life (HLE) portion; and

[0975] (c) Payloads other than antisense oligonucleotides (ASO).

[0976] It should be understood that the payload can be any suitable agent desired to be delivered across the blood-brain barrier and into the brain. For example, the bicyclic peptide ligand complex of the present invention can be considered as a medium for delivering the linked payload across the blood-brain barrier via a transcytosis pathway involving Tfr1. Therefore, the payload can be any agent required to be delivered to the brain. Such purposes are contemplated, particularly including therapeutic, diagnostic, or imaging applications. For example, in one embodiment, the payload comprises a therapeutic agent other than an antisense oligonucleotide (ASO). In an alternative embodiment, the payload comprises a diagnostic agent other than an antisense oligonucleotide (ASO). In an alternative embodiment, the payload comprises an imaging agent other than an antisense oligonucleotide (ASO). In some embodiments, the payload is an effector portion as described herein. In some embodiments, the effector portion is selected from one or more cytotoxic agents, radiochelates, or chromophores.

[0977] Similar to the HLE portion, it should be understood that the payload can be directly bonded to the bicyclic peptide ligand / HLE portion complex, i.e., via a covalent bond, or a spacer or linker can exist between the bicyclic peptide ligand / HLE portion complex and the payload. The design and construction of such spacer or linker will be apparent to those skilled in the art.

[0978] treat

[0979] It should be understood that when the payload contains a therapeutic agent, it may contain any suitable agent (other than antisense oligonucleotides (ASOs)) capable of preventing, inhibiting, or treating a disease or condition via TfR1-mediated therapeutic agent delivery. Examples of suitable therapeutic agents include: small molecules (inhibitors, agonists, and antagonists, ion channel modulators), peptides and bicyclic peptide ligands, antibodies and their fragments (e.g., Fv, Fab, and Fc), and protein and lipid nanobodies.

[0980] Therefore, this document provides a bicyclic peptide ligand as described herein for delivering a payload to a subject in need. A complex comprising a bicyclic peptide ligand as described herein and a half-life extension (HLE) moiety as described herein is also provided for delivering a payload to a subject in need. In some embodiments, the payload is the effector portion as described in more detail herein.

[0981] Therefore, according to another aspect of the present invention, a bicyclic peptide ligand complex is provided, comprising:

[0982] (a) One or more bicyclic peptide ligands specifically targeting transferrin receptor 1 (TfR1), the bicyclic peptide ligand comprising a polypeptide and a molecular scaffold, the polypeptide comprising at least three reactive groups separated by at least two ring sequences, the molecular scaffold forming covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular scaffold.

[0983] (b) The extended half-life (HLE) portion; and

[0984] (c) The payload, which is a therapeutic agent other than an antisense oligonucleotide (ASO),

[0985] It is used to prevent, inhibit or treat diseases or conditions through TfR1-mediated therapeutic delivery.

[0986] Also provided is the use in the preparation of a complex comprising (a) one or more bicyclic peptide ligands as described herein; (b) a half-life-extended (HLE) moiety as described herein; and (c) a payload as described herein, for the prevention, inhibition, or treatment of a disease or condition via TfR1-mediated therapeutic agent delivery. A method for delivering a therapeutic agent to a subject in need is also provided, the method comprising administering to the subject an effective amount of a complex comprising (a) one or more bicyclic peptide ligands as described herein; (b) a half-life-extended (HLE) moiety as described herein; and (c) a payload as described herein.

[0987] Examples of diseases or conditions that can be prevented, suppressed, or treated via TfR1-mediated therapeutic delivery include neurological disorders.

[0988] Examples of such neurological disorders include, but are not limited to:

[0989] Neurological disorders, neurodegenerative diseases, cancer, eye diseases, epileptic seizures, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, and CNS inflammation.

[0990] In one embodiment, the neurological symptoms occur in a human subject. It should be understood that the dosage and / or frequency of administration is adjusted to reduce the concentration of peptide ligands exposed to red blood cells. In another embodiment, the treatment further includes a step of monitoring red blood cell depletion in the human subject.

[0991] The term "prevention" as used in this article refers to the application of a protective composition before the onset of disease. "Inhibition" refers to the application of a composition after an precipitating event but before the clinical manifestation of disease. "Treatment" refers to the application of a protective composition after the onset of disease symptoms.

[0992] Animal model systems are available for screening the effectiveness of peptide ligands in the prevention or treatment of diseases. This invention facilitates the use of animal model systems that allow for the development of peptide ligands that can cross-react with human and animal targets, thereby enabling the use of animal models.

[0993] diagnosis

[0994] It should be understood that when the payload contains a diagnostic agent, it may contain any suitable agent (other than antisense oligonucleotides (ASO)) that can provide information about an individual's condition (i.e., a diagnosis).

[0995] Therefore, according to another aspect of the present invention, a bicyclic peptide ligand complex is provided, comprising:

[0996] (a) One or more bicyclic peptide ligands specifically targeting transferrin receptor 1 (TfR1), the bicyclic peptide ligand comprising a polypeptide and a molecular scaffold, the polypeptide comprising at least three reactive groups separated by at least two ring sequences, the molecular scaffold forming covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular scaffold.

[0997] (b) The extended half-life (HLE) portion; and

[0998] (c) The payload, which is a diagnostic agent other than antisense oligonucleotides (ASO),

[0999] The bicyclic peptide ligand complex is used for diagnosis.

[1000] Also provided is the use of a complex comprising (a) one or more bicyclic peptide ligands as described herein; (b) a half-life extension (HLE) moiety as described herein; and (c) a payload as described herein in the preparation of a pharmaceutical agent for diagnosing a disease or condition in a subject of need. In some embodiments, the payload is an effect moiety as described herein. A method for diagnosing a disease or condition in a subject is also provided, the method comprising administering to the subject an effective amount of a complex comprising (a) one or more bicyclic peptide ligands as described herein; (b) a half-life extension (HLE) moiety as described herein; and (c) a payload as described herein. In some embodiments, the diagnostic step includes obtaining one or more characteristic measurements of the subject, such as measurements characterizing the subject's TfR1 function.

[1001] A specific example of a diagnostic reagent includes a detectable component, which typically contains a dye, radiolabel, or chromophore. Such reagents have the advantage of providing diagnostic information from within the brain (and may also include imaging information).

[1002] As used herein, the terms "detectable portion" and "marking" are used interchangeably and refer to any portion that can be detected, such as primary and secondary markings. Primary markings include, for example, radioactive isotopes (e.g., tritium, etc.). 225 Ac、 227 Ac、 241 Am、 72 As、 74 As、 211 At、 198 Au、 11 B. 7 Be、 212 Bi、 213 Bi、 75 Br、 77 Br、 11 C 14 C 48 Ca, 109 Cd, 139 Ce、 141 Ce、 252 Cf、 55 Co、 57 Co、 60 Co、 51 Cr 130 Cs、 131 Cs、 137 Cs、 61 Cu、 62 Cu、 64 Cu、 67 Cu、 165 Dy、 152 Eu、 155 Eu、 18 F, 55 Fe、 59 Fe、 64 Ga、 67 Ga、 68 Ga、 153 Gd, 68 Ge 122 I, 123 I, 124 I, 125 I, 131 I, 132 I, 111 In、 115m In、 191m Ir、 192 Ir、 81m Kr、 177 Lu、 51 Mn, 52 Mn, 99 Mo、 13 N、95 Nb, 15 O、 191 Os、 194 Os、 32 P, 33 P, 203 Pb, 212 Pb, 103 Pd, 109 Pd, 238 Pu、 223 Ra、 226 Ra、 82 Rb、 186 Re、 188 Re、 105 Rh、 97 Ru、 103 Ru、 35 S, 46 Sc、 47 Sc、 72 Se、 75 Se、 28 Si、 145 Sm、 153 Sm、 117m Sn、 85 Sr、 89 Sr、 90 Sr、 178 Ta、 179 Ta、 182 Ta、 149 Tb, 96 Tc, 99m Tc, 228 Th、 229 Th、 201 Tl、 170 Tm、 171 Tm、 188 W, 127 Xe, 133 Xe, 88 Y、 90 Y、 91 Y、 169 Yb、 62 Zn, 65 Zn, 89 Zr or 95 Zr (where the superscript m indicates the metastable state), quality tags, and fluorescent labels are signal-generating reporter groups that can be detected without further modification. The detectable portion also includes luminescent and phosphorescent groups.

[1003] As used herein, the term "secondary label" refers to a component that requires the presence of a second intermediate to generate a detectable signal, such as biotin and various protein antigens. For biotin, the secondary intermediate may include streptavidin-enzyme conjugates. For antigen labels, the secondary intermediate may include antibody-enzyme conjugates. Some fluorescent groups function as secondary labels because they transfer energy to another group during nonradiative fluorescence resonance energy transfer (FRET), and this second group generates the detected signal.

[1004] As used in this article, the terms “fluorescent label,” “fluorescent dye,” and “fluorophore” refer to the part that absorbs light energy at a specific excitation wavelength and emits light energy at different wavelengths. Examples of fluorescent labels include, but are not limited to: Alexa Fluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY630 / 650, BODIPY 650 / 665), carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue, CascadeYellow, Coumarin 343, Anthocyanin dyes (Cy3, Cy5, Cy3.5, Cy5.5, Cy7, Cy7.5), Dansyl, Dapoxyl, Dialkylaminocoumarin, 4',5'-Dichloro-2',7'-Dimethoxyfluorescein, DM-NERF, Eosin, Erythrosine, Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD700, IRD800), JOE, Rhodamine B, Marina Blue, Methoxycoumarin, Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2',4',5',7'-Tetrabromosulfone-fluorescein, Tetramethyl-Rhodamine (TMR), CarboxytetramethylRhodamine (TAMRA), Texas Red, Texas Red-X.

[1005] As used herein, the term "quality tag" refers to any component that can be uniquely detected by mass spectrometry (MS) based on its quality. Examples of quality tags include electrophoretic release tags, such as N-[3-[4'-[(p-methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceryl]isopiperidinic acid, 4'-[2,3,5,6-tetrafluoro-4-(pentafluorophenoxy)]methylacetophenone, and derivatives thereof. The synthesis and use of these quality tags are described in U.S. Patents 4,650,750, 4,709,016, 5,360,8191, 5,516,931, 5,602,273, 5,604,104, 5,610,020, and 5,650,270. Other examples of quality tags include, but are not limited to, nucleotides, dideoxynucleotides, oligonucleotides of varying lengths and base compositions, oligopeptides, oligosaccharides, and other synthetic polymers of varying lengths and monomer compositions. A variety of organic molecules with appropriate mass ranges (100-2000 Daltons), whether neutral or charged (biomolecules or synthetic compounds), can also be used as mass labels.

[1006] As used herein, the term “quantum dot” refers to any part of a highly luminescent semiconductor nanocrystal (e.g., zinc sulfide-terminated cadmium selenide). The synthesis and uses of these quantum dots are described in U.S. Patents 6,326,144, 6,468,808, 7,192,785, and 7,151,047, as well as in the scientific literature (see: Chan and Nie (1998) Science 281(5385) 2016–2018).

[1007] Imaging

[1008] It should be understood that when the payload contains an imaging agent, it may contain any suitable reagent (other than antisense oligonucleotides (ASO)) that can provide individual-related imaging information.

[1009] Therefore, according to another aspect of the present invention, a bicyclic peptide ligand complex is provided, comprising:

[1010] (a) One or more bicyclic peptide ligands specifically targeting transferrin receptor 1 (TfR1), the bicyclic peptide ligand comprising a polypeptide and a molecular scaffold, the polypeptide comprising at least three reactive groups separated by at least two ring sequences, the molecular scaffold forming covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular scaffold.

[1011] (b) The extended half-life (HLE) portion; and

[1012] (c) The payload, which is an imaging agent other than antisense oligonucleotides (ASO),

[1013] The bicyclic peptide ligand complex is used for diagnosis.

[1014] A specific example of an imaging agent includes a detectable portion, which typically contains dyes, radiolabels, or chromophores. Such agents have the advantage of providing imaging information from inside the brain.

[1015] The present invention is further described below with reference to the following embodiments.

[1016] Example

[1017] Preparation of HLE-containing Tfr1 bicyclic peptide ligand complex

[1018] Example 1: Preparation of B192

[1019]

[1020] Step 1

[1021] The title compound was synthesized using standard Fmoc chemistry. Rink amide MBHA resin (0.5 mmol, 1 equivalent, 0.3 mmol / g, 1.67 g resin) was stirred in DMF at 20 °C with N2 for 2 hours, followed by filtration to collect the resin. For each step of the peptide synthesis, deprotection was performed sequentially, followed by coupling.

[1022] Deprotection: Add 50 ml of DMF containing 20% ​​piperidine to the resin and stir with N2 for 15 minutes. Wash the resin with DMF (50 ml × 5) and filter to separate the resin.

[1023] Coupling: A solution of Fmoc-Lys(Dde)-OH (1.5 equivalents), diisopropylethylamine (DIEA) (3.0 equivalents), and HATU (1.425 equivalents) in DMF (15.0 mL) was added to the resin, and the mixture was stirred with N2 at 20 °C for 30 minutes. The resin was then washed with DMF (50 mL × 5).

[1024] The following amino acids were repeatedly deprotected / coupled: Fmoc-Trp(Boc)-OH, Fmoc-Pro-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-tBuGly-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Ala-OH, Fmoc-Asp(OtBu)-OH, Fmoc-HyP-OH, Fmoc-Pro-OH, and Fmoc-Cys(Trt)-OH. Ac₂O (3 equivalents) and DIEA (4 equivalents) were added, and the reaction mixture was stirred for 15 minutes. The mixture was washed with DMF (50 ml × 5).

[1025] Deprotection: Add DMF (50 ml) containing 3% hydrazine and stir the resin again with N2 for 15 minutes. Wash the resin with DMF (50 ml × 5) and filter to obtain the resin.

[1026] Coupling: Fmoc-PEG2-OH (1.5 equivalents), DIEA (3.0 equivalents), and HATU (1.425 equivalents) in DMF were added to the resin, and the mixture was stirred with N2 at 20°C for 30 minutes. The resin was then washed with DMF (50 m × 5).

[1027] Deprotection: Add DMF (50 ml) containing 20% ​​piperidine to the resin and stir with N2 for 15 minutes.

[1028] Coupling: Add Fmoc-PEG2-OH (1.5 equivalents), DIEA (3.0 equivalents), and HATU (1.425 equivalents) in DMF to the resin and stir with N2 at 20°C for 30 minutes. Then wash the resin with DMF (50 ml × 5).

[1029] Deprotection: Add DMF (50 ml) containing 20% ​​piperidine to the resin and stir with N2 for 15 minutes.

[1030] Coupling: A solution of Fmoc-g-Glu-OH (3 equivalents), DIEA (6.0 equivalents), and HATU (1.425 equivalents) in DMF (15.0 ml) was added to the resin, and the mixture was stirred with N2 at 20°C for 30 minutes. The resin was then washed with DMF (50 ml × 5).

[1031] Deprotection: Add DMF (50 ml) containing 20% ​​piperidine to the resin and stir with N2 for 15 minutes.

[1032] Coupling: A solution of 18-(tert-butoxy)-18-oxooctadecanoic acid (1.5 equivalents), DIEA (6.0 equivalents), and HATU (1.425 equivalents) in DMF (15.0 ml) was added to the resin, and the mixture was stirred with N2 at 20°C for 30 minutes. The resin was then washed with DMF (50 ml × 5).

[1033] At room temperature, a lysis solution (45 ml, 85% TFA / 2.50% TIS / 2.50% H2O / 10% 3-mercaptopropionic acid) was added to a resin-containing flask and stirred for 2 hours. The peptide was precipitated with cold isopropyl ether (230 ml). The precipitate was filtered and the filter cake was collected. The filter cake was washed with isopropyl ether (230 ml × 2). The crude peptide was dried under vacuum for 2 hours to obtain a crude solid (900.0 mg).

[1034] Step 2

[1035] To a solution of linear peptide (230 mg) and TATB (36.5 mg, 0.95 equivalents) in H2O (160.0 mL) and acetonitrile (70 mL), CS2CO3 (aqueous solution, 1 M) was slowly added to adjust the pH of the reaction mixture to 8–9. The mixture was stirred at 25 °C for 90 min. 1 M HCl (aqueous solution) was added dropwise until the solution reached pH 6–7. The mixture was lyophilized to obtain the crude bicyclic peptide. The crude peptide was purified by preparative HPLC (TFA conditions: A: aqueous solution containing 0.075% TFA, B: MeCN) to give the final product B192 (64.1 mg, 97.61% purity, TFA salt), a white solid, confirmed by LCMS (Rt = 1.266 min, MS calculated value: 2899.35, MS measured value: [M+2H]). 2+ = 1450.17).

[1036] Example 2: Preparation of B193

[1037]

[1038] B193 can also be described as follows:

[1039]

[1040] Step 1: Preparation of B194

[1041]

[1042] Linear sequence: [Ac]CP[HyP]DAHLGC[tBuGly]SYCEPW[K(PYA)] (SEQ ID NO: 120). The title compound was prepared by standard Fmoc chemistry, cyclization, and purification according to method B192 (step 1).

[1043] Step 2 : Preparation of B195

[1044]

[1045] Linear sequence: [Ac][K(N3)]GRLIEDICLPRWGCLWEDD (SEQ ID NO: 119).

[1046] The linear peptide was prepared by standard Fmoc chemistry according to the B192 (step 1) method.

[1047] The crude peptide (1 equivalent) was dissolved in 2 M urea (35 ml), and the pH of the resulting solution was adjusted to 8.5 by adding 1 M ammonium bicarbonate. Hydrogen peroxide (30% w / w, aqueous solution, 25 µl) was added, and the reaction mixture was stirred for 30 min. The reaction was monitored by LCMS to determine the presence of any linear peptides; if present, one part hydrogen peroxide was added and stirring continued. The reaction mixture was evaporated, and the residue was purified by HPLC (MeCN / H2O + 0.1% TFA, 20% to 50% over 10 min, flow rate 20 ml / min) to obtain the title compound.

[1048] Step 3

[1049] A mixture of B194 (40.0 mg, 17.6 µmol, 1.00 equivalent), B195 (43.8 mg, 17.6 µmol, 1.10 equivalent), and THPTA (7.68 mg, 17.6 µmol, 1.00 equivalent) was dissolved in t-BuOH (0.30 ml) and H2O (0.30 ml) (pre-degassed and purged three times with N2). Then, an aqueous solution of CuSO4 (0.40 M, 44.1 µl, 1.00 equivalent) and VcNa (7.00 mg, 35.3 µmol, 2.00 equivalent) was added under N2. The pH of the solution was adjusted to 8 by dropwise addition of 0.20 M NH4HCO3 (in a 1:1 t-BuOH / H2O), resulting in a pale yellow solution. The reaction mixture was stirred at 20 °C under a N2 atmosphere for 1 hour. LCMS showed that B194 was completely consumed and detected a main peak with the desired m / z (MW: 4746.37). m / zMeasured value: 1583.2 ([M / 3+H]) + ), 1187.7 ([M / 4+H] + ), 950.4 ([M / 5+H] + The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC (TFA conditions) to give B193 (48.8 mg, 9.66 µmol, yield 54.6%, purity 94.0%) as a white solid, confirmed by LC-MS (Rt = 1.266 min, MS calculated value: 4745.4, MS measured value: [M+H)). + = 4746.52).

[1050] Example 3: In vivo half-life analysis of the Tfr1 bicyclic peptide ligand complex containing HLE

[1051] B191 is a bicyclic peptide specific to Tfr1. As a control (to demonstrate in vivo clearance in the absence of HLE), B191 was administered intravenously to mice (according to the methods described below) to determine plasma concentration-time curves and related pharmacokinetic parameters, as shown below. Figure 1 See Table 1.

[1052] Table 1: Mean pharmacokinetic parameters of B191 after administration of 2.69 mg / kg via intravenous infusion over 15 minutes

[1053]

[1054] from Figure 1 As shown in Table 1, the observed clearance (CL) was 31 ml / min / kg, and the half-life (t½) was 0.28 hours. The plasma unbound fraction of B191 was 0.772.

[1055] B192 was prepared by conjugating B191 with a C-18-diacid-gGlu-PEG2-PEG2 albumin conjugate, as described in Example 1. The resulting HLE-containing complex B192 was administered intravenously to mice (according to the methods described below) to determine plasma concentration-time curves and related pharmacokinetic parameters, as shown below. Figure 2 And Table 2.

[1056] Table 2: Mean pharmacokinetic parameters of B192 after administration of 3.45 mg / kg via intravenous infusion over 15 minutes

[1057]

[1058] from Figure 2As shown in Table 2, B192 unexpectedly exhibited a significant increase in plasma protein binding (plasma unbound fraction 0.005). Consequently, the clearance (CL) decreased to 0.25 ml / min / kg, the volume of distribution (Vd) decreased to 0.16 L / kg, and the half-life (t½) was prolonged to 7.8 hours.

[1059] B193 was prepared by conjugating B191 with the albumin-binding peptide SA21 (J Biol Chem 2002, 277(38), 35035-43). The resulting HLE-containing complex B193 was administered intravenously to mice (according to the methods described below) to determine plasma concentration-time curves and related pharmacokinetic parameters, which are shown in [the table below]. Figure 3 And Table 3.

[1060] Table 3: Mean PK parameters of B193 after administration of 0.28 mg / kg via intravenous infusion over 15 minutes

[1061]

[1062] from Figure 2 As shown in Table 2, B193 also unexpectedly led to a significant increase in plasma protein binding (unbound fraction <0.015). Consequently, CL decreased to 0.10 ml / min / kg, Vd decreased to 0.18 L / kg, resulting in a prolonged half-life (t½) of 22.6 hours.

[1063] Figure 2 and Figure 3 and the results shown in Tables 2 and 3 (with) Figure 1 Compared to the results shown in Table 1, it is clear that the addition of the albumin-binding moiety to the parent molecule B191 significantly increased plasma protein binding. This reduced plasma CL by preventing glomerular filtration and increased the half-life by preventing distribution outside the systemic circulation, thus reducing the volume of distribution.

[1064] Pharmacokinetic studies and bioanalytical methods

[1065] All research and bioanalysis were conducted by WuXi AppTec (Hong Kong) Limited.

[1066] The plasma protein binding of all compounds was assessed by ultracentrifugation.

[1067] In vivo studies were conducted on 7-9 week old male CD-1 (ICR) mice. Animals were housed in groups of up to four in polysulfone cages lined with poplar wood bedding. The environment was maintained at 20-26°C and 40-70% humidity, with a 12-hour light / dark cycle. Animals had free access to sterilized drinking water and certified rodent feed. Animals were acclimatized to the testing facility for at least 3 days prior to entering the study.

[1068] Three animals in each group were administered 2.69 mg / kg of B191 or 3.45 mg / kg of B192 (at 25 mM histidine). In HCl containing 10% sucrose, pH 7, neutralized with NaOH, the dosage is 1 mg / ml, or 0.28 mg / kg of B193 (at 25 mM histidine). The drug was administered in HCl containing 10% sucrose, pH 7, neutralized with NaOH at a concentration of 0.1 mg / ml. All administration solutions were sterile filtered before intravenous infusion over 15 minutes via an indwelling jugular catheter. For B191 and B192, blood samples were collected from the saphenous vein of the animals into low-binding polypropylene tubes at 0.25, 0.33, 0.5, 1, 2, 4, 6, 8, 10, and 24 hours after the start of infusion; for B193, at 0.25, 0.33, 0.5, 1, 2, 4, 6, 8, 10, 24, and 48 hours after the start of infusion. All blood samples were immediately transferred to pre-chilled low-binding microcentrifuge tubes (containing 2 μL K2-EDTA (0.5 M) as an anticoagulant) and placed on wet ice until the plasma was processed by centrifugation at 3200 g for 10 minutes at 4°C. The resulting plasma was transferred to a 1.5 mL low-binding tube, rapidly frozen on dry ice, and stored at -60°C or below until extraction was performed prior to LC-MS / MS analysis.

[1069] Samples were extracted by quenching 15 µL (B192) or 30 µL (B191) of sample (unknown, blank, calibration, or QC) with 150 µL of internal standard (a methanol solution containing 0.5% Triton X-100, and 100 ng / ml each of labetalol, tolbutamide, verapamil, dexamethasone, glibenclamide, and celecoxib). For B193, samples were extracted by adding 15 µL of an aqueous solution containing 4% H3PO4 to 15 µL of sample (unknown, blank, calibration, or QC), vortexing, and then quenching with 300 µL of internal standard (a methanol solution containing 100 ng / ml each of labetalol, tolbutamide, verapamil, dexamethasone, glibenclamide, and celecoxib). After quenching, all samples were vortexed and centrifuged at 12000 g for 15 min at 4 °C. Do not dilute the supernatant, or dilute it 10- or 20-fold in the supernatant from the blank sample and vortex. Transfer aliquots to 96-well plates, centrifuge at 3220 g for 5 minutes at 4°C, and then directly inject the supernatant for LC-MS / MS analysis.

[1070] LC-MS / MS analysis was performed using single-reaction monitoring on a Sciex Triple Quad 6500 (B191) or 6500+ (B192 and B193) equipped with an electrospray ionization source. Chromatographic separations were achieved using the columns and conditions described in Table 4. Unknown samples were quantified using an eight-point calibration curve, which also included four quality control samples within the range. Internal standards selected for quantification and calibration ranges are also listed in Table 4.

[1071] Table 4: LC conditions for compound bioanalysis and quantification

[1072]

[1073] Pharmacokinetic parameters for all compounds were derived from noncompartmental analysis using the slow injection-noncompartmental model 202 (IV infusion input) in Phoenix WinNonlin version 6.3 (B191) or 8.3.5 (B192 and B193).

[1074] The following are the numbering aspects of this disclosure:

[1075] 1. A bicyclic peptide ligand complex comprising:

[1076] (a) One or more bicyclic peptide ligands specifically targeting transferrin receptor 1 (TfR1), the bicyclic peptide ligand comprising a polypeptide and a molecular scaffold, the polypeptide comprising at least three reactive groups separated by at least two ring sequences, the molecular scaffold forming covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular scaffold; and

[1077] (b) Half-life extension (HLE) portion.

[1078] 2. The bicyclic peptide ligand complex according to aspect 1, wherein the bicyclic peptide ligand specifically targeting transferrin receptor 1 (TfR1) is selected from any one of B1 to B191 or B196 to B198.

[1079] 3. The bicyclic peptide ligand complex according to aspect 1 or aspect 2, wherein the HLE portion is a portion capable of binding albumin, said albumin being, for example, serum albumin, particularly human serum albumin (HSA).

[1080] 4. The bicyclic peptide ligand complex according to aspect 3, wherein the albumin-binding portion is selected from:

[1081] (i) Short-chain fatty acids (i.e. fatty acids with 5 or fewer carbon atoms), such as butyric acid (C4);

[1082] (ii) Medium-chain fatty acids (i.e. fatty acids with 6 to 12 carbon atoms), such as hexanoic acid (C6), octanoic acid (C8), decanoic acid (C10), or lauric acid (C12);

[1083] (iii) Long-chain fatty acids (i.e. fatty acids with 13 to 21 carbon atoms), such as myristic (C14) acid, palmitic (C16) acid, stearic (C18) acid, and arachidic (C20) acid;

[1084] (iv) Very long-chain fatty acids (i.e. fatty acids with 22 or more carbon atoms), such as behenic acid (C22), ceramide (C24) and ceramide (C26);

[1085] (v) Genentech’s small molecule FVIIa peptide-albumin binding inhibitor (Bioorg Med Chem Lett, 2002, 12(20), 2883-6 and Bioorg Med Chem Lett, 2003, 13(9), 1513-5), which greatly increased plasma half-life in rabbits;

[1086] (vi) The Fmoc protecting group has been shown to significantly reduce the hydrolysis of compounds in the presence of HSA (Int J PepRes Ther, 2007, 13(1-2), 12);

[1087] (vii) Coumarin derivatives, such as warfarin-type coumarin derivatives, have been shown to improve in vivo half-life in rats;

[1088] (viii) Evans blue, which binds to the Sudlow site I of HSA with low molar affinity (2.5 µM) and has a long half-life in blood (Theranostics, 2016, 6(2), 243-53);

[1089] (ix) Diflunisal, which binds to sites I and II of HSA with an affinity of about 3 µM (J Med Chem 2017, 60(17), 7434-46);

[1090] (x) Indomethacin, which binds primarily to site I of HSA with an affinity of 0.7 µM (J Med Chem 2017, 60(17), 7434-46);

[1091] (xi) Lithocholic acid (bile acid), an example of an endogenous molecule, can control blood glucose levels for more than 24 hours when conjugated with insulin (Pharm Res 2006, 23(1), 49-55);

[1092] (xii) 4-(p-iodophenyl)butyric acid (Albutag) and related phenylbutyric acid examples have been found to be stable in mouse and human serum albumin (Angew Chem Int Ed, 2008, 47(17), 3196-3201);

[1093] (xiii) PSMA derivatives, such as RPS-068 (Eur J Nucl Med Mol Imaging 2018, 45(11), 1841-51), RPS-072 (J Nucl Med 2019, 60(5), 656-63), PSMA-ALB-56 (Mol Pharmaceutics 2018, 15, 2297-306), NODAGA analogs (Cu) (Mol Pharmaceutics 2018, 15, 5556-64), or those described in EP 3 778 592;

[1094] (xiv) peptides, such as F-Tag (Nat Commun 2017, 8, 16092), 89D03 (WO 2011 / 095545), [Ac][K(N3)]GRLIEDICLPRWGCLWEDD (SEQ ID NO: 119; SA21; J Biol Chem2002, 277(38), 35035-43);

[1095] (xv) Liraglutide (Diabetes Care 2002; 25:1398-1404);

[1096] (xvi) Smegglutinin (Med Chem; 2015, 58, 7370-7380);

[1097] (xvii) Knob domain antibody fragment;

[1098] (xviii) Polyethylene glycolated compounds, such as those described in WO 2020 / 205948;

[1099] (xix) Compounds containing small molecule aromatic compounds, such as those described in WO 2008 / 053360; and

[1100] (xx) PPB03 (Bioconjugate Chem 2017, 28(9), 2372-2383).

[1101] 5. The bicyclic peptide ligand complex according to aspect 4, wherein the HLE moiety is a fatty acid, such as a long-chain fatty acid, particularly a C18 diacid, especially octadecanoic acid.

[1102] 6. The bicyclic peptide ligand complex according to aspect 4, wherein the HLE moiety is an albumin-binding peptide, such as SA21([Ac][K(N3)]GRLIEDICLPRWGCLWEDD (SEQ ID NO: 119)).

[1103] 7. The bicyclic peptide ligand complex according to any one of aspects 1 to 6, further comprising a spacer region or linker between the bicyclic peptide ligand and the HLE portion.

[1104] 8. The peptide ligand complex according to any one of aspects 1 to 7, further comprising a payload in addition to an antisense oligonucleotide (ASO).

[1105] 9. The peptide ligand complex according to aspect 8, further comprising a spacer region or linker between the bicyclic peptide ligand / HLE partial complex and the payload.

[1106] 10. The peptide ligand complex according to aspect 4, wherein the payload is selected from therapeutic agents, diagnostic agents, and imaging agents.

[1107] 11. The peptide-ligand complex according to aspect 10, wherein the therapeutic agent is selected from small molecules (inhibitors, agonists and antagonists, ion channel modulators), peptides and bicyclic peptide ligands, antibodies and fragments thereof (e.g., Fv, Fab and Fc), proteins and lipid nanobodies.

[1108] 12. The peptide ligand complex according to aspect 10, wherein the diagnostic agent or imaging agent is a detectable portion, such as a dye, radiolabel, or chromophore.

[1109] 13. A bicyclic peptide ligand specifically targeting transferrin receptor 1 (TfR1), wherein the peptide ligand is B191 or B196 to B198.

[1110] 14. A pharmaceutical composition comprising a bicyclic peptide ligand complex according to any one of aspects 1 to 12 or a bicyclic peptide ligand according to aspect 13, in combination with one or more pharmaceutically acceptable excipients.

[1111] 15. A peptide ligand complex according to any one of aspects 1 to 11, or a bicyclic peptide ligand according to aspect 13, or a pharmaceutical composition according to aspect 14, for the prevention, inhibition, or treatment of a disease or condition via TfR1-mediated therapeutic delivery.

[1112] 16. The peptide ligand complex, bicyclic peptide ligand, or pharmaceutical composition used according to aspect 15, wherein the disease or condition delivered via TfR1-mediated therapeutic agent delivery is selected from neurological disorders, such as neuropathic disorders, neurodegenerative diseases, cancer, eye diseases, epileptic seizures, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, and CNS inflammation.

Claims

1. A bicyclic peptide ligand complex comprising: (a) One or more bicyclic peptide ligands specifically targeting transferrin receptor 1 (TfR1), the bicyclic peptide ligand comprising a polypeptide and a molecular scaffold, the polypeptide comprising at least three reactive groups separated by at least two ring sequences, the molecular scaffold forming covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular scaffold; and (b) Half-life extension (HLE) portion.

2. The bicyclic peptide ligand complex according to claim 1, wherein the polypeptide comprises a first circular sequence containing 7 amino acids and a second circular sequence containing 3 amino acids; The first ring sequence has an amino acid sequence. -AA1-AA2-AA3-AA4-AA5-AA6-AA7; in: A1 is selected from S, P, A, [K(N3)], [HyP], [Oxa], and [Cis-HyP]; A2 is selected from A, S, P, Q, G, [HyP], [Aib], N, I, [Aze], [dA], [K(N3)], [Oxa], and [Cis-HyP]. A3 is selected from D, A, E, and [Gla]; A4 is selected from D, A, S, [Aib], [Abu], and [K(N3)]. A5 is selected from W, Y, H, A, [DOPA], [hTyr], [pCaPhe], and [pCoPhe]; A6 is selected from L, Q, [tBuAla], [Cba], [Abu], [Aib], A, S, T, D, E, and N; and A7 is selected from G, [dA], A, [dS], [dT], [dD], [dE], [dN], [dQ], [dY], S, D, Y, and N; And the second ring sequence has an amino acid sequence. -AA1-AA2-AA3-; in: AA1 is selected from [tBuGly], [EPA], [Chg], [tBuAla], [C5g], [Cbg], [Cpg], [B-MeIle], I, A, Y and [3HyV]; AA2 is selected from S, A, [HSer], T, D, E, N, and Q; and AA3 is selected from W, Y, [2Nal], [3tBuTyr], [1Nal], [4Pal], A, [DOPA], [pCaPhe], [pCoPhe], and [hTyr]; Or its variants, or its pharmaceutically acceptable salts.

3. The bicyclic peptide complex according to claim 2, The first ring sequence has an amino acid sequence. -AA1-AA2-AA3-AA4-AA5-AA6-AA7; in: A1 is selected from S, P, A, [K(N3)], [HyP], [Oxa], and [Cis-HyP]; preferably, A1 is selected from S and P; A2 is selected from A, S, P, Q, G, [HyP], [Aib], N, I, [Aze], [dA], [K(N3)], [Oxa], and [Cis-HyP]; preferably, A2 is selected from P and [HyP]. A3 is D; A4 is A; A5 is selected from H and Y; A6 is L; and A7 is G; And the second ring sequence has an amino acid sequence. -AA1-AA2-AA3-; in: AA1 is selected from [tBuGly] and I; AA2 is S; and AA3 is Y; Or its pharmaceutically acceptable salt.

4. The bicyclic peptide ligand complex according to any one of claims 1 to 3, wherein the first ring sequence is selected from: SADDWLG; SSDAYLG; PPDAHLG; PQDAYLG; PPDSWQG; SPDAHLG; PGDAHLG; PPDSHLG; P[HyP]DAYLG; S[HyP]DAHLG; P[Aib]DAHLG; P[Aib]DAYLG; SADAHLG; S[Aib]DAHLG; PPDAYLG; S[Aib]DAYLG; APDAHLG; SPDAYLG; PNDAHLG; PIDAHLG; SPD[Aib]HLG; SPDAH[tBuAla]G; SPDAH[Cba]G; SPD[Abu]HLG; S[Aze]DAHLG; SPDDHLG; SPDSHLG; SPDAH[Abu]G; P[dA]DAHLG; SPDAHL[dA]; SPDAH[Aib]G; SPAAHLG; SPDAALG; SPDAHAG; SPDAHLA; [K(N3)]PDAHLG; S[K(N3)]DAHLG; SPD[K(N3)]HLG; P[HyP]DAHLG; [HyP][HyP]DAYLG [Oxa][HyP]DAYLG [Cis-HyP][HyP]DAYLG P[Oxa]DAYLG P[Cis-HyP]DAYLG P[HyP]DA[DOPA]LG P[HyP]DA[hTyr]LG P[HyP]DA[pCaPhe]LG P[HyP]DA[pCoPhe]LG P[HyP]DAYL[dS] P[HyP]DAYL[dT] P[HyP]DAYL[dD] P[HyP]DAYL[dE] P[HyP]DAYL[dN] P[HyP]DAYL[dQ] P[HyP]DAYL[dY] P[HyP]DAYLS P[HyP]DAYLD P[HyP]DAYLY; P[HyP]DAYLN; P[HyP]EAYLG; P[HyP][Gla]AYLG; P[HyP]DAYSG; P[HyP]DAYTG; P[HyP]DAYDG; P[HyP]DAYEG; P[HyP]DAYNG; P[HyP]DAYQG; and YLPDW[tBuAla]; and its variants; and its pharmaceutically acceptable salts; And / or wherein the second ring sequence is selected from ISW; ISY; [tBuGly]SY; [EPA]SY; [Chg]SY; IS[2Nal]; IS[3tBuTyr]; IS[1Nal]; IS[4Pal]; [tBuAla]SY; [C5g]SY; [Cbg]SY; [Cpg]SY; [B-MeIle]SY; ASY; IAY; ISA; [tBuGly]S[DOPA]; [tBuGly]S[pCaPhe]; [tBuGly]S[pCoPhe]; [tBuGly]S[hTyr]; [tBuGly][HSer]Y; [tBuGly]TY; [tBuGly]DY; [tBuGly]EY; [tBuGly]NY; [tBuGly]QY; YSY; [3HyV]SY; and GDEY; and its variants; And its pharmaceutically acceptable salts.

5. The bicyclic peptide ligand complex according to any one of claims 1 to 4, wherein the peptide comprises one or more N-terminal and / or C-terminal additions.

6. The bicyclic peptide ligand complex according to any one of claims 1 to 5, wherein the molecular scaffold is selected from 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tripropyl-2-en-1-one (also known as triacryloylhexahydro-s-triazine (TATA)) and 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethylone) (TATB).

7. The bicyclic peptide ligand complex according to any one of claims 1 to 6, wherein the bicyclic peptide ligand specifically targeting transferrin receptor 1 (TfR1) is selected from any one of B1 to B191 or B196 to B198.

8. The bicyclic peptide ligand complex according to any one of claims 1 to 7, wherein the HLE portion is a portion capable of binding albumin, said albumin being, for example, serum albumin, particularly human serum albumin (HSA).

9. The bicyclic peptide ligand complex according to claim 8, wherein the albumin-binding portion is selected from: (i) Short-chain fatty acids (i.e. fatty acids with 5 or fewer carbon atoms), such as butyric acid (C4); (ii) Medium-chain fatty acids (i.e. fatty acids with 6 to 12 carbon atoms), such as hexanoic acid (C6), octanoic acid (C8), decanoic acid (C10), or lauric acid (C12); (iii) Long-chain fatty acids (i.e. fatty acids with 13 to 21 carbon atoms), such as myristic (C14) acid, palmitic (C16) acid, stearic (C18) acid, and arachidic (C20) acid; (iv) Very long-chain fatty acids (i.e. fatty acids with 22 or more carbon atoms), such as behenic acid (C22), ceramide (C24) and ceramide (C26); (v) Genentech’s small molecule FVIIa peptide-albumin binding inhibitor (Bioorg Med Chem Lett, 2002, 12(20), 2883-6 and Bioorg Med Chem Lett, 2003, 13(9), 1513-5), which greatly increased plasma half-life in rabbits; (vi) The Fmoc protecting group has been shown to significantly reduce the hydrolysis of compounds in the presence of HSA (Int J Pep ResTher, 2007, 13(1-2), 12); (vii) Coumarin derivatives, such as warfarin-type coumarin derivatives, have been shown to improve in vivo half-life in rats; (viii) Evans blue, which binds to the Sudlow site I of HSA with low molar affinity (2.5 µM) and has a long half-life in blood (Theranostics, 2016, 6(2), 243-53); (ix) Diflunisal, which binds to sites I and II of HSA with an affinity of about 3 µM (J Med Chem 2017, 60(17), 7434-46); (x) Indomethacin, which binds primarily to site I of HSA with an affinity of 0.7 µM (J Med Chem 2017, 60(17), 7434-46); (xi) Lithocholic acid (bile acid), an example of an endogenous molecule, can control blood glucose levels for more than 24 hours when conjugated with insulin (Pharm Res 2006, 23(1), 49-55); (xii) 4-(p-iodophenyl)butyric acid (Albutag) and related phenylbutyric acid examples have been found to be stable in mouse and human serum albumin (Angew Chem Int Ed, 2008, 47(17), 3196-3201); (xiii) PSMA derivatives, such as RPS-068 (Eur J Nucl Med Mol Imaging 2018, 45(11), 1841-51), RPS-072 (J Nucl Med 2019, 60(5), 656-63), PSMA-ALB-56 (MolPharmaceutics 2018, 15, 2297-306), NODAGA analogs (Cu) (Mol Pharmaceutics 2018, 15, 5556-64), or those described in EP 3 778 592; (xiv) peptides, such as F-Tag (Nat Commun 2017, 8, 16092), 89D03 (WO 2011 / 095545), [Ac][K(N3)]GRLIEDICLPRWGCLWEDD (SEQ ID NO: 119; SA21; J Biol Chem 2002, 277(38), 35035-43); (xv) Liraglutide (Diabetes Care 2002; 25:1398-1404); (xvi) Smegglutinin (Med Chem; 2015, 58, 7370-7380); (xvii) Knob domain antibody fragment; (xviii) Polyethylene glycolated compounds, such as those described in WO 2020 / 205948; (xix) Compounds containing small molecule aromatic compounds, such as those described in WO 2008 / 053360; as well as (xx) PPB03 (Bioconjugate Chem 2017, 28(9), 2372-2383).

10. The bicyclic peptide ligand complex according to any one of the preceding claims, wherein the HLE portion is a fatty acid, such as a long-chain fatty acid, particularly a C18 diacid, especially octadecanoic acid.

11. The bicyclic peptide ligand complex according to any one of the preceding claims, wherein the HLE moiety is an albumin-binding peptide, such as SA21: [Ac]RLIEDICLPRWGCLWEDD[CONH2] Or its derivatives that bind to albumin.

12. The bicyclic peptide ligand complex according to claim 11, wherein the HLE portion is [Ac][K(N3)]GRLIEDICLPRWGCLWEDD (SEQ ID NO: 119).

13. The bicyclic peptide ligand complex according to any one of claims 1 to 12, further comprising a spacer region or linker between the bicyclic peptide ligand and the HLE portion.

14. The peptide ligand complex according to any one of claims 1 to 13, further comprising a payload in addition to an antisense oligonucleotide (ASO).

15. The peptide ligand complex of claim 14, further comprising a spacer region or linker between the bicyclic peptide ligand / HLE partial complex and the payload.

16. The peptide ligand complex according to claim 14 or 15, wherein the payload is selected from therapeutic agents, diagnostic agents, and imaging agents.

17. The peptide-ligand complex of claim 16, wherein the therapeutic agent is selected from small molecules (inhibitors, agonists and antagonists, ion channel modulators), peptides and bicyclic peptide ligands, antibodies and fragments thereof (e.g., Fv, Fab and Fc), proteins and lipid nanobodies.

18. The peptide ligand complex of claim 16, wherein the diagnostic agent or imaging agent is a detectable portion, such as a dye, radiolabel, or chromophore.

19. A bicyclic peptide ligand specifically targeting transferrin receptor 1 (TfR1), wherein the peptide ligand comprises a polypeptide as defined in claim 2 or 3.

20. A bicyclic peptide ligand specifically targeting transferrin receptor 1 (TfR1), wherein the peptide ligand is B191 or B196 to B198.

21. A pharmaceutical composition comprising a bicyclic peptide ligand complex according to any one of claims 1 to 18 or a combination of a bicyclic peptide ligand according to claim 19 or 20 with one or more pharmaceutically acceptable excipients.

22. The peptide ligand complex according to any one of claims 1 to 18, or the bicyclic peptide ligand according to claim 19 or 20, or the pharmaceutical composition according to claim 21, for the prevention, inhibition, or treatment of a disease or condition via TfR1-mediated therapeutic delivery.

23. The peptide ligand complex, bicyclic peptide ligand, or pharmaceutical composition used according to claim 22, wherein the disease or condition is selected from neurological disorders, such as neuropathic disorders, neurodegenerative diseases, cancer, eye diseases, epileptic seizures, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, and CNS inflammation.

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