Bicyclic peptide ligand complexes specific for TFR1

Abstract

Provided herein are bicyclic peptide ligand complexes specific for transferrin receptor 1 (TfR1). Also provided are pharmaceutical compositions comprising said bicyclic peptide ligand complexes and the use of said bicyclic peptide ligand complexes and pharmaceutical compositions in preventing, suppressing or treating a disease or disorder through TfR1 mediated delivery of a therapeutic agent.

Description

[0001] BICYCLIC PEPTIDE LIGAND COMPLEXES SPECIFIC FOR TFR1

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to bicyclic peptide ligand complexes, such as bicyclic peptide ligand complexes, specific for 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 preventing, suppressing or treating a disease or disorder through TfR1 mediated delivery of a therapeutic agent.

[0004] BACKGROUND OF THE INVENTION

[0005] Transferrins are glycoproteins found in vertebrates which bind to and consequently mediate the transport of Iron (Fe) through blood plasma. Transferrins are produced in the liver and contain binding sites for two Fe3+atoms. Human transferrin is encoded by the TF gene and produced as a 76 kDa glycoprotein.

[0006] Transferrin glycoproteins bind iron tightly, but reversibly. Although iron bound to transferrin is less than 0.1% (4 mg) of total body iron, it forms the most vital iron pool with the highest rate of turnover (25 mg / 24 h). Transferrin has a molecular weight of around 80 kDa and contains two specific high-affinity Fe(lll) binding sites. The affinity of transferrin for Fe(lll) is extremely high (association constant is 1020M"1at pH 7.4) but decreases progressively with decreasing pH below neutrality. Transferrins are not limited to only binding to iron but also bind to different metal ions. These glycoproteins are located in various bodily fluids of vertebrates. When not bound to iron or any other metal atom, transferrin is known as "apotransferrin".

[0007] Transferrin receptor protein 1 (TfR1), also known as CD71 , is a protein that in humans is encoded by the TFRC gene. TfR1 is required for iron import from transferrin into cells by endocytosis. TfR1 is a transmembrane glycoprotein composed of two disulfide-linked monomers joined by two disulfide bonds. Each monomer binds one holo-transferrin molecule creating an iron-Tf-TfR complex which enters the cell by endocytosis.

[0008] TfR1 is a potential new target in cases of human leukemia and lymphoma. TfR1 expressed on the endothelial cells of the blood-brain barrier has been used in preclinical research to allow the delivery of large molecules including antibodies into the brain. Without being bound by theory it is believed that such delivery is achieved via transcytotic transport over endothelial cells of the blood-brain barrier.

[0009] SUBSTITUTE SHEET (RULE 26) There is thus a need to provide alternative complexes which are specific for Tfr1 in order to facilitate optimal delivery to the brain.

[0010] SUMMARY OF THE INVENTION

[0011] According to a first aspect of the invention, there is provided a bicyclic peptide ligand complex which comprises:

[0012] (a) one or more bicyclic peptide ligands specific for transferrin receptor 1 (TfR1) comprising a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold; and

[0013] (b) a half-life extending (HLE) moiety.

[0014] According to a further aspect of the invention, there is provided a polypeptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence as defined herein. According to a further aspect of the invention, there is provided a peptide ligand comprising a polypeptide as described herein, or a pharmaceutically acceptable salt thereof, attached to a molecular scaffold as described herein.

[0015] According to a yet further aspect of the invention, there is provided a pharmaceutical composition comprising the bicyclic peptide ligand or bicyclic peptide ligand complex as defined herein in combination with one or more pharmaceutically acceptable excipients.

[0016] According to a further aspect of the invention, there is provided a bicyclic peptide ligand, peptide ligand complex or pharmaceutical composition as defined herein for use in preventing, suppressing or treating a disease or disorder through TfR1 mediated delivery of a therapeutic agent.

[0017] BRIEF DESCRIPTION OF THE FIGURES

[0018] Figure 1 : Mean plasma concentration time profile for B191 after dosing 2.69 mg / kg by iv infusion for 15 min.

[0019] Figure 2: Individual and mean plasma concentration time profiles for B192 after dosing 3.45 mg / kg by iv infusion for 15 min.

[0020] Figure 3: Individual and mean plasma concentration time profiles for B193 after dosing 0.28 mg / kg by iv infusion for 15 min. DETAILED DESCRIPTION OF THE INVENTION

[0021] Definitions

[0022] Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., 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), for definitions and terms of the art.

[0023] All publications, patents and patent applications cited herein are incorporated by reference in their entirety.

[0024] The term “amino acid” in the context of the present disclosure is used in its broadest sense and is meant to include organic compounds containing amine (NH2) and carboxyl (COOH) functional groups, along with a side chain (e.g., a R group) specific to each amino acid. In some embodiments, the amino acids refer to naturally occurring L a-amino acids or residues. The commonly used one 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; l=lle; 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, A. L., (1975) Biochemistry, 2d ed., pp. 71-92, Worth Publishers, New York). The general term “amino acid” further includes D-amino acids, retro-inverso amino acids as well as chemically modified amino acids such as amino acid analogues, naturally occurring amino acids that are not usually incorporated into proteins such as norleucine, and chemically synthesised compounds having properties known in the art to be characteristic of an amino acid, such as p-amino acids. For example, analogues or mimetics of phenylalanine or proline, which allow the same conformational restriction of the peptide compounds as do natural Phe or Pro, are included within the definition of amino acid. Such analogues and mimetics are referred to herein as "functional equivalents" of the respective amino acid. Other examples of amino acids are listed by Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., N.Y. 1983, which is incorporated herein by reference. The chemical properties of the 20 main amino acids are provided in Table A below. Table A - Chemical properties of amino acids

[0025] The terms “polypeptide”, and “peptide” are interchangeably used herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. Polypeptides can also undergo maturation or post-translational modification processes that may include, but are not limited to: glycosylation, proteolytic cleavage, lipidization, signal peptide cleavage, propeptide cleavage, phosphorylation, and such like.

[0026] As used herein, Abu represents aminobutyric acid, Aib represents aminoisobutyric acid, Aze represents azetidine, B-Melle represents beta-methyl isoleucine, C5g represents cyclopentyl glycine, Cba represents p-cyclobutylalanine, Cbg represents cyclobutyl glycine, Chg represents cyclohexyl glycine, Cpg represents cyclopropryl glycine, EPA represents 2- amino-3-ethyl-pentanoic acid, HyP represents trans-4-hydroxy-L-proline, [K(Na)] represents 6-azido lysine, 1 Nal represents 1-naphthylalanine, 2Nal represents 2-naphthylalanine, 4Pal represents 4-pyridylalanine, tBuAla represents t-butyl-alanine, tBuGly represents t-butyl- glycine, 3tBuTyr represents 3-t-Butyl-Tyrosine; AzPro represents azidopropyl, Aze represents azetidine, 1 Nal represents 1-naphthylalanine, NMeTrp represents N-methyl- tryptophan, K(PYA) represents lysine, s-4-pcntynoyl. Peg represents polyethylene glycol, Pip represents pipecolic acid, Sar represents sarcosine, Fl represents fluorescein and [K(N3)(PYA-Maleimide)] represents a modified lysine having

[0027] Peptide ligand complexes

[0028] The present invention provides complexes comprising a polypeptide which is capable of binding to TfR1, and a half-life extending moiety. Half-life extending moieties are described in more detail herein.

[0029] A peptide capable of binding to TfR1 as provided herein may be comprised, for example, in a peptide ligand comprising said polypeptide covalently bound to a molecular scaffold (e.g. a molecular scaffold as described in more detail herein) such that two or more polypeptide loops are formed on the molecular scaffold. A peptide or peptide ligand may be attached directly or indirectly (e.g. via a linker) to one or more effector groups and / or functional groups, such as one or more cytotoxic agents, radiochelators, or chromophores.

[0030] According to a first aspect of the invention, there is provided a bicyclic peptide ligand complex which comprises:

[0031] (a) one or more bicyclic peptide ligands specific for transferrin receptor 1 (TfR1) comprising a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold; and

[0032] (b) a half-life extending (HLE) moiety.

[0033] It will be appreciated that the term “specific for TfR1” refers to the ability of the peptide ligand to bind to transferrin receptor 1 (TfR1). It will also be appreciated that the peptide ligand will have a differing affect upon TfR1 depending on the precise epitope of binding. For example, the affect will either be inhibitory (i.e. the peptide ligand impedes / inhibits the binding of transferrin to TfR1) or non-inhibitory (i.e. the peptide ligand does not impede / inhibit the binding of transferrin to TfR1.

[0034] Bicyclic Peptide Ligands

[0035] It will be appreciated that the present invention relates to both “monomeric” bicyclic peptides, i.e. those which contain a single (monomeric) bicyclic peptide ligand and “multimeric” bicyclic peptides, i.e. those which contain more than one bicyclic peptide ligand (such as 2, 3 or 4 bicyclic peptide ligands) conjugated via one or more linkers. Monomeric Bicyclic Peptide Ligands

[0036] In one embodiment, the bicyclic peptide ligand complex comprises a single (monomeric) bicyclic peptide ligand.

[0037] A bicyclic peptide ligand for use in the invention typically comprises a polypeptide of 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 natural or non-natural amino acids, and X2 represents a second chain of natural or non-natural amino acids. Typically, when the polypeptide is bound to a molecular scaffold at the three reactive groups to form a peptide ligand, X1 joins with said RG1 and RG2 groups to form a first polypeptide loop, and X2 joins with said RG2 and RG3 groups to form a second polypeptide loop. Accordingly, in some embodiments X1 defines a first loop sequence and X2 defines a second loop sequence.

[0038] The reactive groups RGi, RG2 and RG3 provide attachment points to a molecular scaffold, such as a molecular scaffold as defined herein; and typically comprise functional groups found within the side chains of particular amino acids found in the peptide. Such reactive groups may be e.g. cysteine, homocysteine (hCys, (S)-2-Amino-4-sulfanylbutanoic acid), PGys ((R)-3-amino-3-mercaptopropanoic acid), penicillamine (Pen, (R)-2-amino-3-mercapto- 3-methylbutanoic acid), Dap ((S)-2,3-diaminopropanoic acid), N-alkyl-Dap (e.g. N-methyl- Dap, (S)-2-amino-3-(methylamino)propanoic acid, or lysine, or any other suitable reactive group. The reactive groups RG1, RG2 and RG3 may be the same or different. In one embodiment, said reactive groups comprise cysteine residues.

[0039] Inhibitory Peptide Ligands

[0040] In one embodiment, the peptide ligand is specific for TfR1 and binds to TfR1 in a manner which impedes / inhibits the binding of transferrin to TfR1.

[0041] In a further embodiment, said loop sequences each comprise 2, 3, 4, 5, 6, 7, 8 or 9 amino acids. In a further embodiment, said loop sequences comprise 2, 3, 6, 8 or 9 amino acids. In some embodiments the bicycle peptide ligand comprises a first loop sequence comprising

[0042] 2, 3, 4, 5, 6, 7, 8 or 9 amino acids and a second loop sequence comprising 2, 3, 4, 5, 6, 7, 8 or 9 amino acids. In some embodiments the bicycle peptide ligand comprises a first loop sequence comprising 2, 3, 6, 8 or 9 amino acids and a second loop sequence comprising 2,

[0043] 3, 6, 8 or 9 amino acids. In some embodiments the length of the polypeptide comprising the first and second loop sequences and the reactive groups is from about 12 to about 16 amino acids, e.g. 13, 14 or 15 amino acids.

[0044] In one embodiment, said polypeptide comprises three reactive groups, e.g. three cysteine residues separated by two loop sequences the first of which consists of 2 amino acids and the second of which consists of 9 amino acids.

[0045] In one embodiment, said polypeptide comprises three reactive groups, e.g. three cysteine residues separated by two loop sequences both of which consist of 6 amino acids.

[0046] In one embodiment, said polypeptide comprises three reactive groups, e.g. three cysteine residues separated by two loop sequences the first of which consists of 3 amino acids and the second of which consists of 8 amino acids.

[0047] In one embodiment of such peptide ligands, a loop sequence contains 2 amino acids. In some embodiments when the loop sequence contains 2 amino acids, the loop sequence contains a moiety of formula -AA1-AA2-. In some embodiments AA1 and AA2 are each independently non-polar 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 moiety of formula -AA1-AA2- is -AL-.

[0048] In some embodiments of such peptide ligands, a loop sequence contains 3 amino acids. In some embodiments when the loop sequence contains 3 amino acids, the loop sequence contains a moiety of formula -AA1-AA2-AA3-. In some embodiments AA1, AA2 and AA3 are each independently non-polar amino acids and / or charged amino acids. In some embodiments each of 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 moiety of formula -AA1-AA2-AA3- is -AEL-.

[0049] In some embodiments of such peptide ligands, a loop sequence contains 6 amino acids. In some embodiments when the loop sequence contains 6 amino acids, the loop sequence contains a moiety of formula -AA1-AA2-AA3-AA4-AA5-AA6-. In some embodiments each amino acid in said 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 said loop sequence is independently selected from R, E, F, D, T, G, L, A and I. In some embodiments each amino acid in said loop sequence is independently selected from R, K, E, F, D, T and S. In some embodiments each amino acid in said loop sequence is independently selected from R, E, F, D and T. In some embodiments each amino acid in said loop sequence is independently selected from G, L, A, V, F, I, E and D. In some embodiments each amino acid in said loop sequence is independently selected from G, L, A, F, I and E. In some embodiments the moiety of formula -AA1-AA2-AA3-AA4-AA5-AA6- is selected from -REFFDT- and -GLAFIE-.

[0050] In some embodiments of such peptide ligands, a loop sequence contains 8 amino acids. In some embodiments when the loop sequence contains 8 amino acids, the loop sequence contains a moiety of formula -AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-. In some embodiments each amino acid in said 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 said loop sequence is independently selected from Y, D, G, V, W, and S. In some embodiments the moiety of formula -AA1-AA2- AA3-AA4-AA5-AA6-AA7-AA8- is -YDGVYWYS-.

[0051] In some embodiments of such peptide ligands, a loop sequence contains 9 amino acids. In some embodiments when the loop sequence contains 9 amino acids, the loop sequence contains a moiety of formula -AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-. In some embodiments each amino acid in said 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 said loop sequence is independently selected from N, D, W, T, L, P, W, and H. In some embodiments the moiety of formula -AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9- is -NDWTLPWH H-

[0052] In some embodiments of such peptide ligands, a loop sequence contains 4 amino acids. In some embodiments when a loop sequence contains 4 amino acids, the loop sequence corresponds to a loop sequence containing 2 or 3 amino acids, and contains 2 or 1 additional amino acids (respectively) which may be inserted anywhere within said amino acid loop sequence. In some embodiments the loop sequence corresponds to a loop sequence containing 6, 8 or 9 amino acids, from which 2, 4 or 5 amino acids (respectively) have been removed.

[0053] In some embodiments of such peptide ligands, a loop sequence contains 5 amino acids. In some embodiments when a loop sequence contains 5 amino acids, the loop sequence corresponds to a loop sequence containing 2 or 3 amino acids, and contains 3 or 2 additional amino acids (respectively) which may be inserted anywhere within said amino acid loop sequence. In some embodiments the loop sequence corresponds to a loop sequence containing 6, 8 or 9 amino acids, from which 1 , 3 or 4 amino acids (respectively) have been removed.

[0054] In some embodiments of such peptide ligands, a loop sequence contains 7 amino acids. In some embodiments when a loop sequence contains 7 amino acids, the loop sequence corresponds to a loop sequence containing 2, 3 or 6 amino acids, and contains 5, 4 or 1 additional amino acids (respectively) which may be inserted anywhere within said amino acid loop sequence. In some embodiments the loop sequence corresponds to a loop sequence containing 8 or 9 amino acids, from which 1 or 2 amino acids (respectively) have been removed.

[0055] In one embodiment, the peptide ligand comprises a polypeptide having an amino acid sequence selected from:

[0056] RG1-AL-RG2-NDWTLPWHH-RG3;

[0057] RG1-REFFDT-RG2-GLAFIE-RG3; and RG1-LEA-RG2-YDGVYWYS-RG3 or a variant thereof as described herein; wherein RGi, RG2 and RG3 are as defined herein; or a pharmaceutically acceptable salt thereof.

[0058] In one embodiment, the peptide ligand comprises an polypeptide having an amino acid sequence of:

[0059] CALCN DWTLPWH HC (SEQ ID NO: 1);

[0060] CREFFDTCGLAFIEC (SEQ ID NO: 2); and CLEACYDGVYWYSC (SEQ ID NO: 3); or a variant thereof as described herein, or a pharmaceutically acceptable salt thereof.

[0061] In one embodiment, the peptide ligand comprises an amino acid sequence of:

[0062] CALCN DWTLPWH HC (SEQ ID NO: 1);

[0063] CREFFDTCGLAFIEC (SEQ ID NO: 2); and CLEACYDGVYWYSC (SEQ ID NO: 3); or a pharmaceutically acceptable salt thereof.

[0064] In one embodiment the peptide ligand comprises a polypeptide as described herein, wherein the polypeptide comprises N- and / or C-terminal additions as described in more detail herein. In some embodiments the N-terminal additions comprise an N-terminal alanine. In some embodiments an inhibitory peptide ligand comprises a polypeptide as described herein attached (e.g. by being covalently bonded) to a molecular scaffold as described herein. In some embodiments the polypeptide is attached (e.g. covalently bonded) to the molecular scaffold by bonds between the molecular scaffold and each of RGi, RG2 and RG3. When RG1, RG2 and RG3 are cysteine residues, the polypeptide is typically bonded to the molecular scaffold by covalent bonds between the thiol groups of said cysteine residues and the scaffold. In some embodiments the scaffold is 1 , 1 1 "-(1 ,3,5-triazinane-1 ,3,5-triyl)triprop- 2-en-1-one (TATA) .

[0065] In a further 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 N- and / or C-terminal additions and is selected from:

[0066] A-(SEQ ID NO: 1)-A (herein referred to as B1);

[0067] A-(SEQ ID NO: 1)-A-[Sar6]-[K-FI] (herein referred to as B2);

[0068] A-(SEQ ID NO: 2)-A (herein referred to as B3);

[0069] A-(SEQ ID NO: 2)-A-[Sar6]-[K-FI] (herein referred to as B4);

[0070] A-(SEQ ID NO: 3)-A (herein referred to as B5); and A-(SEQ ID NO: 3)-A-[Sar6]-[K-FI] (herein referred to as B6). wherein Sar represents sarcosine and Fl represents fluorescein; i.e. [K-FI] represents lysine with fluorescein attached at the 6-position.

[0071] For the purpose of this description, inhibitory bicyclic peptides are assumed to be cyclised with TATA and yielding a tri-substituted structure. However, as will be clear from the descriptions of the invention presented herein, cyclisation may be performed with any suitable molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed. Cyclisation occurs on the first, second and third reactive groups (RG1, RG2 and RG3), respectively. When the reactive groups are cysteines, cyclisation occurs on the first, second and third cysteine residues, respectively.

[0072] Non-lnhibitory Peptide Ligands

[0073] In one embodiment, the peptide ligand is specific for TfRIand binds to TfRIin a manner which does not inhibit / impede the binding of transferrin to TfR1.

[0074] In one embodiment, the peptide ligand comprises a polypeptide comprising two loop sequences each comprising 3, 4, 5, 6, or 7 amino acids. In one embodiment, the peptide ligand comprises a polypeptide comprising two loop sequences each comprising 3, 4, 6, or 7 amino acids. In one embodiment the bicycle peptide ligand comprises a polypeptide comprising a loop sequence comprising about 7 amino acids and a loop sequence comprising about 3 amino acids. In one embodiment the bicycle peptide ligand comprises a polypeptide comprising a loop sequence comprising about 6 amino acids and a loop sequence comprising about 4 amino acids. In some embodiments the length of the polypeptide comprising the first and second loop sequences and the reactive groups is from about 12 to about 16 amino acids, e.g. 13, 14 or 15 amino acids.

[0075] In a further embodiment, said loop sequences comprise 3 or 7 amino acids.

[0076] In one embodiment, said polypeptide comprises three reactive groups, e.g. three cysteine residues separated by two loop sequences the first of which consists of 7 amino acids and the second of which consists of 3 amino acids. In one embodiment, said polypeptide comprises three reactive groups, e.g. three cysteine residues separated by two loop sequences the first of which consists of 6 amino acids and the second of which consists of 4 amino acids.

[0077] In some embodiments a loop sequence contains 3 amino acids. In some embodiments a loop sequence containing 3 amino acids has an amino acid sequence -AA1-AA2-AA3-. In some embodiments each of AA1 to AA3 is any amino acid, such as any natural amino acid or unnatural amino acid. In some embodiments AA1 is selected from [tBuGly], [EPA], [Chg], [tBuAla], [C5g], [Cbg], [Cpg], [B-Melle], I, A, Y and [3HyV], In some embodiments AA1 is selected from [tBuGly], [EPA], [Chg], [tBuAla], [C5g], [Cbg], [Cpg], [B-Melle], 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 an unnatural amino acid. In some embodiments AA3 is W, Y, [2Nal], [3tBuTyr], [1 Nal], [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 the moiety -AA1-AA2-AA3- is -l / A / D / [tBuGly]-S / A / E-W / Y / A-. In some embodiments the moiety - AA1-AA2-AA3- is -l / [tBuGly]-S-Y-. In some embodiments the moiety -AA1-AA2-AA3- is -l-S- Y-. In some embodiments the moiety -AA1-AA2-AA3- is -[tBuGly]-S-Y-.

[0078] In some embodiments of such peptide ligands, a loop sequence contains 4 amino acids. In some embodiments when a loop sequence contains 4 amino acids, the loop sequence corresponds to a loop sequence containing 3 amino acids, and contains 1 additional amino acids which may be inserted anywhere within said amino acid loop sequence. In some embodiments a loop sequence containing 4 amino acids has an amino acid sequence -AAi- AA2-AA3-AA4-. In some embodiments each of AA1 to AA4 is any amino acid, e.g. any natural or unnatural amino acid. In some embodiments each of AA1 to AA4 is independently selected from G, D, E, W, Y, [2Nal], [3tBuTyr], [1 Nal], [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], [1 Nal], [4Pal], or A. In some embodiments AA4 is W, Y, or A. In some embodiments AA4 is Y. In some embodiments the moiety -AA1-AA2-AA3-AA4- is -G-D-E- W / Y / A-. In some embodiments the moiety -AA1-AA2-AA3-AA4- is -GDEY-.

[0079] In some embodiments a loop sequence contains 7 amino acids. In some embodiments a loop sequence containing 7 amino acids has an 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)J, E, [Aib], [Cba], [Gia], [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 [Gia], 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, As is selected from W, Y, H, A, [DOPA], [hTyr], [pCaPhe] and [pCoPhe], Typically, As is H or Y. In one embodiment As is H. In one embodiment As is Y. In some embodiments, As is selected from L, Q, [tBuAla], [Cba], [Abu], [Aib], A, S, T, D, E and N. Typically, As 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 moiety -AA1-AA2-AA3-AA4-AA5- AA6-AA7- has the sequence -AA1-AA2-D-A-H / Y-L-G-. In some embodiments the moiety -AA1- AA2-AA3-AA4-AA5-AA6-AA7- has the sequence -S / P / A / [K(N3)] / [HyP] / [Oxa] / [Cis-HyP]-AA2-D- A-H / Y-L-G-. In some embodiments the moiety -AA1-AA2-AA3-AA4-AA5-AA6-AA7- has the sequence -AAi-A / S / P / Q / G / [HyP] / [Aib] / N / l / [Aze] / [dA] / [K(N3)] / [Oxa] / [Cis-HyP]-D-A-H / Y-L-G-.

[0080] In some embodiments the moiety -AA1-AA2-AA3-AA4-AA5-AA6-AA7- has the sequence -S / P-P / [HyP]-D-A-H / Y-L-G-. In some embodiments the moiety -AA1-AA2-AA3-AA4-AA5-AA6- AA7- has the sequence -SPDAHLG-. In some embodiments the moiety -AA1-AA2-AA3-AA4- AA5-AA6-AA7- has the sequence -P[HyP]DAHLG-. In some embodiments the moiety -AA1- AA2-AA3-AA4-AA5-AA6-AA7- has the sequence -SPDAYLG-. In some embodiments the moiety -AA1-AA2-AA3-AA4-AA5-AA6-AA7- has the sequence -P[HyP]DAYLG-.

[0081] In some embodiments of such peptide ligands, a loop sequence contains 6 amino acids. In some embodiments when a loop sequence contains 6 amino acids, the loop sequence corresponds to a loop sequence containing 7 amino acids, wherein one amino acid is deleted from said sequence. In some embodiments a loop sequence containing 6 amino acids has an amino acid sequence -AA1-AA2-AA3-AA4-AA5-AA6-.

[0082] In some embodiments each of AA1 to AAe is any amino acid, e.g. any natural or unnatural amino acid. In some embodiments each of AA1 to AAe 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 AAe is independently selected from Y, L, P, D, W, and [tBuAla],

[0083] 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 often 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 AAe is [tBuAla], In some embodiments the moiety -AA1-AA2-AA3-AA4-AA5-AA6- is -YLPDW- [tBuAla]-.

[0084] In some embodiments, the first loop sequence contains 6 or 7 amino acids and is selected from:

[0085] SADDWLG;

[0086] SSDAYLG;

[0087] PPDAHLG;

[0088] PQDAYLG;

[0089] PPDSWQG;

[0090] SPDAHLG;

[0091] PGDAHLG;

[0092] PPDSHLG;

[0093] P[HyP]DAYLG;

[0094] S[HyP]DAHLG;

[0095] P[Aib]DAHLG;

[0096] P[Aib]DAYLG;

[0097] SADAHLG; S[Aib]DAHLG; PPDAYLG; S[Aib]DAYLG; APDAHLG; SPDAYLG; PNDAHLG; PIDAHLG; SPD[Aib]HLG; SPDAH[tBuAla]G; SPDAH[Cba]G;

[0098] SPD[Abu]HLG; S[Aze]DAHLG; SPDDHLG;

[0099] SPDSHLG; SPDAH[Abu]G; P[dA]DAHLG;

[0100] SPDAHL[dA]; SPDAH[Aib]G; SPAAHLG;

[0101] SPDAALG; SPDAHAG; SPDAHLA;

[0102] [K(N3)]PDAHLG; S[K(N3)]DAHLG;

[0103] SPD[K(N3)]HLG; P[HyP]DAHLG;

[0104] [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]

[0105] P[HyP]DAYL[dE]

[0106] P[HyP]DAYL[dN]

[0107] P[HyP]DAYL[dQ]

[0108] P[HyP]DAYL[dY]

[0109] P[HyP]DAYLS

[0110] P[HyP]DAYLD

[0111] P[HyP]DAYLY;

[0112] P[HyP]DAYLN;

[0113] P[HyP]EAYLG;

[0114] P[HyP][Gla]AYLG;

[0115] P[HyP]DAYSG;

[0116] P[HyP]DAYTG;

[0117] P[HyP]DAYDG;

[0118] P[HyP]DAYEG;

[0119] P[HyP]DAYNG;

[0120] P[HyP]DAYQG; and

[0121] YLPDW[tBuAla]; and variants thereof as described herein; and pharmaceutically acceptable salts thereof.

[0122] In some embodiments the second loop sequence contains 3 or 4 amino acids and is selected from

[0123] ISW;

[0124] I SY;

[0125] [tBuGly]SY;

[0126] [EPA]SY;

[0127] [Chg]SY;

[0128] IS[2Nal];

[0129] IS[3tBuTyr];

[0130] IS[1Nal];

[0131] IS[4Pal];

[0132] [tBuAla]SY;

[0133] [C5g]SY;

[0134] [Cbg]SY;

[0135] [Cpg]SY;

[0136] [B-Melle]SY; ASY;

[0137] IAY;

[0138] ISA;

[0139] [tBuGly]S[DOPA];

[0140] [tBuGly]S[pCaPhe];

[0141] [tBuGly]S[pCoPhe];

[0142] [tBuGly]S[hTyr];

[0143] [tBuGly][HSer]Y;

[0144] [tBuGly]TY;

[0145] [tBuGly]DY;

[0146] [tBuGly]EY;

[0147] [tBuGly]NY;

[0148] [tBuGly]QY;

[0149] YSY;

[0150] [3HyV]SY; and

[0151] GDEY; and variants thereof as described herein; and pharmaceutically acceptable salts thereof.

[0152] In some embodiments the peptide ligand comprises a polypeptide having an amino acid sequence selected from:

[0153] RG1-SADDWLG-RG2-ISW-RG3;

[0154] RG1-SSDAYLG-RG2-ISW-RG3;

[0155] RG1-PPDAHLG-RG2-ISW-RG3;

[0156] RG1-PQDAYLG-RG2-ISW-RG3;

[0157] RG1-PPDSWQG-RG2-ISY-RG3;

[0158] RG1-SPDAHLG-RG2-ISY-RG3;

[0159] RG1-PGDAHLG-RG2-ISY-RG3;

[0160] RG1-PPDSHLG-RG2-ISY-RG3;

[0161] RG1-SADDWLG-RG2-ISY-RG3;

[0162] RGi-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3;

[0163] RGi-P[HyP]DAYLG-RG2-ISY-RG3;

[0164] RGi-S[HyP]DAHLG-RG2-ISY-RG3;

[0165] RGi-P[Aib]DAHLG-RG2-[tBuGly]SY-RG3;

[0166] RG1-PPDAHLG-RG2-ISY-RG3;

[0167] RGi-P[Aib]DAYLG-RG2-[tBuGly]SY-RG3;

[0168] RG1-SADAHLG-RG2-ISY-RG3; RGi-S[Aib]DAHLG-RG2-[tBuGly]SY-RG3;

[0169] RGI-SPDAHLG-RG2-[EPA]SY-RG3;

[0170] RGi-PPDAYLG-RG2-[tBuGly]SY-RG3;

[0171] RGi-S[Aib]DAYLG-RG2-[tBuGly]SY-RG3;

[0172] RGI-APDAHLG-RG2-ISY-RG3;

[0173] RGi-P[Aib]DAHLG-RG2-ISY-RG3;

[0174] RGi-SPDAYLG-RG2-[tBuGly]SY-RG3;

[0175] RGi-SPDAHLG-RG2-[tBuGly]SY-RG3;

[0176] RGI-PNDAHLG-RG2-ISY-RG3;

[0177] RGI-PIDAHLG-RG2-ISY-RG3;

[0178] RGI-SPDAYLG-RG2-ISY-RG3;

[0179] RGI-PPDAYLG-RG2-ISY-RG3;

[0180] RGi-S[Aib]DAHLG-RG2-ISY-RG3;

[0181] RGi-SPDAHLG-RG2-[Chg]SY-RG3;

[0182] RGI-APDAHLG-RG2-ISY-RG3;

[0183] RGi-SPDAHLG-RG2-IS[2Nal]-RG3;

[0184] RGi-SPDAHLG-RG2-IS[3tBuTyr]-RG3;

[0185] RGi-SPD[Aib]HLG-RG2-ISY-RG3;

[0186] RGI-SPDAHLG-RG2-IS[1 Nal]-RG3;

[0187] RGi-SPDAH[tBuAla]G-RG2-ISY-RG3;

[0188] RGi-SPDAH[Cba]G-RG2-ISY-RG3;

[0189] RGI-SPDAHLG-RG2-ISW-RG3;

[0190] RGi-SPD[Abu]HLG-RG2-ISY-RG3;

[0191] RGi-S[Aze]DAHLG-RG2-ISY-RG3;

[0192] RGI-SPDDHLG-RG2-ISY-RG3;

[0193] RGI-SPDSHLG-RG2-ISY-RG3;

[0194] RGi-SPDAH[Abu]G-RG2-ISY-RG3;

[0195] RGi-SPDAHLG-RG2-IS[4Pal]-RG3;

[0196] RGi-P[dA]DAHLG-RG2-ISY-RG3;

[0197] RGi-SPDAYLG-RG2-[tBuAla]SY-RG3;

[0198] RGi-SPDAHLG-RG2-[C5g]SY-RG3;

[0199] RGi-SPDAHLG-RG2-[Cbg]SY-RG3;

[0200] RGi-SPDAHL[dA]-RG2-ISY-RG3;

[0201] RGi-SPDAH[Aib]G-RG2-ISY-RG3;

[0202] RGi-SPDAHLG-RG2-[Cpg]SY-RG3;

[0203] RGi-SPDAHLG-RG2-[B-Melle]SY-RG3;

[0204] RGI-SADAHLG-RG2-ISY-RG3; RGI-SPAAHLG-RG2-ISY-RG3;

[0205] RG1-SPDAALG-RG2-ISY-RG3;

[0206] RG1-SPDAHAG-RG2-ISY-RG3;

[0207] RG1-SPDAHLA-RG2-ISY-RG3;

[0208] RG1-SPDAHLG-RG2-ASY-RG3;

[0209] RG1-SPDAHLG-RG2-IAY-RG3;

[0210] RG1-SPDAHLG-RG2-ISA-RG3;

[0211] RGI-[K(N3)]PDAHLG-RG2-ISY-RG3;

[0212] RGI-S[K(N3)]DAHLG-RG2-ISY-RG3;

[0213] RGI-SPD[K(N3)]HLG-RG2-ISY-RG3;

[0214] RGi-P[HyP]DAHLG-RG2-[tBuGly]SY-RG3;

[0215] RG1-[HyP][HyP]DAYLG-RG2-[tBuGly]SY-RG3-EPW;

[0216] RG1-[Oxa][HyP]DAYLG-RG2-[tBuGly]SY-RG3-EPW;

[0217] RG1-[Cis-HyP][HyP]DAYLG-RG2-[tBuGly]SY-RG3-EPW;

[0218] RG1-P[Oxa]DAYLG-RG2-[tBuGly]SY-RG3-EPW;

[0219] RG1-P[Cis-HyP]DAYLG-RG2-[tBuGly]SY-RG3-EPW;

[0220] RG1-P[HyP]DA[DOPA]LG-RG2-[tBuGly]SY-RG3-EPW;

[0221] RG1-P[HyP]DA[hTyr]LG-RG2-[tBuGly]SY-RG3-EPW;

[0222] RG1-P[HyP]DA[pCaPhe]LG-RG2-[tBuGly]SY-RG3-EPW;

[0223] RG1-P[HyP]DA[pCoPhe]LG-RG2-[tBuGly]SY-RG3-EPW;

[0224] RG1-P[HyP]DAYL[dS]-RG2-[tBuGly]SY-RG3-EPW;

[0225] RG1-P[HyP]DAYL[dT]-RG2-[tBuGly]SY-RG3-EPW;

[0226] RG1-P[HyP]DAYL[dD]-RG2-[tBuGly]SY-RG3-EPW;

[0227] RG1-P[HyP]DAYL[dE]-RG2-[tBuGly]SY-RG3-EPW;

[0228] RG1-P[HyP]DAYL[dN]-RG2-[tBuGly]SY-RG3-EPW;

[0229] RG1-P[HyP]DAYL[dQ]-RG2-[tBuGly]SY-RG3-EPW;

[0230] RG1-P[HyP]DAYL[dY]-RG2-[tBuGly]SY-RG3-EPW;

[0231] RG1-P[HyP]DAYLS-RG2-[tBuGly]SY-RG3-EPW;

[0232] RG1-P[HyP]DAYLD-RG2-[tBuGly]SY-RG3-EPW;

[0233] RG1-P[HyP]DAYLY-RG2-[tBuGly]SY-RG3-EPW;

[0234] RG1-P[HyP]DAYLN-RG2-[tBuGly]SY-RG3-EPW;

[0235] RG1-P[HyP]DAYLG-RG2-[tBuGly]S[DOPA]-RG3-EPW;

[0236] RG1-P[HyP]DAYLG-RG2-[tBuGly]S[pCaPhe]-RG3-EPW;

[0237] RG1-P[HyP]DAYLG-RG2-[tBuGly]S[pCoPhe]-RG3-EPW;

[0238] RG1-P[HyP]DAYLG-RG2-[tBuGly]S[hTyr]-RG3-EPW;

[0239] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-E[HyP]W;

[0240] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-E[Oxa]W; RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-E[Cis-HyP]W;

[0241] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-EPY;

[0242] RG1-P[HyP]EAYLG-RG2-[tBuGly]SY-RG3-EPW;

[0243] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-EP[DOPA];

[0244] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-EP[pCaPhe];

[0245] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-EP[pCoPhe];

[0246] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-EP[hTyr];

[0247] RG1-P[HyP][Gla]AYLG-RG2-[tBuGly]SY-RG3-EPW;

[0248] RG1-P[HyP]DAYSG-RG2-[tBuGly]SY-RG3-EPW;

[0249] RG1-P[HyP]DAYTG-RG2-[tBuGly]SY-RG3-EPW;

[0250] RG1-P[HyP]DAYDG-RG2-[tBuGly]SY-RG3-EPW;

[0251] RG1-P[HyP]DAYEG-RG2-[tBuGly]SY-RG3-EPW;

[0252] RG1-P[HyP]DAYNG-RG2-[tBuGly]SY-RG3-EPW;

[0253] RG1-P[HyP]DAYQG-RG2-[tBuGly]SY-RG3-EPW;

[0254] RG1-P[HyP]DAYLG-RG2-[tBuGly][HSer]Y-RG3-EPW;

[0255] RG1-P[HyP]DAYLG-RG2-[tBuGly]TY-RG3-EPW;

[0256] RG1-P[HyP]DAYLG-RG2-[tBuGly]DY-RG3-EPW;

[0257] RG1-P[HyP]DAYLG-RG2-[tBuGly]EY-RG3-EPW;

[0258] RG1-P[HyP]DAYLG-RG2-[tBuGly]NY-RG3-EPW;

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

[0260] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-DPW;

[0261] RG1-P[HyP]DAYLG-RG2-[tBuGly]SY-RG3-[Gla]PW;

[0262] RG1-P[HyP]DAYLG-RG2-YSY-RG3-EPW;

[0263] RG1-P[HyP]DAYLG-RG2-[3HyV]SY-RG3-EPW; and RGi-YLPDW[tBuAla]-RG2-GDEY-RG3; or a variant thereof as described herein; wherein RGi, RG2and RG3are as defined herein; or a pharmaceutically acceptable salt thereof.

[0264] In one embodiment, the peptide ligand comprises an polypeptide having an amino acid sequence of:

[0265] CSADDWLGCISWC (SEQ ID NO: 4);

[0266] CSSDAYLGCISWC (SEQ ID NO: 5);

[0267] CPPDAHLGCISWC (SEQ ID NO: 6);

[0268] CPQDAYLGCISWC (SEQ ID NO: 7);

[0269] CPPDSWQGCISYC (SEQ ID NO: 8);

[0270] CSPDAHLGCISYC (SEQ ID NO: 9) (herein referred to as B7);

[0271] CPGDAHLGCISYC (SEQ ID NO: 10); CPPDSHLGCISYC (SEQ ID NO: 11);

[0272] CSADDWLGCISYC (SEQ ID NO: 12);

[0273] CP[HyP]DAYLGC[tBuGly]SYC (SEQ ID NO: 13);

[0274] CP[HyP]DAYLGCISYC (SEQ ID NO: 14);

[0275] CS[HyP]DAHLGCISYC (SEQ ID NO: 15);

[0276] CP[Aib]DAHLGC[tBuGly]SYC (SEQ ID NO: 16);

[0277] CPPDAHLGCISYC (SEQ ID NO: 17);

[0278] CP[Aib]DAYLGC[tBuGly]SYC (SEQ ID NO: 18);

[0279] CSADAHLGCISYC (SEQ ID NO: 19);

[0280] CS[Aib]DAHLGC[tBuGly]SYC (SEQ ID NO: 20);

[0281] CSPDAHLGC[EPA]SYC (SEQ ID NO: 21);

[0282] CPPDAYLGC[tBuGly]SYC (SEQ ID NO: 22);

[0283] CS[Aib]DAYLGC[tBuGly]SYC (SEQ ID NO: 23);

[0284] CAPDAHLGCISYC (SEQ ID NO: 24);

[0285] CP[Aib]DAHLGCISYC (SEQ ID NO: 25);

[0286] CSPDAYLGC[tBuGly]SYC (SEQ ID NO: 26);

[0287] CSPDAHLGC[tBuGly]SYC (SEQ ID NO: 27);

[0288] CPNDAHLGCISYC (SEQ ID NO: 28);

[0289] CPIDAHLGCISYC (SEQ ID NO: 29);

[0290] CSPDAYLGCISYC (SEQ ID NO: 30);

[0291] CPPDAYLGCISYC (SEQ ID NO: 31);

[0292] CS[Aib]DAHLGCISYC (SEQ ID NO: 32);

[0293] CSPDAHLGC[Chg]SYC (SEQ ID NO: 33);

[0294] CAPDAHLGCISYC (SEQ ID NO: 34);

[0295] CYLPDW[tBuAla]CGDEYC (SEQ ID NO: 35);

[0296] CSPDAHLGCIS[2Nal]C (SEQ ID NO: 36);

[0297] CSPDAHLGCIS[3tBuTyr]C (SEQ ID NO: 37);

[0298] CSPD[Aib]HLGCISYC (SEQ ID NO: 38);

[0299] CSPDAHLGCIS[1 Nal]C (SEQ ID NO: 39);

[0300] CSPDAH[tBuAla]GCISYC (SEQ ID NO: 40);

[0301] CSPDAH[Cba]GCISYC (SEQ ID NO: 41);

[0302] CSPDAHLGCISWC (SEQ ID NO: 42);

[0303] CSPD[Abu]HLGCISYC (SEQ ID NO: 43);

[0304] CS[Aze]DAHLGCISYC (SEQ ID NO: 44);

[0305] CSPDDHLGCISYC (SEQ ID NO: 45);

[0306] CSPDSHLGCISYC (SEQ ID NO: 46);

[0307] CSPDAH[Abu]GCISYC (SEQ ID NO: 47); CSPDAHLGCIS[4Pal]C (SEQ ID NO: 48);

[0308] CP[dA]DAHLGCISYC (SEQ ID NO: 49);

[0309] CSPDAYLGC[tBuAla]SYC (SEQ ID NO: 50);

[0310] CSPDAHLGC[C5g]SYC (SEQ ID NO: 51);

[0311] CSPDAHLGC[Cbg]SYC (SEQ ID NO: 52);

[0312] CSPDAHL[dA]CISYC (SEQ ID NO: 53);

[0313] CSPDAH[Aib]GCISYC (SEQ ID NO: 54);

[0314] CSPDAHLGC[Cpg]SYC (SEQ ID NO: 55);

[0315] CSPDAHLGC[B-Melle]SYC (SEQ ID NO: 56);

[0316] CSADAHLGCISYC (SEQ ID NO: 57);

[0317] CSPAAHLGCISYC (SEQ ID NO: 58);

[0318] CSPDAALGCISYC (SEQ ID NO: 59);

[0319] CSPDAHAGCISYC (SEQ ID NO: 60);

[0320] CSPDAHLACISYC (SEQ ID NO: 61);

[0321] CSPDAHLGCASYC (SEQ ID NO: 62);

[0322] CSPDAHLGCIAYC (SEQ ID NO: 63);

[0323] CSPDAHLGCISAC (SEQ ID NO: 64);

[0324] C[K(N3)]PDAHLGCISYC (SEQ ID NO: 65);

[0325] CS[K(N3)]DAHLGCISYC (SEQ ID NO: 66);

[0326] CSPD[K(N3)]HLGCISYC (SEQ ID NO: 67);

[0327] CP[HyP]DAHLGC[tBuGly]SYC (SEQ ID NO: 68); or a variant thereof as described herein, or a pharmaceutically acceptable salt thereof.

[0328] In one embodiment, the peptide ligand comprises an amino acid sequence of:

[0329] CSADDWLGCISWC (SEQ ID NO: 4);

[0330] CSSDAYLGCISWC (SEQ ID NO: 5);

[0331] CPPDAHLGCISWC (SEQ ID NO: 6);

[0332] CPQDAYLGCISWC (SEQ ID NO: 7);

[0333] CPPDSWQGCISYC (SEQ ID NO: 8);

[0334] CSPDAHLGCISYC (SEQ ID NO: 9) (herein referred to as B7);

[0335] CPGDAHLGCISYC (SEQ ID NO: 10);

[0336] CPPDSHLGCISYC (SEQ ID NO: 11);

[0337] CSADDWLGCISYC (SEQ ID NO: 12);

[0338] CP[HyP]DAYLGC[tBuGly]SYC (SEQ ID NO: 13);

[0339] CP[HyP]DAYLGCISYC (SEQ ID NO: 14);

[0340] CS[HyP]DAHLGCISYC (SEQ ID NO: 15);

[0341] CP[Aib]DAHLGC[tBuGly]SYC (SEQ ID NO: 16); CPPDAHLGCISYC (SEQ ID NO: 17);

[0342] CP[Aib]DAYLGC[tBuGly]SYC (SEQ ID NO: 18);

[0343] CSADAHLGCISYC (SEQ ID NO: 19);

[0344] CS[Aib]DAHLGC[tBuGly]SYC (SEQ ID NO: 20);

[0345] CSPDAHLGC[EPA]SYC (SEQ ID NO: 21);

[0346] CPPDAYLGC[tBuGly]SYC (SEQ ID NO: 22);

[0347] CS[Aib]DAYLGC[tBuGly]SYC (SEQ ID NO: 23);

[0348] CAPDAHLGCISYC (SEQ ID NO: 24);

[0349] CP[Aib]DAHLGCISYC (SEQ ID NO: 25);

[0350] CSPDAYLGC[tBuGly]SYC (SEQ ID NO: 26);

[0351] CSPDAHLGC[tBuGly]SYC (SEQ ID NO: 27);

[0352] CPNDAHLGCISYC (SEQ ID NO: 28);

[0353] CPIDAHLGCISYC (SEQ ID NO: 29);

[0354] CSPDAYLGCISYC (SEQ ID NO: 30);

[0355] CPPDAYLGCISYC (SEQ ID NO: 31);

[0356] CS[Aib]DAHLGCISYC (SEQ ID NO: 32);

[0357] CSPDAHLGC[Chg]SYC (SEQ ID NO: 33);

[0358] CAPDAHLGCISYC (SEQ ID NO: 34);

[0359] CYLPDW[tBuAla]CGDEYC (SEQ ID NO: 35);

[0360] CSPDAHLGCIS[2Nal]C (SEQ ID NO: 36);

[0361] CSPDAHLGCIS[3tBuTyr]C (SEQ ID NO: 37);

[0362] CSPD[Aib]HLGCISYC (SEQ ID NO: 38);

[0363] CSPDAHLGCIS[1 Nal]C (SEQ ID NO: 39);

[0364] CSPDAH[tBuAla]GCISYC (SEQ ID NO: 40);

[0365] CSPDAH[Cba]GCISYC (SEQ ID NO: 41);

[0366] CSPDAHLGCISWC (SEQ ID NO: 42);

[0367] CSPD[Abu]HLGCISYC (SEQ ID NO: 43);

[0368] CS[Aze]DAHLGCISYC (SEQ ID NO: 44);

[0369] CSPDDHLGCISYC (SEQ ID NO: 45);

[0370] CSPDSHLGCISYC (SEQ ID NO: 46);

[0371] CSPDAH[Abu]GCISYC (SEQ ID NO: 47);

[0372] CSPDAHLGCIS[4Pal]C (SEQ ID NO: 48);

[0373] CP[dA]DAHLGCISYC (SEQ ID NO: 49);

[0374] CSPDAYLGC[tBuAla]SYC (SEQ ID NO: 50);

[0375] CSPDAHLGC[C5g]SYC (SEQ ID NO: 51);

[0376] CSPDAHLGC[Cbg]SYC (SEQ ID NO: 52);

[0377] CSPDAHL[dA]CISYC (SEQ ID NO: 53); CSPDAH[Aib]GCISYC (SEQ ID NO: 54);

[0378] CSPDAHLGC[Cpg]SYC (SEQ ID NO: 55);

[0379] CSPDAHLGC[B-Melle]SYC (SEQ ID NO: 56);

[0380] CSADAHLGCISYC (SEQ ID NO: 57);

[0381] CSPAAHLGCISYC (SEQ ID NO: 58);

[0382] CSPDAALGCISYC (SEQ ID NO: 59);

[0383] CSPDAHAGCISYC (SEQ ID NO: 60);

[0384] CSPDAHLACISYC (SEQ ID NO: 61);

[0385] CSPDAHLGCASYC (SEQ ID NO: 62);

[0386] CSPDAHLGCIAYC (SEQ ID NO: 63);

[0387] CSPDAHLGCISAC (SEQ ID NO: 64);

[0388] C[K(N3)]PDAHLGCISYC (SEQ ID NO: 65);

[0389] CS[K(N3)]DAHLGCISYC (SEQ ID NO: 66);

[0390] CSPD[K(N3)]HLGCISYC (SEQ ID NO: 67);

[0391] CP[HyP]DAHLGC[tBuGly]SYC (SEQ ID NO: 68); wherein Abu represents aminobutyric acid, Aib represents aminoisobutyric acid, Aze represents azetidine, B-Melle represents beta-methyl isoleucine, C5g represents cyclopentyl glycine, Cba represents p-cyclobutylalanine, Cbg represents cyclobutyl glycine, Chg represents cyclohexyl glycine, Cpg represents cyclopropryl glycine, EPA represents 2- amino-3-ethyl-pentanoic acid, HyP represents trans-4-hydroxy-L-proline, [K(N3)] represents 6-azido lysine, 1Nal represents 1-naphthylalanine, 2Nal represents 2-naphthylalanine, 4Pal represents 4-pyridylalanine, tBuAla represents t-butyl-alanine, tBuGly represents t-butyl- glycine, 3tBuTyr represents 3-t-Butyl-Tyrosine, or a pharmaceutically acceptable salt thereof.

[0392] In some embodiments, the first loop sequence contains 7 amino acids and is selected from:

[0393] SADDWLG;

[0394] SSDAYLG;

[0395] PPDAHLG;

[0396] PQDAYLG;

[0397] PPDSWQG;

[0398] SPDAHLG;

[0399] PGDAHLG; and PPDSHLG; and variants thereof as described herein; and pharmaceutically acceptable salts thereof. In some embodiments the second loop sequence contains 3 amino acids and is selected from ISW and I SY; and variants thereof as described herein; and pharmaceutically acceptable salts thereof.

[0400] In some embodiments the peptide ligand comprises a polypeptide having an amino acid sequence selected from:

[0401] RG1-SADDWLG-RG2-ISW-RG3;

[0402] RG1-SSDAYLG-RG2-ISW-RG3;

[0403] RG1-PPDAHLG-RG2-ISW-RG3;

[0404] RG1-PQDAYLG-RG2-ISW-RG3;

[0405] RG1-PPDSWQG-RG2-ISY-RG3;

[0406] RG1-SPDAHLG-RG2-ISY-RG3;

[0407] RG1-PGDAHLG-RG2-ISY-RG3;

[0408] RG1-PPDSHLG-RG2-ISY-RG3;

[0409] RG1-SADDWLG-RG2-ISY-RG3; and

[0410] RGi-P[HyP]DAHLG-RG2-[tBuGly]SY-RG3or a variant thereof as described herein; wherein RG1, RG2 and RG3 are as defined herein; or a pharmaceutically acceptable salt thereof.

[0411] In a further embodiment, the peptide ligand comprises an amino acid sequence of:

[0412] CSADDWLGCISWC (SEQ ID NO: 4);

[0413] CSSDAYLGCISWC (SEQ ID NO: 5);

[0414] CPPDAHLGCISWC (SEQ ID NO: 6);

[0415] CPQDAYLGCISWC (SEQ ID NO: 7);

[0416] CPPDSWQGCISYC (SEQ ID NO: 8);

[0417] CSPDAHLGCISYC (SEQ ID NO: 9);

[0418] CPGDAHLGCISYC (SEQ ID NO: 10);

[0419] CPPDSHLGCISYC (SEQ ID NO: 11);

[0420] CSADDWLGCISYC (SEQ ID NO: 12); and

[0421] CP[HyP]DAHLGC[tBuGly]SYC or a variant thereof as described herein; pharmaceutically acceptable salt thereof.

[0422] In a further embodiment, the peptide ligand comprises an amino acid sequence of:

[0423] CSADDWLGCISWC (SEQ ID NO: 4);

[0424] CSSDAYLGCISWC (SEQ ID NO: 5); CPPDAHLGCISWC (SEQ ID NO: 6);

[0425] CPQDAYLGCISWC (SEQ ID NO: 7);

[0426] CPPDSWQGCISYC (SEQ ID NO: 8);

[0427] CSPDAHLGCISYC (SEQ ID NO: 9);

[0428] CPGDAHLGCISYC (SEQ ID NO: 10);

[0429] CPPDSHLGCISYC (SEQ ID NO: 11); and CSADDWLGCISYC (SEQ ID NO: 12); or a pharmaceutically acceptable salt thereof.

[0430] In another embodiment, the peptide ligand comprises a polypeptide having an amino acid sequence RGi-P[HyP]DAHLG-RG2-[tBuGly]SY-RG3, or a variant thereof as described herein, or a pharmaceutically acceptable salt thereof, wherein RGi, RG2 and RG3 are as defined herein. In a still yet further embodiment, the peptide ligand comprises an amino sequence of CP[HyP]DAHLGC[tBuGly]SYC (SEQ ID NO: 68), wherein HyP represents trans-4-hydroxy-L-proline and tBuGly represents t-butyl-glycine.

[0431] Also provided herein is a peptide ligand specific for transferrin receptor 1 (TfR1), wherein the peptide ligand comprises a polypeptide having an amino acid sequence as described herein. For avoidance of doubt, in some embodiments the peptide ligand further comprises a halflife extending moiety, e.g. a half-life extending moiety as described herein. In some embodiments the peptide ligand does not further comprises a half-life extending moiety, e.g. a half-life extending moiety as described herein. It will be appreciated that the peptide ligand of SEQ ID NO: 68 constitutes a novel peptide ligand with specificity for Tfr1 . Accordingly, provided herein is a peptide ligand specific for transferrin receptor 1 (TfR1), wherein the peptide ligand comprises a polypeptide having an amino acid sequence RGi-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. Thus according to a further aspect of the invention there is provided a peptide ligand specific for transferrin receptor 1 (TfR1), wherein the peptide ligand comprises an amino sequence of CP[HyP]DAHLGC[tBuGly]SYC (SEQ ID NO: 68), wherein HyP represents trans-4-hydroxy- L-proline and tBuGly represents t-butyl-glycine.

[0432] In some embodiments the peptide comprises N- and / or C-terminal additions. In some embodiments the N- and / or C-terminal additions comprise one or more natural or nonnatural amino acids. In some embodiments the N- and / or C-terminal additions comprise one or more groups selected from A, G, [Sar], N, [1 Nal], W, [dW], H, M, E, V, T, S, [NMeTrp], F, [AzPro], [dY], [Pip], P, [Aze], [dP], I, Q, [K(N3)J, [K(N3)(PYA-maleimide], PEG (e.g. PEG5 to PEG 30, such as PEG10, PEG12, PEG14, PEG16, PEG18, PEG20, PEG22, PEG24, PEG26, PEG28, PEG30). In some embodiments the one or more N- and / or C-terminal additions comprise from about 1 to about 10, e.g. from about 2 to about 5 e.g. 2, 3, or 4 such groups. Examples of N-terminal modifications include extension of the peptide by one or more amino acids or amino acid analogues such as alanine (A) or a variant thereof, or N- terminal acetylation, represented by “Ac”. Examples of C-terminal modifications include extension of the peptide by one or more amino acids or amino acid analogues such as alanine (A), or C-terminal amidation, i.e. the conversion of a C-terminal carboxylic acid group (-CO(O)H or -CO(O)- to an amide -C(O)NH2. In some embodiments the N- and / or C- terminal additions comprise one or more N-terminal additions selected from

[0433] A-;

[0434] Ac-;

[0435] [FI]G[Sar5]-A-;

[0436] N[1 NalJN-;

[0437] NWN-;

[0438] Ac-NWN-;

[0439] Ac-N[1 NalJN-;

[0440] N[dW]N-;

[0441] Ac-N[dW]N-;

[0442] HWM-;

[0443] NEV-;

[0444] HTS-;

[0445] Ac-N[NMeTrp]N-;

[0446] N[NMeTrp]N-;

[0447] Ac-A-;

[0448] ESF-;

[0449] [AzPro]-NWN-;

[0450] [AzPro]-; and Ac-N[dY]N-; and / or one or more C-terminal additions selected from

[0451] -A;

[0452] -A-[Sar6]-[K-FIJ;

[0453] -[Sar6]-[K-FIJ;

[0454] -E[Pip]W;

[0455] -EPW;

[0456] -E[Aze]W;

[0457] -E[dP]W; -PHP;

[0458] -PIVH;

[0459] -EHQE;

[0460] -[K(N3)J;

[0461] -EPW-[K(N3)];

[0462] -E-[dP]-W-[K(N3)];

[0463] -E-[Aze]-W-[K(N3)];

[0464] -E-[Pip]-W-[K(N3)];

[0465] -[K(N3)(PYA-maleimide];

[0466] -EPW-[Peg10]-[K(N3)];

[0467] -EPW-[Peg24]-[K(N3)]; and -EPWGGSGGS-[K(N3)].

[0468] In some embodiments the peptide ligand comprises a polypeptide of SEQ ID NO: 68 and comprising one or more N- and / or C-terminal additions as described herein. Exemplary peptides include: Ac-(RGi-P[HyP]DAHLG-RG2-[tBuGly]SY-RG3); e.g. Ac-(SEQ ID NO: 68) (herein referred to as B191). In some embodiments the peptide ligand comprises a polypeptide of SEQ ID NO: 13 and comprises one or more N- and / or C-terminal additions as described herein.

[0469] In some embodiments a non-inhibitory peptide ligand comprises a polypeptide as described herein attached (e.g. by being covalently bonded) to a molecular scaffold as described herein. In some embodiments the polypeptide is attached (e.g. covalently bonded) to the molecular scaffold by bonds between the molecular scaffold and each of RGi, RG2and RG3. When RGi, RG2and RG3are cysteine residues, the polypeptide is typically bonded to the molecular scaffold by covalent bonds between the thiol groups of said cysteine residues and the scaffold. In some embodiments the scaffold is 1 , 1 1 "-(1 ,3,5-triazinane-1 ,3,5-triyl)tris(2- bromoethanone) (TATB).

[0470] In a further embodiment, the molecular scaffold is 1 , 1 ', 1 "-(1 ,3,5-triazinane-1 ,3,5-triyl)tris(2- bromoethanone) (TATB) and the peptide ligand comprises N- and / or C-terminal additions and is selected from:

[0471] A-(SEQ ID NO: 4)-A (herein referred to as B8);

[0472] A-(SEQ ID NO: 4)-A-[Sar6]-[K-FI] (herein referred to as B9);

[0473] A-(SEQ ID NO: 5)-A (herein referred to as B10);

[0474] A-(SEQ ID NO: 5)-A-[Sar6]-[K-FI] (herein referred to as B11);

[0475] A-(SEQ ID NO: 6)-A (herein referred to as B12); Ac-(SEQ ID NO: 6) (herein referred to as B13);

[0476] A-(SEQ ID NO: 7)-A (herein referred to as B14);

[0477] Ac-(SEQ ID NO: 7) (herein referred to as B15);

[0478] A-(SEQ ID NO: 8)-A (herein referred to as B16);

[0479] A-(SEQ ID NO: 8)-A-[Sar6]-[K-FI] (herein referred to as B17);

[0480] A-(SEQ ID NO: 9)-A (herein referred to as B18);

[0481] A-(SEQ ID NO: 9)-A-[Sar6]-[K-FI] (herein referred to as B19); (SEQ ID NO: 9)-[Sar6]-[K-FI] (herein referred to as B20);

[0482] Ac-(SEQ ID NO: 9)-A-[Sar6]-[K-FI] (herein referred to as B21);

[0483] Ac-(SEQ ID NO: 9)-[Sar6]-[K-FI] (herein referred to as B22); [FI]G[Sar5]-A-(SEQ ID NO: 9)-A (herein referred to as B23); N[1 Nal]N-(SEQ ID NO: 9) (herein referred to as B24);

[0484] Ac-(SEQ ID NO: 9)-E[Pip]W (herein referred to as B25);

[0485] Ac-(SEQ ID NO: 9)-EPW (herein referred to as B26);

[0486] NWN-(SEQ ID NO: 9) (herein referred to as B27);

[0487] NWN-(SEQ ID NO: 9)-A (herein referred to as B28);

[0488] Ac-(SEQ ID NO: 9)-E[Aze]W (herein referred to as B29);

[0489] Ac-NWN-(SEQ ID NO: 9) (herein referred to as B30);

[0490] Ac-(SEQ ID NO: 9)-E[dP]W (herein referred to as B31);

[0491] Ac-N[1 Nal]N-(SEQ ID NO: 9) (herein referred to as B32);

[0492] N[dW]N-(SEQ ID NO: 9) (herein referred to as B33);

[0493] Ac-N[dW]N-(SEQ ID NO: 9) (herein referred to as B34);

[0494] HWM-(SEQ ID NO: 9)-A (herein referred to as B35);

[0495] A-(SEQ ID NO: 9)-PHP (herein referred to as B36);

[0496] A-(SEQ ID NO: 9)-EPW (herein referred to as B37);

[0497] NEV-(SEQ ID NO: 9)-A (herein referred to as B38);

[0498] A-(SEQ ID NO: 9)-PIVH (herein referred to as B39);

[0499] Ac-(SEQ ID NO: 9) (herein referred to as B40);

[0500] HTS-(SEQ ID NO: 9)-A (herein referred to as B41);

[0501] Ac-N[NMeTrp]N-(SEQ ID NO: 9) (herein referred to as B42);

[0502] N[NMeTrp]N-(SEQ ID NO: 9) (herein referred to as B43);

[0503] Ac-A-(SEQ ID NO: 9)-A (herein referred to as B44);

[0504] A-(SEQ ID NO: 9)-EHQE (herein referred to as B45);

[0505] ESF-(SEQ ID NO: 9)-A (herein referred to as B46);

[0506] NWN-(SEQ ID NO: 9)-[K(N3)J (herein referred to as B47);

[0507] Ac-NWN-(SEQ ID NO: 9)-[K(N3)] (herein referred to as B48); [AzPro]-NWN-(SEQ ID NO: 9) (herein referred to as B49); Ac-(SEQ ID NO: 9)-EPW-[K(N3)] (herein referred to as B50);

[0508] [AzPro]-(SEQ ID NO: 9)-EPW (herein referred to as B51);

[0509] Ac-(SEQ ID NO: 9)-[K(N3)J (herein referred to as B52);

[0510] [AzPro]-(SEQ ID NO: 9) (herein referred to as B53);

[0511] Ac-N[dY]N-(SEQ ID NO: 9)-[K(N3)] (herein referred to as B54);

[0512] Ac-(SEQ ID NO: 9)-E-[dP]-W-[K(N3)] (herein referred to as B55);

[0513] Ac-(SEQ ID NO: 9)-E-[Aze]-W-[K(N3)] (herein referred to as B56);

[0514] Ac-(SEQ ID NO: 9)-E-[Pip]-W-[K(N3)] (herein referred to as B57);

[0515] Ac-(SEQ ID NO: 9)-[K(N3)(PYA-maleimide] (herein referred to as B58);

[0516] Ac-(SEQ ID NO: 9)-EPW-[Peg ]-[K(N3)] (herein referred to as B59);

[0517] Ac-(SEQ ID NO: 9)-EPW-[Peg24]-[K(N3)] (herein referred to as B60);

[0518] Ac-(SEQ ID NO: 9)-EPWGGSGGS-[K(N3)] (herein referred to as B61);

[0519] A-(SEQ ID NO: 10)-A (herein referred to as B62);

[0520] Ac-(SEQ ID NO: 10) (herein referred to as B63);

[0521] A-(SEQ ID NO: 11)-A (herein referred to as B64);

[0522] Ac-(SEQ ID NO: 11) (herein referred to as B65);

[0523] A-(SEQ ID NO: 12)-A (herein referred to as B66);

[0524] Ac-(SEQ ID NO: 12) (herein referred to as B67);

[0525] Ac-(SEQ ID NO: 13) (herein referred to as B68);

[0526] Ac-(SEQ ID NO: 13)-EPW (herein referred to as B69);

[0527] Ac-NWN-(SEQ ID NO: 13) (herein referred to as B70);

[0528] NWN-(SEQ ID NO: 13) (herein referred to as B71);

[0529] A-(SEQ ID NO: 13)-A (herein referred to as B72);

[0530] Ac-(SEQ ID NO: 13)-[K(N3)] (herein referred to as B73);

[0531] Ac-NWN-(SEQ ID NO: 13)-[K(N3)] (herein referred to as B74);

[0532] Ac-(SEQ ID NO: 13)-EPW-[K(N3)] (herein referred to as B75);

[0533] Ac-(SEQ ID NO: 14) (herein referred to as B76);

[0534] Ac-(SEQ ID NO: 14)-[K(N3)] (herein referred to as B77);

[0535] [AzPro]-(SEQ ID NO: 14) (herein referred to as B78);

[0536] Ac-(SEQ ID NO: 15) (herein referred to as B79);

[0537] A-(SEQ ID NO: 15)-A (herein referred to as B80);

[0538] Ac-(SEQ ID NO: 15)-[K(N3)] (herein referred to as B81);

[0539] [AzPro]-(SEQ ID NO: 15) (herein referred to as B82);

[0540] A-(SEQ ID NO: 16)-A (herein referred to as B83);

[0541] TYMN-(SEQ ID NO: 17)-A (herein referred to as B84);

[0542] A-(SEQ ID NO: 17)-A (herein referred to as B85);

[0543] A-(SEQ ID NO: 18)-A (herein referred to as B86); Ac-(SEQ ID NO: 19) (herein referred to as B87);

[0544] A-(SEQ ID NO: 20)-A (herein referred to as B88);

[0545] A-(SEQ ID NO: 21)-A (herein referred to as B89);

[0546] A-(SEQ ID NO: 22)-A (herein referred to as B90);

[0547] A-(SEQ ID NO: 23)-A (herein referred to as B91);

[0548] Ac-(SEQ ID NO: 24) (herein referred to as B92);

[0549] A-(SEQ ID NO: 25)-A (herein referred to as B93);

[0550] A-(SEQ ID NO: 26)-A (herein referred to as B94);

[0551] A-(SEQ ID NO: 27)-A (herein referred to as B95);

[0552] IDSN-(SEQ ID NO: 28)-A (herein referred to as B96);

[0553] WGKS-(SEQ ID NO: 29)-A (herein referred to as B97);

[0554] A-(SEQ ID NO: 30)-A (herein referred to as B98);

[0555] A-(SEQ ID NO: 31)-A (herein referred to as B99);

[0556] A-(SEQ ID NO: 32)-A (herein referred to as B100);

[0557] A-(SEQ ID NO: 33)-A (herein referred to as B101);

[0558] A-(SEQ ID NO: 34)-A-[Sar6]-[K-FI] (herein referred to as B102);

[0559] A-(SEQ ID NO: 35)-A (herein referred to as B103);

[0560] A-(SEQ ID NO: 36)-A (herein referred to as B104);

[0561] A-(SEQ ID NO: 37)-A (herein referred to as B105);

[0562] A-(SEQ ID NO: 38)-A (herein referred to as B106);

[0563] A-(SEQ ID NO: 39)-A (herein referred to as B107);

[0564] A-(SEQ ID NO: 40)-A (herein referred to as B108);

[0565] A-(SEQ ID NO: 41)-A (herein referred to as B109);

[0566] A-(SEQ ID NO: 42)-A (herein referred to as B110);

[0567] A-(SEQ ID NO: 43)-A (herein referred to as B111);

[0568] A-(SEQ ID NO: 44)-A (herein referred to as B112);

[0569] Ac-(SEQ ID NO: 44) (herein referred to as B113);

[0570] Ac-(SEQ ID NO: 44)-[K(N3)J (herein referred to as B114);

[0571] [AzPro]-(SEQ ID NO: 44) (herein referred to as B115);

[0572] A-(SEQ ID NO: 45)-A (herein referred to as B116);

[0573] A-(SEQ ID NO: 46)-A (herein referred to as B117);

[0574] A-(SEQ ID NO: 47)-A (herein referred to as B118);

[0575] A-(SEQ ID NO: 48)-A (herein referred to as B119);

[0576] A-(SEQ ID NO: 49)-A (herein referred to as B120);

[0577] A-(SEQ ID NO: 50)-A (herein referred to as B121);

[0578] A-(SEQ ID NO: 51)-A (herein referred to as B122);

[0579] A-(SEQ ID NO: 52)-A (herein referred to as B123); A-(SEQ ID NO: 53)-A (herein referred to as B124);

[0580] A-(SEQ ID NO: 54)-A (herein referred to as B125);

[0581] A-(SEQ ID NO: 55)-A (herein referred to as B126);

[0582] A-(SEQ ID NO: 56)-A (herein referred to as B127);

[0583] A-(SEQ ID NO: 57)-A-[Sar6]-[K-FI] (herein referred to as B128);

[0584] A-(SEQ ID NO: 58)-A-[Sar6]-[K-FI] (herein referred to as B129);

[0585] A-(SEQ ID NO: 59)-A-[Sar6]-[K-FI] (herein referred to as B130);

[0586] A-(SEQ ID NO: 60)-A-[Sar6]-[K-FI] (herein referred to as B131);

[0587] A-(SEQ ID NO: 61)-A-[Sar6]-[K-FI] (herein referred to as B132);

[0588] A-(SEQ ID NO: 62)-A-[Sar6]-[K-FI] (herein referred to as B133);

[0589] A-(SEQ ID NO: 63)-A-[Sar6]-[K-FI] (herein referred to as B134);

[0590] A-(SEQ ID NO: 64)-A-[Sar6]-[K-FI] (herein referred to as B135);

[0591] Ac-(SEQ ID NO: 65) (herein referred to as B136);

[0592] Ac-(SEQ ID NO: 66) (herein referred to as B137);

[0593] Ac-(SEQ ID NO: 67) (herein referred to as B138);

[0594] Ac-(SEQ ID NO: 68) (herein referred to as B191);

[0595] Ac-(SEQ ID NO: 68)-EPW (herein referred to as B196);

[0596] Ac-(SEQ ID NO: 68)-EPW-[K(N3)] (herein referred to as B197); and

[0597] Ac-(SEQ ID NO: 68)-EPW-[K(PYA)] (herein referred to as B198); wherein AzPro represents azidopropyl, Aze represents azetidine, 1 Nal represents 1- naphthylalanine, NMeTrp represents N-methyl-tryptophan, K(PYA) represents lysine, £-4- pentynoyl, [K(N3)] represents 6-azido lysine, Peg represents polyethylene glycol, Pip represents pipecolic acid, Sar represents sarcosine, Fl represents fluorescein and [K(N3)(PYA-Maleimide)] represents a modified lysine having the following structure:

[0598] In a further embodiment, the molecular scaffold is 1 , 1 ', 1 "-(1 ,3,5-triazinane-1 ,3,5-triyl)tris(2- bromoethanone) (TATB) and the peptide ligand comprises N- and / or C-terminal additions and is selected from:

[0599] Ac-(SEQ ID NO: 68) (herein referred to as B191);

[0600] Ac-(SEQ ID NO: 68)-EPW (herein referred to as B196);

[0601] Ac-(SEQ ID NO: 68)-EPW-[K(N3)J (herein referred to as B197); and

[0602] Ac-(SEQ ID NO: 68)-EPW-[K(PYA)] (herein referred to as B198); wherein [K(N3)] represents 6-azido lysine and K(PYA) represents lysine, £-4-pentynoyl. It will be appreciated that the bicyclic peptide ligands of B191 and B196 to B198 constitutes novel bicyclic peptide ligands with specificity for Tfrl. Thus, according to a further aspect of the invention there is provided a bicyclic peptide ligand specific for transferrin receptor 1 (TfR1), wherein the peptide ligand is B191 or B196 to B198.

[0603] In particular, B191 was tested in the SPR assay described in WO 2022 / 101633 and demonstrated a Kd of 20nM with human Tfr1 and 379nM with cynomolgus TfR1 (Kd). Thus, according to a further aspect of the invention there is provided a bicyclic peptide ligand specific for transferrin receptor 1 (TfR1), wherein the peptide ligand is B191.

[0604] 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 N- and / or C-terminal additions and is:

[0605] Ac-(SEQ ID NO: 13) (herein referred to as B139).

[0606] Further examples of bicyclic peptides specific for Tfr1 include:

[0607] C[HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 69, herein referred to as B140); C[Oxa][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 70, herein referred to as B141); C[Cis-HyP][HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 71, herein referred to as B142); CP[Oxa]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 72, herein referred to as B143);

[0608] CP[Cis-HyP]DAYLGC[tBuGly]SYCEPW (SEQ ID NO: 73, herein referred to as B144); CP[HyP]DA[DOPA]LGC[tBuGly]SYCEPW (SEQ ID NO: 74, herein referred to as B145); CP[HyP]DA[pCaPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 75, herein referred to as B146); CP[HyP]DA[pCoPhe]LGC[tBuGly]SYCEPW (SEQ ID NO: 76, herein referred to as B147);

[0609] CP[HyP]DA[hTyr]LGC[tBuGly]SYCEPW (SEQ ID NO: 77, herein referred to as B148); CP[HyP]DAYL[dS]C[tBuGly]SYCEPW (SEQ ID NO: 78, herein referred to as B149); CP[HyP]DAYL[dT]C[tBuGly]SYCEPW (SEQ ID NO: 79, herein referred to as B150); CP[HyP]DAYL[dD]C[tBuGly]SYCEPW (SEQ ID NO: 80, herein referred to as B151);

[0610] CP[HyP]DAYL[dE]C[tBuGly]SYCEPW (SEQ ID NO: 81, herein referred to as B152); CP[HyP]DAYL[dN]C[tBuGly]SYCEPW (SEQ ID NO: 82, herein referred to as B153); CP[HyP]DAYL[dQ]C[tBuGly]SYCEPW (SEQ ID NO: 83, herein referred to as B154); CP[HyP]DAYL[dY]C[tBuGly]SYCEPW (SEQ ID NO: 84, herein referred to as B155);

[0611] CP[HyP]DAYLSC[tBuGly]SYCEPW (SEQ ID NO: 85, herein referred to as B156);

[0612] CP[HyP]DAYLDC[tBuGly]SYCEPW (SEQ ID NO: 86, herein referred to as B157); CP[HyP]DAYLYC[tBuGly]SYCEPW (SEQ ID NO: 87, herein referred to as B158); CP[HyP]DAYLNC[tBuGly]SYCEPW (SEQ ID NO: 88, herein referred to as B159); CP[HyP]DAYLGC[tBuGly]S[DOPA]CEPW (SEQ ID NO: 89, herein referred to as B160); CP[HyP]DAYLGC[tBuGly]S[pCaPhe]CEPW (SEQ ID NO: 90, herein referred to as B161); CP[HyP]DAYLGC[tBuGly]S[pCoPhe]CEPW (SEQ ID NO: 91, herein referred to as B162); CP[HyP]DAYLGC[tBuGly]S[hTyr]CEPW (SEQ ID NO: 92, herein referred to as B163); CP[HyP]DAYLGC[tBuGly]SYCE[HyP]W (SEQ ID NO: 93, herein referred to as B164); CP[HyP]DAYLGC[tBuGly]SYCE[Oxa]W (SEQ ID NO: 94, herein referred to as B165); CP[HyP]DAYLGC[tBuGly]SYCE[Cis-HyP]W (SEQ ID NO: 95, herein referred to as B166); CP[HyP]DAYLGC[tBuGly]SYCEPY (SEQ ID NO: 96, herein referred to as B167);

[0613] CP[HyP]DAYLGC[tBuGly]SYCEP[DOPA] (SEQ ID NO: 97, herein referred to as B168); CP[HyP]DAYLGC[tBuGly]SYCEP[pCaPhe] (SEQ ID NO: 98, herein referred to as B169); CP[HyP]DAYLGC[tBuGly]SYCEP[pCoPhe] (SEQ ID NO: 99, herein referred to as B170); CP[HyP]DAYLGC[tBuGly]SYCEP[hTyr] (SEQ ID NO: 100, herein referred to as B171); CP[HyP]EAYLGC[tBuGly]SYCEPW (SEQ ID NO: 101 , herein referred to as B172); CP[HyP][Gla]AYLGC[tBuGly]SYCEPW (SEQ ID NO: 102, herein referred to as B173); CP[HyP]DAYSGC[tBuGly]SYCEPW (SEQ ID NO: 103, herein referred to as B174); CP[HyP]DAYTGC[tBuGly]SYCEPW (SEQ ID NO: 104, herein referred to as B175);

[0614] CP[HyP]DAYDGC[tBuGly]SYCEPW (SEQ ID NO: 105, herein referred to as B176); CP[HyP]DAYEGC[tBuGly]SYCEPW (SEQ ID NO: 106, herein referred to as B177); CP[HyP]DAYNGC[tBuGly]SYCEPW (SEQ ID NO: 107, herein referred to as B178); CP[HyP]DAYQGC[tBuGly]SYCEPW (SEQ ID NO: 108, herein referred to as B179); CP[HyP]DAYLGC[tBuGly][HSer]YCEPW (SEQ ID NO: 109, herein referred to as B180); CP[HyP]DAYLGC[tBuGly]TYCEPW (SEQ ID NO: 110, herein referred to as B181); CP[HyP]DAYLGC[tBuGly]DYCEPW (SEQ ID NO: 111, herein referred to as B182); CP[HyP]DAYLGC[tBuGly]EYCEPW (SEQ ID NO: 112, herein referred to as B183);

[0615] CP[HyP]DAYLGC[tBuGly]NYCEPW (SEQ ID NO: 113, herein referred to as B184); CP[HyP]DAYLGC[tBuGly]QYCEPW (SEQ ID NO: 114, herein referred to as B185); CP[HyP]DAYLGC[tBuGly]SYCDPW (SEQ ID NO: 115, herein referred to as B186); CP[HyP]DAYLGC[tBuGly]SYC[Gla]PW (SEQ ID NO: 116, herein referred to as B187); CP[HyP]DAYLGCYSYCEPW (SEQ ID NO: 117, herein referred to as B188); and CP[HyP]DAYLGC[3HyV]SYCEPW (SEQ ID NO: 118, herein referred to as B189), wherein Cis-HyP represents cis- L-4- hydroxyproline, DOPA represents 3,4-dihydroxy- phenylalanine, Gia represents L-y-carboxyglutamic acid, HyP represents hydroxyproline, HSer represents homoserine, hTyr represents homo-tyrosine, 3HyV represents 3-hydroxy-L- valine, Oxa represents oxazolidine-4-carboxylic acid, pCaPhe represents L-4- carbamoylphenylalanine, pCoPhe represents 4-carboxy-L-phenylalanine, tBuGly represents t-butyl-glycine. The polypeptide may have an amino acid sequence as defined herein (e.g. a polypeptide according to any one of SEQ ID NOs: 1 to 118) wherein one, two, three, four, or five of the amino acids, typically one, two or three of the amino acids, are modified as described herein. In some embodiments, the polypeptide may have at least 70% sequence identity with any one of the peptides described herein (e.g. any one of SEQ ID NOs: 1 - 118), such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. It will be appreciated that references herein to “sequence identity” are to be understood as meaning the percentage identity between two protein sequences, e.g. SEQ ID NO: X and SEQ ID NO: Y, for example as measured using the methods described herein.

[0616] In some embodiments an inhibitory peptide ligand comprises a polypeptide as described herein attached (e.g. by being covalently bonded) to a molecular scaffold as described herein. In some embodiments the polypeptide is attached (e.g. covalently bonded) to the molecular scaffold by bonds between the molecular scaffold and each of RGi, RG2 and RG3. When RG1, RG2 and RG3 are cysteine residues, the polypeptide is typically bonded to the molecular scaffold by covalent bonds between the thiol groups of said cysteine residues and the scaffold. In some embodiments the scaffold is 1 , 1 ', 1 "-(1 ,3,5-triazinane-1 ,3,5-triyl)triprop- 2-en-1-one (TATA) or 1 ,T,1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (TATB).

[0617] For the purpose of this description, non-inhibitory bicyclic peptides are assumed to be cyclised with TATA or TATB and yielding a tri-substituted structure. However, as will be clear from the descriptions of the invention presented herein, cyclisation may be performed with any suitable molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed. In some embodiments the polypeptide is attached (e.g. covalently bonded) to the molecular scaffold by bonds between the molecular scaffold and each of RG1, RG2 and RG3. When RG1, RG2 and RG3 are cysteine residues, cyclisation occurs on the first, second and third cysteine residues, respectively.

[0618] In a further embodiment, the pharmaceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium or ammonium salt.

[0619] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as in the arts of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (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) 4thed., John Wiley & Sons, Inc.), which are incorporated herein by reference.

[0620] Multimeric Bicyclic Peptide Ligands

[0621] In one embodiment, the bicyclic peptide ligand complex comprises more than one bicyclic peptide (such as 2, 3 or 4) conjugated via one or more linkers. The bicycle peptides may the same or different. In some embodiments at least one of the bicycle peptides in the complex is a peptide ligand (e.g. a monomeric bicyclic peptide ligand) as described in more detail herein. In some embodiments the bicyclic peptide ligand complex comprises at least two (i.e. 2, 3 or 4) of any of the monomeric bicyclic peptide ligands as defined herein.

[0622] This aspect of the invention describes a series of multimerized bicyclic peptides with various chemical linkers and hinges of various lengths and rigidity using different sites of attachments within said bicyclic peptide which bind and activate TfR1 with a wide range of potency and efficacy.

[0623] It will be appreciated by the skilled person that this aspect of the invention presents multiply arranged (multimeric) bicyclic peptides which provide a synergistic benefit by virtue of the resultant properties of said multimeric binding complexes compared to the corresponding monomeric binding complexes which contain a single bicyclic peptide. For example, the multimeric binding complexes of this aspect of the invention typically have greater levels of binding potency or avidity (as measured herein by Kd values) than their monomeric counterparts. Furthermore, the multimeric binding complexes of the invention are designed to be sufficiently small enough to be cleared by the kidneys.

[0624] Without being bound by theory it is believed that multimerized bicyclic peptides are able to activate receptors by homo-crosslinking more than one of the same receptor. Thus, in one embodiment, said bicyclic peptide ligands are specific for the same target within TfR1. In a further embodiment, the multimeric binding complex comprises at least two identical bicyclic peptide ligands. By “identical” it is meant bicyclic peptides having the same amino acid sequence, most critically the same amino acid sequence refers to the binding portion of said bicyclic peptide (for example, the sequence may vary in attachment position). In this embodiment, each of the bicyclic peptides within the multimeric binding complex will bind exactly the same epitope upon the same target of TfR1- the resultant target bound complex will therefore create a homodimer (if the multimeric complex comprises two identical bicyclic peptides), homotrimer (if the multimeric complex comprises three identical bicyclic peptides) or homotetramer (if the multimeric complex comprises four identical bicyclic peptides), etc. In an alternative embodiment, the multimeric binding complex comprises at least two differing bicyclic peptide ligands. By “differing” it is meant bicyclic peptides having a different amino acid sequence. In this embodiment, the differing bicyclic peptide ligands within the multimeric binding complex will bind to different epitopes on TfR1 - the resultant target bound complex will therefore create a biparatopic (if the multimeric complex comprises two differing bicyclic peptides), triparatopic (if the multimeric complex comprises three differing bicyclic peptides) or tetraparatopic (if the multimeric complex comprises four differing bicyclic peptides), etc.

[0625] Without being bound by theory it is believed that multimerized bicyclic peptides are able to activate receptors by hetero-crosslinking differing targets, such as differing target sites on TfR1. Thus, in one embodiment, said bicyclic peptide ligands are specific for different targets on TfR1. It will be appreciated that in this embodiment, the multimeric binding complex comprises at least two differing bicyclic peptide ligands (i.e. bicyclic peptide ligands having differing amino acid sequences). In this embodiment, each of the bicyclic peptides within the multimeric binding complex will bind a differing epitope upon TfR1- the resultant target bound complex will therefore create a bispecific multimeric binding complex (if the multimeric complex comprises two differing bicyclic peptides), trispecific multimeric binding complex (if the multimeric complex comprises three differing bicyclic peptides), tetraspecific multimeric binding complex (if the multimeric complex comprises four differing bicyclic peptides), etc.

[0626] It will be appreciated that the multimeric binding complexes of the invention may be designed to be capable of binding to a range of different targets on TfR1.

[0627] The bicyclic peptides within the multimeric binding complexes of the invention may be assembled via a number of differing options. For example, there may be a central hinge or branching moiety with spacer or arm elements radiating from said hinge or branch point each of which will contain a bicyclic peptide. Alternatively, it could be envisaged that a circular support member may hold a number of inwardly or outwardly projecting bicyclic peptides.

[0628] In one embodiment, each bicyclic peptide ligand is connected to a central hinge moiety by a spacer group. It will be appreciated that the spacer group may be linear and connect a single bicyclic peptide with the central hinge moiety. Thus, in one embodiment, the multimeric binding complex comprises a compound of formula (I): wherein CHM represents a central hinge moiety;

[0629] Bicycle represents a bicyclic peptide ligand as defined herein; and m represents an integer selected from 2 to 10.

[0630] In one embodiment, m represents an integer selected from 2, 3 or 4.

[0631] In a further embodiment, m represents 2.

[0632] When m represents 2, it will be appreciated that the central hinge moiety will require 2 points of attachment. Thus, in one embodiment, m represents 2 and CHM is a motif of formula (A):

[0633] (A).

[0634] This and other groups which may be used to link bicycle peptide ligands together in multimeric binding complexes are described in more detail herein.

[0635] Dimers

[0636] In one embodiment, the multimeric binding complex comprises two identical bicyclic peptides and comprises a dimeric binding complex described in the following Table A:

[0637] Table A: Exemplified Dimeric Binding Complexes of the Invention

[0638] In one embodiment, the bicyclic peptide ligand specific for transferrin receptor 1 (TfR1) is selected from any of B1 to B191 or B196 to B198.

[0639] In one embodiment the multimeric binding complex additional comprises one or more effector groups and / or functional groups, such as one or more cytotoxic agents, radiochelators, or chromophores. In one embodiment, the multimeric binding complex additionally comprises a fluorophore.

[0640] Linkers

[0641] In embodiments which provide a multimeric binding complex comprising at least one bicycle peptide ligand as described herein (e.g. at least 2, 3, or 4 bicycle peptide ligands as described herein), the peptide ligands may be conjugated together via any suitable linker. Such linkers may also be used to link a bicycle peptide ligand as described herein (or a complex of bicycle peptide ligands as described herein) to a payload such as an effector group as described in more detail herein. Linkers as described herein may also be used to link a bicyclic peptide ligand to a HLE moiety as described in more detail herein.

[0642] In one embodiment the linker is a linear linker or a branched linker. In some embodiments the linker is a branched linker and comprises three or four branches. In some embodiments the linker is capable of binding to three or four bicyclic peptide ligands. In some embodiments the linker comprises three branches and is capable of binding to three bicyclic peptide ligands. In some embodiments the linker comprises four branches and is capable of binding to four bicyclic peptide ligands. In some embodiments the linker comprises one or more repeating monomer groups.

[0643] In some embodiments, the linker is a bidentate or polydentate group having a length of from about 0.3 nm to about 300 nm. In some embodiments the linker has a length of from about 0.5 nm to about 200 nm, such as from about 1 nm to about 100 nm, e.g. from about 1.5 nm to about 50 nm, e.g. from about 2 nm to about 20 nm, such as from about 3 nm to about 10 nm. In some embodiments the linker length is the persistence length. In some embodiments the length is determined when linker is in aqueous solution under physiological conditions, (e.g. phosphate buffered saline, pH 7.4 at 37 °C) and in some embodiments can be determined using atomic force microscopy. In some embodiments the linker comprises an alkyl, alkenyl or alkynyl group, such as a C1.30 alkyl (e.g. C2-12, e.g. C2-6 alkyl), C2-30 alkenyl (e.g. C2-12, e.g. C2-6 alkenyl) or C2-30 alkynyl (e.g. C2-12, e.g. C2-6 alkynyl) group. When a linker comprises an alkyl, alkenyl or alkynyl group the group may be straight- or branched-chain and may be optionally substituted.

[0644] In some embodiments, the linker comprises one or more linking moieties such as one or more poly(alkyleneglycol) groups such as poly(ethyleneglycol) or poly(propyleneglycol). In some embodiments the linker may comprise one or more groups such as an amine group, an amide group; an alkylene group; a carbamate group; an ether group; an ester group; a disulphide bond; a hydrazone group; a sulfonamide group; a thioether group; or a cyclic group, preferably a 4-12 membered carbocyclic or heterocyclic group, a 5-12 membered heteroaryl group or a C6-12 aryl group; wherein said alkylene, alkenylene, alkynylene, poly(alkyleneglycol), amine and cyclic group is each independently optionally substituted.

[0645] In some embodiments the linker comprises one or more amino acids or amino acid analogs. In some embodiments the linker comprises from 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 attached together. In some embodiments the linker comprises a moiety of form wherein each R1is H or C1-4 alkyl; each R2is selected from the side chain of an amino acid (e.g. a canonical amino acid) or is CM alkyl which may be substituted e.g. with OH, SH, SC1-4 alkyl, aryl (which may be substituted with OH), heteroaryl, C(O)OH, C(O)NH2, N+Hs, NH(C=N+H2)NH2, for example, R2may comprise the side chain of arginine or homo-arginine; and wherein LINK is a linker, wherein LINK is optionally a C2-8 hydrocarbylene (e.g. alkylene) linker optionally terminated by or substituted with one or more groups such as an amine group, an amide group; an alkylene group; a carbamate group; an ether group; an ester group; a disulphide bond; a hydrazone group; a sulfonamide group; a thioether group; or a cyclic group as defined herein; for example, LINK may comprise a C3-6 alkylene group which terminates in or is interrupted by an amide group; e.g. LINK may comprise a moiety of form -Ci-4alkylene-NHC(O)-Ci-4alkylene-, e.g. -C4alkylene-NHC(O)-Cialkylene-. In some embodiments said moiety is attached to a polypeptide comprised in a peptide ligand or bicyclic peptide ligand as defined herein, e.g. at the N- or C- terminus of a polypeptide comprised in a peptide ligand or bicyclic peptide ligand as defined herein. In some embodiments the linker comprises one or more reactive groups for reaction with a bicyclic peptide ligand as described herein. Exemplary reactive groups include azide goups (which may react with alkyne groups on a bicyclic peptide ligand as described herein, e.g. in the presence of suitable conditions, for example in the presence of an azide-alkyne cycloaddition catalyst, e.g. thereby forming a 1 ,2,3-triazole group); carboxylic acids and activated derivatives thereof (such as NHS-esters) (which may react with amine groups on a bicyclic peptide ligand as described herein, e.g. in the presence of suitable conditions, e.g. thereby forming an amide bond), and the like.

[0646] In some embodiments when the linker comprises a poly(alkyleneglycol), the poly(alkyleneglycol) is poly(ethyleneglycol) (PEG) or poly(propyleneglycol) (PPG). In some embodiments the linker comprises one or more group PEGn, wherein n represents the number of contiguous ethyleneglycol units in each said PEG group. In some embodiments n is an integer from about 2 to about 25, such as about 3 to about 10. In some embodiments the linker comprises one or more branches e.g. three branches and each branch comprises a PEGn group.

[0647] In some embodiments when the linker comprises an amide group, said amide group is of formula -NHC(O)- or -C(O)NH-. In some embodiments when the linker comprises an amine group, the amine group is of formula N(R)a, wherein each R may be the same or different. In some embodiments each R comprises a bicyclic peptide ligand as described herein, for example attached to the amine nitrogen via a linking moiety comprising one or more group PEGn as described herein. In some embodiments when the linker comprises a cyclic group the cyclic group is a Ge aryl group. In some embodiments when the linker comprises an alkylene group the alkylene group is a C1-3 alkylene group.

[0648] In one embodiment, the linker is a linear linker. Without being bound by theory it is believed that a linear linker has the advantage of allowing the presence of one first peptide ligand at one end and one second peptide ligand and / or an HLE moiety e.g as described herein at the other end.

[0649] In some embodiments a linear linker comprises a group of formula

[0650] (Alk)m— PEGn— (Alk)mwherein each Aik is independently a C1-4 alkylene group; each m is independently 0 or 1 ; n is an integer from about 2 to about 25. In some embodiments a linear linker may comprise (e.g. may be interrupted by) an amide group. In some embodiments a linear linker may comprise a group of formula

[0651] PEGn-(Alk)m-arnide-(Alk)m-PEGnwherein each Aik is independently a C1-4 alkylene group; 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 a linear linker may further comprise an amino acid or artificial amino acid such as gGlu. In some embodiments a linker may comprise a group for reacting with a first bicyclic peptide ligand as described herein and a group for reacting with a second peptide ligand and / or an HLE moiety e.g. as described herein.

[0652] In some embodiments a linker comprises a group of formula

[0653] (Alk)m-arnide-(Alk)m wherein each Aik is independently a C1-4 alkylene group; each m is independently 0 or 1 ; and amide is an amide group (-NHC(O)- or -C(O)NH-). In some embodiments the linker further comprises a terminating group such as an azide (N3) group (which may be used to react with an alkyne group e.g. on a bicyclic peptide ligand or HLE moiety, e.g. comprised in a PYA group such as K(PYA)) or an alkyne group (which may be used to react with an azide group e.g. on a bicyclic peptide ligand or HLE moiety, e.g. comprised in a K(N3) group).

[0654] In some embodiments the linear linker is a group of formula

[0655] N3— (Alk)m— PEGn— (Alk)m— Q wherein each Aik is independently a C1-3 alkylene group; 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 activated derivative thereof (e.g. an NHS-ester group).

[0656] Those skilled in the art will appreciate that when conjugated with bicyclic peptide ligands as described herein, the azide group will typically react with an alkyne group on one bicyclic peptide ligand (e.g. to an alkyne group comprised in a K(PYA) moiety as described herein) and the carboxylic acid or NHS-ester will typically react with an amine group on another bicyclic peptide ligand (e.g. to the N-terminal amino group of an polypeptide as described herein). Similarly, when conjugated to a payload such as an effector group, an azide or carboxylic acid group may react with a complementary group (such as an alkyne group or an amide group on the payload).

[0657] In one embodiment, the linker is a branched linker. Without being bound by theory it is believed that the branched linker has the advantage of allowing the presence of one first peptide ligand at one end and the two or more second peptide ligands and / or HLE moieties at the other end.

[0658] In some embodiments a branched linker comprises a group of formula

[0659] [(Alk)m— (PEGn)v— (Alk)m]x-W-[(Alk)m— (PEGn)v— (Alk)m]ywherein each Aik is independently a C1-4 alkylene group which independently optionally terminates in 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 N or a benzene ring; and x and y are each independently 0 or an integer from 1 to about 4; wherein x + y is equal to the number of branches in the branched linker; for example a linker comprising three branches may have x = 1 and y = 2 or x = 2 and y = 1. In some embodiments a branched linker an alkylene group may be interrupted by or terminate in an amide group (-NHC(O)- or -C(O)NH-). In some embodiments a linker may comprise one or more groups for reacting 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 the an azide (N3) group may be used to react with an alkyne group e.g. on a bicyclic peptide ligand or HLE moiety, e.g. comprised in a PYA group such as K(PYA)) and / or an alkyne group may be used to react with an azide group e.g. on a bicyclic peptide ligand or HLE moiety, e.g. comprised in a K(N3) group. In some embodiments the branched linker is a group of formula

[0660] [N3— (Alk)m— PEGn— (Alk)m-]x-W-[-(Alk)m— PEGn— (Alk)m— Q]ywherein each Aik is independently a C1-3 alkylene group which independently optionally terminates in 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 activated derivative thereof (e.g. an NHS-ester group); W is N or a benzene ring; and x and y are each independently 0 or an integer from 1 to about 4; wherein x + y is equal to the number of branches in the branched linker; for example a linker comprising three branches may have x = 1 and y = 2 or x = 2 and y = 1 . For avoidance of doubt, when a linker comprises a plurality of groups [N3 — (Alk)m — PEGn— (Alk)m-] and / or [-(Alk)m— PEGn— (Alk)m— Q], said groups may be the same or different. In some embodiments the branched linker is

[0661] Numbering

[0662] When referring to amino acid residue positions within the peptides of the invention, cysteine residues are omitted from the numbering as they are invariant, therefore, the numbering of amino acid residues within the peptides of the invention is referred to as below:

[0663] -C-A1-L2-C-N3-D4-W5-T6-L7-P8-W9-H10-H11-C- (SEQ ID NO: 1).

[0664] Molecular Format

[0665] N- or C-terminal extensions to the bicycle core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal biotin-G-Sars tail would be denoted as:

[0666] [Biot]-G-[Sar5]-A-(SEQ ID NO: X). Wherein [Biot] represents biotin and Sar represents sarcosine.

[0667] Inversed Peptide Sequences

[0668] In light of the disclosure in Nair et al. (2003) J. Immunol. 170(3), 1362-1373, it is envisaged that the peptide sequences disclosed herein would also find utility in their retro-inverso form. For example, the sequence is reversed (i.e. N-terminus become C-terminus and vice versa) and their stereochemistry is likewise also reversed (i.e. D-amino acids become L-amino acids and vice versa).

[0669] Peptide Ligand Definition

[0670] A peptide ligand, as referred to herein, refers to a peptide, peptidic or peptidomimetic covalently bound to a molecular scaffold. Typically, such peptides, peptidics or peptidomimetics comprise a peptide having natural or non-natural amino acids, two or more reactive groups (i.e. cysteine, homocysteine (hCys, (S)-2-Amino-4-sulfanylbutanoic acid), PCys ((R)-3-amino-3-mercaptopropanoic acid), or penicillamine (Pen, (R)-2-amino-3- mercapto-3-methylbutanoic acid), Dap ((S)-2,3-diaminopropanoic acid) or N-alkyl-Dap (e.g. N-methyl-Dap, (S)-2-amino-3-(methylamino)propanoic acid) which are capable of forming covalent bonds to the scaffold, and a sequence subtended between said reactive groups which is referred to as the loop sequence, since it forms a loop when the peptide, peptidic or peptidomimetic is bound to the scaffold. In the present case, the peptides, peptidics or peptidomimetics typically comprise at least three cysteine residues (referred to herein as Ci, Cii and Ciii), and form at least two loops on the scaffold.

[0671] Accordingly, in some embodiments the present disclosure provides a peptide, peptide ligand or peptide ligand complex as provided herein, wherein one or more of the cysteine residues of one or more of the polypeptides comprised in said peptide, peptide ligand, or peptide ligand complex is replaced with homocysteine (hCys), pCys, penicillamine (Pen), Dap or N- methyl-Dap. In some embodiments one or more (e.g. 1 , 2 or 3) of the cysteine residues in any one of the polypeptides described herein are replaced with homocysteine (hCys), pCys, penicillamine (Pen), Dap or N-methyl-Dap.

[0672] Peptide specificity

[0673] As explained above, in some embodiments the provided peptides and ligands comprising such (described in more detail herein) are specific for TfR1.

[0674] As used herein, the term “specific” (“specific binding”, etc) refers in its broadest sense to a peptide or peptide ligand which 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 non-specific. In some embodiments a peptide which exhibits non-specific binding is promiscuous; that is the peptide is capable of binding to multiple different biological species, typically including the target of interest and off-target binding sites, such as binding sites on cell types other than a target cell type. Accordingly, in some embodiments a peptide or peptide ligand which is chosen or designed to bind specifically to its intended target does not exhibit promiscuous binding to off-target binding sites.

[0675] In some embodiments the binding to the target is a binding to a particular epitope on the target. A peptide or peptide ligand may be designed to be specific for a particular epitope or may be identified by suitable screening methods, such as display techniques (e.g. phage display) which can be used to develop high-affinity binders against given targets (e.g. epitopes). Alternatively, a peptide or peptide ligand can be identified with specific binding to a biological target (such as a cellular target) without knowledge of the specific epitope to which it binds. In some embodiments a peptide or peptide ligand which specifically binds to a target or epitope has a high affinity for the target or epitope. In some embodiments the binding affinity of a peptide or peptide ligand to its epitope may be expressed in terms of its dissociation constant (KD, also written as Kd). Typically, a peptide or peptide ligand which specifically binds to a biological target will have a KD against that target of less than 10 pM, e.g. less than 1 pM. Often, a peptide or peptide ligand which specifically binds to a biological target will have a nanomolar KD against 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 affinities can be determined by methods known in the art, such as SPR and competition assays. Some suitable assays are described in the examples.

[0676] In some embodiments a peptide or peptide ligand which specifically binds to a biological target will have a higher affinity (lower KD) for the specific biological binding site than for other biological binding site. For example, a peptide or peptide ligand which specifically binds to TfR1 will typically bind to TfR1 with a higher affinity than to other binding sites. In some embodiments a peptide or peptide ligand which specifically binds to a biological target will bind to the desired binding site with an affinity at least twice as strong, e.g. at least 5 times, e.g. at least 10 times, e.g. at least 20 time, e.g. at least 50 times, e.g. at least 100 times, e.g. at least 1000 times or more as strong as the binding of the peptide to any other off-target binding sites.

[0677] Advantages of the Peptide Ligands

[0678] Certain bicyclic peptides of the present invention have a number of advantageous properties which enable them to be considered as suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include: - Species cross-reactivity. This is a typical requirement for preclinical pharmacodynamics and pharmacokinetic evaluation;

[0679] - Protease stability. Bicyclic peptide ligands should in most circumstances demonstrate stability to plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases and the like. Protease stability should be maintained between different species such that a bicyclic peptide lead candidate can be developed in animal models as well as administered with confidence to humans;

[0680] - Desirable solubility profile. This is a function of the proportion of charged and hydrophilic versus hydrophobic residues and intra / inter-molecular H-bonding, which is important for formulation and absorption purposes; and

[0681] - An optimal plasma half-life in the circulation. Depending upon the clinical indication and treatment regimen, it may be required to develop a bicyclic peptide with short or prolonged in vivo exposure times for the management of either chronic or acute disease states. The optimal exposure time will be governed by the requirement for sustained exposure (for maximal therapeutic efficiency) versus the requirement for short exposure times to minimise toxicological effects arising from sustained exposure to the agent.

[0682] Pharmaceutically Acceptable Salts

[0683] It will be appreciated that salt forms are within the scope of this invention, and references to peptide ligands include the salt forms of said ligands.

[0684] The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.

[0685] Acid addition salts (mono- or di-salts) may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include mono- or di-salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic, 2- hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic), isethionic, lactic (e.g. (+)-L- lactic, (±)-DL-lactic), lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1 ,5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L- pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L- tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

[0686] One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

[0687] If the compound is anionic, or has a functional group which may be anionic (e.g. -COOH may be -COO'), then a salt may be formed with an organic or inorganic base, generating a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li+, Na+and K+, alkaline earth metal cations such as Ca2+and Mg2+, and other cations such as Al3+or Zn+. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH4+) and substituted ammonium ions (e.g. NHaR+, NH2R2+, NHRa+, NR4+). Examples of some suitable substituted ammonium ions are 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. An example of a common quaternary ammonium ion is N(CH3)4+.

[0688] In one embodiment, the pharmaceutically acceptable salt is selected from the sodium, potassium, calcium or ammonium salt.

[0689] Where the peptides of the invention contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the peptides of the invention. The peptides (including peptide ligands comprising said peptides) of the invention may exist as a zwitterion. Such compounds may also be provided in the form of a pharmaceutically acceptable salt. Suitable salts include those formed with pharmaceutically acceptable acids, which provide a proton to a negatively charged group such as a COO- group, and a counterion to balance the positive charge on a positively charged group such as a quaternary nitrogen atom. Suitable pharmaceutically acceptable acids include hydrochloric acid, sulphonic acids including methanesulphonic acid and toluene sulphonic acid, ascorbic acid and citric acid. Hydrochloric acid and sulphonic acids are preferred, in particular hydrochloric acid. Alternatively, zwitterions can be combined with pharmaceutically acceptable bases, for example, alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides.

[0690] Modified Derivatives

[0691] It will be appreciated that modified derivatives of the peptide ligands as defined herein are within the scope of the present invention.

[0692] In some embodiments, modified derivatives include functional fragments, derivatives and variants of sequences provided herein.

[0693] As those skilled in the art will appreciate, fragments of amino acid sequences include deletion variants of such sequences wherein one or more, such as at least 1, 2, 3, 4 or 5 amino acids are deleted. Deletion may occur at the C- terminus or N-terminus of the reference sequence or within the reference sequence.

[0694] Derivatives of amino acid sequences include modified sequences including sequences which are modified in vivo or ex vivo. Many different protein modifications are known to those skilled in the art and include modifications to introduce new functionalities to amino acid residues, modifications to protect reactive amino acid residues or modifications to couple amino acid residues to chemical moieties such as reactive functional groups on linkers for attachment to such amino acid residues. Exemplary modifications that can be made to the provided peptides and ligands are described in more detail herein.

[0695] Derivatives of amino acid sequences include addition variants of such sequences wherein one or more, such as at least 1, 2, 3, 4, or 5 amino acids are added or introduced into the reference sequence. Addition may occur at the C- terminus or N-terminus of the reference sequence or within the reference sequence. Variants of amino acid sequences include sequences wherein one or more amino acid such as at least 1, 2, 3, 4, or 5 amino acid residues in the reference sequence are exchanged for one or more alternative residues. Variants of amino acid sequences include sequences carrying naturally occurring amino acids and / or unnatural amino acids.

[0696] Variants, derivatives and fragments of the aforementioned amino acid sequences typically 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 function(s) 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 to NPR3. In one such embodiment, the variants, derivatives and fragments bind to the same epitope as the reference sequence. In another embodiment, the variants, derivatives and fragments retain the binding affinity of the reference sequence. Typically, variants, derivatives and fragments of a reference sequence have increased / improved activity / functionality when compared to the reference sequence.

[0697] In some embodiments a variant, derivative or fragment of an amino acid sequence is expressed in terms of its percentage identity to the reference sequence. Protocols to determine percent identity are routine procedures within the scope of those skilled in the art. Suitable methods include CLUSTAL W (Thompson et al., Nucleic Acids Research, 22(22) 4673-4680 (1994)) and iterative refinement (Gotoh, J. Mol. Biol. 264(4) 823-838 (1996)); and methods described by Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. In an exemplary method, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.), with percentage identity then calculated as: [100 x (T / L)]; wherein T = total number of identical matches and L = length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences.

[0698] In some embodiments a variant, derivative or fragment of a reference sequence has 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 more identity to the reference sequence.

[0699] Examples of such suitable modified derivatives include one or more modifications selected from: N-terminal and / or C-terminal modifications; 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 isolectronic 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 (such as 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 a spacer group; replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues; replacement of one or more amino acid residues with one or more replacement amino acids, such as an alanine, replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds within the bicyclic peptide ligand; replacement of one or more peptide bonds with a surrogate bond; peptide backbone length modification; substitution of the hydrogen on the alpha-carbon of one or more amino acid residues with another chemical group; modification of amino acids such as cysteine, lysine, glutamate / aspartate and tyrosine with suitable amine, thiol, carboxylic acid and phenol-reactive reagents so as to functionalise said amino acids; and introduction or replacement of amino acids that introduce orthogonal reactivities that are suitable for functionalisation, for example azide or alkyne-group bearing amino acids that allow functionalisation with alkyne or azide-bearing moieties, respectively.

[0700] Amino acid residues may typically be replaced with other amino acid residues of similar chemical structure, similar chemical properties or similar side-chain volume (“conservative substitutions”). The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table A above. Where amino acids have similar polarity, this can also be determined by reference to the hydropathy scale for amino acid side chains, which is well-known the person skilled in the art.

[0701] In one embodiment, the modified derivative comprises an N-terminal and / or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N- terminal modification using suitable amino-reactive chemistry, and / or C-terminal modification using suitable carboxy-reactive chemistry. In a further embodiment, said N-terminal or C- terminal modification comprises addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator, or a chromophore. In a further 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, the N-terminal residue is capped with acetic anhydride or other appropriate reagents during peptide synthesis leading to a molecule which is N-terminally acetylated. This embodiment provides the advantage of removing a potential recognition point for aminopeptidases and avoids the potential for degradation of the bicyclic peptide.

[0702] In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group which facilitates the conjugation of effector groups and retention of potency of the bicyclic peptide to its target.

[0703] In a further 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 leading to a molecule which is C-terminally amidated. This embodiment provides the advantage of removing a potential recognition point for carboxypeptidase and reduces the potential for proteolytic degradation of the bicyclic peptide.

[0704] In one embodiment, the modified derivative comprises replacement of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, nonnatural amino acids may be selected having isosteric / isoelectronic side chains which are neither recognised by degradative proteases nor have any adverse effect upon target potency.

[0705] Alternatively, non-natural amino acids may be used having constrained amino acid side chains, such that proteolytic hydrolysis of the nearby peptide bond is conformationally and sterically impeded. In particular, these concern proline analogues, bulky sidechains, Ca- disubstituted derivatives (for example, aminoisobutyric acid, Aib), and cyclo amino acids, a simple derivative being amino-cyclopropylcarboxylic acid.

[0706] In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine (Ci) and / or the C-terminal cysteine (C ).

[0707] In one embodiment, the modified derivative comprises replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues. In a further embodiment, the modified derivative comprises replacement of a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the advantage of improving the pharmaceutical stability profile of the resultant bicyclic peptide ligand.

[0708] In one embodiment, the modified derivative comprises replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged versus hydrophobic amino acid residues is an important characteristic of the bicyclic peptide ligands. For example, hydrophobic amino acid residues influence the degree of plasma protein binding and thus the concentration of the free available fraction in plasma, while charged amino acid residues (in particular arginine) may influence the interaction of the peptide with the phospholipid membranes on cell surfaces. The two in combination may influence half-life, volume of distribution and exposure of the peptide drug, and can be tailored according to the clinical endpoint. In addition, the correct combination and number of charged versus hydrophobic amino acid residues may reduce irritation at the injection site (if the peptide drug has been administered subcutaneously).

[0709] In one embodiment, the modified derivative comprises replacement of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and by a propensity of D-amino acids to stabilise p-turn conformations (Tugyi et al. (2005) PNAS, 102(2), 413-418).

[0710] In one embodiment, the modified derivative comprises removal of any amino acid residues and substitution with alanines, such as D-alanines. This embodiment provides the advantage of identifying key binding residues and removing potential proteolytic attack site(s).

[0711] It should be noted that each of the above mentioned modifications serve to deliberately improve the potency or stability of the peptide. Further potency improvements based on modifications may be achieved through the following mechanisms:

[0712] - Incorporating hydrophobic moieties that exploit the hydrophobic effect and lead to lower off rates, such that higher affinities are achieved;

[0713] - Incorporating charged groups that exploit long-range ionic interactions, leading to faster on rates and to higher affinities (see for example Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and

[0714] - Incorporating additional constraint into the peptide, by for example constraining side chains of amino acids correctly such that loss in entropy is minimal upon target binding, constraining the torsional angles of the backbone such that loss in entropy is minimal upon target binding and introducing additional cyclisations in the molecule for identical reasons.

[0715] (for reviews see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et a / ., Curr. Medicinal Chem (2009), 16, 4399-418).

[0716] Isotopic Variations

[0717] The present invention includes all pharmaceutically acceptable (radio)isotope-labelled peptide ligands of the invention, wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature, and peptide ligands of the invention, wherein metal chelating groups are attached (termed “effector”) that are capable of holding relevant (radio)isotopes, and peptide ligands of the invention, wherein certain functional groups are covalently replaced with relevant (radio)isotopes or isotopically labelled functional groups.

[0718] Examples of isotopes suitable for inclusion in the peptide ligands of the invention comprise isotopes of hydrogen, such as2H (D) and3H (T), carbon, such as11C,13C and14C, chlorine, such as36CI, fluorine, such as18F, iodine, such as123l,125l and1311, nitrogen, such as13N and15N, oxygen, such as15O,17O and18O, phosphorus, such as32P, sulphur, such as S, copper, such as64Cu, gallium, such as67Ga or68Ga, yttrium, such as90Y and lutetium, such as177Lu, and Bismuth, such as213Bi.

[0719] Certain isotopically-labelled peptide ligands of the invention, for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies, and to clinically assess the presence and / or absence of the target on diseased tissues. The peptide ligands of the invention can further have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes or receptors. The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The radioactive isotopes tritium, i.e.3H (T), and carbon-14, i.e.14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0720] Substitution with heavier isotopes such as deuterium, i.e.2H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

[0721] Substitution with positron emitting isotopes, such as11C,18F,15O and13N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.

[0722] Isotopically-labelled compounds of peptide ligands of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using an appropriate isotopically-labelled reagent in place of the non-labelled reagent previously employed.

[0723] Molecular Scaffold

[0724] In one embodiment, the molecular scaffold comprises a non-aromatic molecular scaffold. References herein to “non-aromatic molecular scaffold” refers to any molecular scaffold as defined herein which does not contain an aromatic (i.e. unsaturated) carbocyclic or heterocyclic ring system.

[0725] Suitable examples of non-aromatic molecular scaffolds are described in Heinis et al. (2014) Angewandte Chemie, International Edition 53(6) 1602-1606.

[0726] As noted in the foregoing documents, the molecular scaffold may be a small molecule, such as a small organic molecule.

[0727] In one embodiment the molecular scaffold may be a macromolecule. In one embodiment the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates.

[0728] In one embodiment the molecular scaffold comprises reactive groups that are capable of reacting with functional group(s) of the polypeptide to form covalent bonds.

[0729] The molecular scaffold may comprise chemical groups which form the linkage with a peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

[0730] In one embodiment, the molecular scaffold is 1 ,1',1"-(1 ,3,5-triazinane-1 ,3,5-triyl)triprop-2-en- 1-one (also known as triacryloylhexahydro-s-triazine (TATA):

[0731] TATA.

[0732] Thus, following cyclisation with the bicyclic peptides of the invention on the reactive groups of the peptide (e.g. on RGi, RG2 and RG3; e.g. on the Ci, CH, and Cui cysteine residues), the molecular scaffold forms a tri-substituted 1 ,T,1"-(1,3,5-triazinane-1,3,5-triyl)tripropan-1-one derivative of TATA having the following structure: wherein * denotes the point of attachment of the three cysteine residues.

[0733] In an alternative embodiment, the molecular scaffold is 1 , 1 1 "-(1 ,3,5-triazinane-1 ,3,5-triyl) tris(2-bromoethanone) (TATB). Thus, following cyclisation with the bicyclic peptides of the invention on the Ci, CH, and C cysteine residues, the molecular scaffold forms a tri-substituted derivative of TATB having the following structure:

[0734] Synthesis

[0735] The peptides of the present invention may be manufactured synthetically by standard techniques followed by reaction with a molecular scaffold in vitro. When this is performed, standard chemistry may be used. This enables the rapid large scale preparation of soluble material for further downstream experiments or validation. Such methods could be accomplished using conventional chemistry such as that disclosed in Timmerman et al. (supra).

[0736] Thus, the invention also relates to the manufacture of polypeptides or conjugates selected as set out herein, wherein the manufacture comprises optional further steps as explained below. In one embodiment, these steps are carried out on the end product polypeptide / conjugate made by chemical synthesis.

[0737] Optionally amino acid residues in the polypeptide of interest may be substituted when manufacturing a conjugate or complex.

[0738] Peptides can also be extended, to incorporate for example another loop and therefore introduce multiple specificities.

[0739] To extend the peptide, it may simply be extended chemically at its N-terminus or C-terminus or within the loops using orthogonally protected lysines (and analogues) using standard solid phase or solution phase chemistry. Standard (bio)conjugation techniques may be used to introduce an activated or activatable N- or C-terminus. Alternatively, additions may be made by fragment condensation or native chemical ligation e.g. as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779), or by enzymes, for example using subtiligase as described in (Chang et al. Proc Natl Acad Sci II S A. 1994 Dec 20; 91 (26): 12544-8 or in Hikari etal. Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).

[0740] Alternatively, the peptides may be extended or modified by further conjugation through disulphide bonds. This has the additional advantage of allowing the first and second peptide to dissociate from each other once within the reducing environment of the cell. In this case, the molecular scaffold (e.g. TATA or TATB) could be added during the chemical synthesis of the first peptide so as to react with the three cysteine groups; a further cysteine or thiol could then be appended to the N- or C-terminus of the first peptide, so that this cysteine or thiol only reacted with a free cysteine or thiol of the second peptide, forming a disulphide-linked bicyclic peptide-peptide conjugate.

[0741] Furthermore, addition of other functional groups or effector groups may be accomplished in the same manner, using appropriate chemistry, coupling at the N- or C-termini or via side chains. In one embodiment, the coupling is conducted in such a manner that it does not block the activity of either entity.

[0742] In some embodiments a polypeptide as provided herein may be synthesised by solid-phase synthesis. In some embodiments, the synthesis of a peptide ligand as provided herein may comprise solid-phase synthesis of a polypeptide as described herein. In some embodiments the solid-phase synthesis comprises Fmoc solid-phase peptide synthesis (e.g. as described in more detail herein). In some embodiments the synthesized polypeptide is cyclised with a molecular scaffold as described herein. In some embodiments the cyclised scaffold is purified e.g. by lyophilisation.

[0743] Half-Life Extending (HLE) Moieties

[0744] It will be appreciated that the HLE moiety may comprise any suitable agent capable of extending the half-life of the resultant complex. References to “extending the half-life” include any agent which is capable of increasing the duration of time before the resultant complex is metabolised within the host compared to the duration of time for a complex where the HLE moiety is absent. The half-life may for example by the plasma half-life following administration of the complex to a subject.

[0745] It will be readily apparent to the skilled person that HLE moieties are well known in the art. Any suitable half-life extender may be used. As those skilled in the art will appreciate, a HLE moiety can also be referred to as an “Exposure Modifier” or “Exposure Modifying moiety” (EM moiety). In one embodiment, the HLE moiety is a moiety capable of binding to albumin, such as serum albumin, in particular human serum albumin (HSA). HSA is the most abundant protein in the plasma (~ 40g / L). It has remarkable solubility and stability and crucially has an approximately 19 day half-life in humans. HSA has the ability to bind a large diversity of hydrophobic small molecules, such as up to 7 long chain fatty acids simultaneously and has two known binding sites for medium chain fatty acids.

[0746] Examples of albumin binders include:

[0747] (i) Short chain fatty acids (i.e. those with five or fewer carbon atoms) such as butyric (C4) acid;

[0748] (ii) Medium chain fatty acids (i.e. those with 6 to 12 carbon atoms) such as caproic (C6) acid, caprylic (C8) acid, capric (C10) acid or lauric (C12) acid;

[0749] (iii) Long chain fatty acids (i.e. those with 13 to 21 carbon atoms) such as myristic (C14) acid, palmitic (C16) acid, stearic (C18) acid, arachidic (C20) acid;

[0750] (iv) Very long chain fatty acids (i.e. those with 22 or more carbon atoms) such as behenic (C22) acid, lignoceric (C24) and cerotic (C26) acid;

[0751] (v) Genentech’s small molecule FVIIa peptide-albumin binding inhibitors (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;

[0752] (vi) Fmoc protecting groups which have been shown to demonstrate a significant reduction in hydrolysis of compounds in the presence of HSA (I nt J Pep Res Ther, 2007, 13(1-2), 12);

[0753] (vii) Coumarin derivatives, such as warfarin type coumarin derivatives which have been shown to improve the in vivo half-life in rats;

[0754] (viii) Evans Blue which binds to the Sudlow site I of HSA with low micromolar affinity (2.5pM) and has a long half-life in blood (Theranostics, 2016, 6(2), 243-53);

[0755] (ix) Diflunisal which binds to sites I and II of HSA with affinities of ~ 3pM (J Med Chem 2017, 60(17), 7434-46);

[0756] (x) Indomethacin which binds mainly to site I of HSA with an affinity of 0.7pM (J Med Chem 2017, 60(17), 7434-46);

[0757] (xi) Lithocholic acid (Bile acid) which is an example of an endogenous molecule and when conjugated to insulin was capable of controlling glucose levels for more than 24 hours (Pharm Res 2006, 23(1), 49-55);

[0758] (xii) 4-(p-lodophenyl)butyric acid (Albutag) and related phenylbutyric acid examples which 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 (Mol Pharmaceutics 2018, 15, 2297-306), a NODAGA analogue (Cu) (Mol Pharmaceutics 2018, 15, 5556-64) or those described in EP 3 778 592;

[0759] (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);

[0760] (xv) Liraglutide (Diabetes Care 2002; 25:1398-1404);

[0761] (xvi) Semaglutide (Med Chem; 2015, 58, 7370-7380);

[0762] (xvii) Knob Domain Antibody Fragments;

[0763] (xviii) Pegylated compounds such as those described in WO 2020 / 205948;

[0764] (xix) Small molecule aromatic containing compounds such as those described in WO 2008 / 053360; and

[0765] (xx) PPB03 (Bioconjugate Chem 2017, 28(9), 2372-2383).

[0766] In one particular embodiment, the HLE is selected from:

[0767] (i) Short chain fatty acids (i.e. those with five or fewer carbon atoms) such as butyric (C4) acid;

[0768] (ii) Medium chain fatty acids (i.e. those with 6 to 12 carbon atoms) such as caproic (C6) acid, caprylic (C8) acid, capric (C10) acid or lauric (C12) acid;

[0769] (iii) Long chain fatty acids (i.e. those with 13 to 21 carbon atoms) such as myristic (C14) acid, palmitic (C16) acid, stearic (C18) acid, arachidic (C20) acid; and

[0770] (iv) Very long chain fatty acids (i.e. those with 22 or more carbon atoms) such as behenic (C22) acid, lignoceric (C24) and cerotic (C26) acid.

[0771] In a further particular embodiment, the HLE is selected from:

[0772] (ii) Medium chain fatty acids (i.e. those with 6 to 12 carbon atoms) such as caproic (C6) acid, caprylic (C8) acid, capric (C10) acid or lauric (C12) acid; and

[0773] (iii) Long chain fatty acids (i.e. those with 13 to 21 carbon atoms) such as myristic (C14) acid, palmitic (C16) acid, stearic (C18) acid, arachidic (C20) acid.

[0774] In a yet further particular embodiment, the HLE is selected from:

[0775] (iii) Long chain fatty acids (i.e. those with 13 to 21 carbon atoms) such as myristic (C14) acid, palmitic (016) acid, stearic (018) acid, arachidic (020) acid.

[0776] In one particular embodiment, the HLE is selected from a peptide, a cyclic peptide, a bicyclic peptide, a fatty acid, or a lipid. In one particular embodiment, the HLE moiety is a fatty acid, such as a long chain fatty acid, in particular 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- or branched-chain fatty acid. Data is presented herein in Example 3, Figure 2 and Table 2 which shows that an HLE containing bicyclic peptide ligand complex containing octadecanoic acid (B192) demonstrated an increased half-life of 7.8 hours (compared with 0.28 hours for an equivalent complex lacking an HLE, B191).

[0777] 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 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 comprises a PEG.

[0778] In some embodiments the HLE moiety comprises 1 , 2, 3 or more fatty acid groups. For example, in some embodiments the HLE moiety comprises a bis-lipid group. In some embodiments the bis-lipid comprises two fatty acid groups conjugated to the bicyclic peptide ligand. In some embodiments a bis-lipid group is of the following form wherein the wiggly line represents the point of attachment to the compound, n is an integer from about 1 to about 10 and each m is independently an integer of from about 6 to about 20, such as about 10 to about 15. Examples of suitable fatty acid groups include C16 fatty acids. In some embodiments a bis-lipid group is of the following form wherein the wiggly line represents the point of attachment to the compound and n is an integer from about 1 to about 10.

[0779] In some embodiments the HLE moiety comprises myristic acid, hexadecenoic acid, octadecanoic acid, octadecanoic diacid, palmitic acid, palmitic diacid, 16-tetrazole- hexadecanoic acid, 16-sulfo-hexadecanoic acid, docosanoic diacid, 1,2-distearoyl-sn- glycero-3-phosphoethanolamine, lauric acid, dimeric lauric acid, tetrameric lauric acid, or a, w-octadecanoic diacid. In some embodiments the HLE moiety comprises a lipid conjugate or and substituent as described in any of WO2016161374, WO2019217459, WO2020257194, WO2021092371, WG2023064530, or WO2024129748.

[0780] In some embodiments the HLE moiety comprises a fatty acid and a PEG. In some embodiments the HLE moiety comprises a fatty acid conjugated to a PEG. In some embodiments the HLE moiety comprises a C4-C26 fatty acid e.g. a 04, 06, 08, 010, 012, 014, 016, 018 or 020 fatty acid conjugated to a PEG. Is the PEG moiety comprises from about 2 to about 50, e.g. from about 3 to about 20 such as from about 4 to about 10 PEG monomer units. In some embodiments the PEG moiety is interrupted by a linking moiety such as an amide group. In some embodiments the PEG moiety comprises a group of formula: (PEG)n-L-(PEG)m wherein n and m are each independently an integer of from about 1 to about 10, e.g. about 2 to about 5, e.g. 2, 3, 4 or 5; and wherein L is a linking group. In some embodiments L is a linking moiety comprising an amide, a carbamate, or a carbonyl group. In some embodiments the PEG moiety comprises (PEG)2-CH2-C(O)NH- (PEG)2.

[0781] In some embodiments the HLE moiety comprises a moiety of formula:

[0782] [Fatty Acid]-[linker 1]-[PEG]-[Terminus]; wherein

[0783] [Fatty Acid] is a C4-C26 fatty acid;

[0784] [linker 1] is a linker; for example [linker 1] may consist or comprise of an amino acid or artificial amino acid, such as gGlu;

[0785] [PEG] is a PEG moiety comprising from 2 to about 50 PEG units and being optionally interrupted by one or more linking moieties; for example wherein [PEG] comprises (PEG)2- CH2-C(O)NH-(PEG)2; [Terminus] is an optionally present terminating group which may be used to link the HLE moiety to a bicyclic peptide ligand as described herein, such as an amino acid or artificial amino acid; such as lysine.

[0786] For example, in some embodiments the HLE moiety comprises a 04, 06, 08, 010, 012, 014, 016, 018, 020, 022, 024, 026, 028 or 030 fatty acid (e.g. a 012, 014, 016, 018, 020, 022 or 024 fatty acid, e.g. a 016, 018 or 020 fatty acid, e.g. a 018 fatty acid) attached via an amino acid (e.g. gGlu) to a PEG moiety of formula (PEG)n-L-(PEG)m, wherein n and m are each independently an integer of 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 formula -CH2-C(O)NH-; and wherein the PEG moiety is attached via an amide bond to the side chain of a lysine amino acid which may be attached to or comprised as the N- or 0- terminus of a polypeptide comprised in a bicyclic peptide ligand as described herein. An example is the HLE moiety used in compound B192 described in example 3:

[0787] Terminus

[0788] Wherein [Terminus] is an optionally present terminating group which may be used to link the

[0789] HLE moiety to a bicyclic peptide ligand as described herein, such as an amino acid or artificial amino acid; such as lysine. When [Terminus] is a lysine residue comprised in or attached to the bicyclic peptide ligand, the HLE moiety may be of the form:

[0790] In one particular embodiment, the HLE moiety is an albumin binding peptide, such as SA21. SA21 has the amino acid sequence [Ac]RLIEDICLPRWGCLWEDD[CONH2] wherein the cysteine residues are disulphide cyclised; the N-terminal acetyl group and / or the C-terminal amide group may be present or absent. In some embodiments wherein the HLE moiety is an albumin binding peptide, the albumin binding peptide is a derivative of SA21 comprising a reactive group for conjugation to a bicyclic peptide ligand as described herein. Any suitable reactive group can be used. In some embodiments a reactive group is present on a reactive amino acid which may be inserted to the sequence of SA21, for example at the N-terminus or the C-terminus, typically the N-terminus. A suitable reactive group is an azide group which may be used for example in a click reaction with a corresponding alkyne group attached to a bicycle moiety, for example an alkyne group comprised in a [K(PYA)] (N- pentynoyl-L-lysine) group. In some embodiments an azide group may be attached to an amino acid such as lysine, e.g. in a [K(N3)] group. The [K(N3)] group may be present at the N- or C- terminus, e.g. the N-terminus, of the SA21 peptide. In some embodiments the amino acid comprising the reactive group, e.g. the [K(N3)] group, is attached to the N- terminus of the SA-21 peptide via a spacer, such as a further amino acid, e.g. glycine or lysine. Accordingly, in some embodiments the HLE moiety is a derivative of SA-21 (i.e. an albumin-binding derivative) such as ([Ac][K(N3)]GRLIEDICLPRWGCLWEDD (SEQ ID NO: 119). Data is presented herein in Example 3, Figure 3 and Table 3 which shows that an HLE containing bicyclic peptide ligand complex containing SA21 (B193) demonstrated an increased half-life of 22.6 hours (compared with 0.28 hours for an equivalent complex lacking an HLE, B191). In some embodiments the HLE moiety is a variant of SA-21 comprising one or more modifications as described herein in the context of variants of polypeptides comprised in bicyclic peptide ligands; for example one or more amino acid modifications may be made to the SA-21 sequence.

[0791] In some embodiments a HLE moiety is ABDcon or a variant thereof. ABDcon has the amino acid sequence LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA. In some embodiments a HLE moiety is ABDcon-azide. ABDcon-azide has an amino acid sequence [K(N3)]LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA. In some embodiments the HLE moiety is a variant of ABDcon or ABDcon-azide comprising one or more modifications as described herein in the context of variants of polypeptides comprised in bicyclic peptide ligands; for example one or more amino acid modifications may be made to the ABDcon-azide sequence.

[0792] In some embodiments a HLE moiety is ABM00001 or a variant thereof. ABM00001 has the amino acid sequence GVSDFYKNLINRAKTVEGVHALIGHI. In some embodiments a HLE moiety is PP001 or a variant thereof. PP001 has the amino acid sequence LASAKEAANAELDAYGVSDFYKRLIDKAKTVEGVEALKDAILAALP. In some embodiments a HLE moiety is PEP or a variant thereof. PEP has the amino acid sequence DITGAALLEAKEAAINELKQYGISDYYVTLINKAKTVEGVNALKAEILSALP. In some embodiments a HLE is PEP-X or a variant thereof. PEP-X has the amino acid sequence DITGAALLEAKDAAINELKQYGISDYYVHLINKADTVEGVNALKDEILSALP. In some embodiments a variant may comprise one or more modifications as described herein in the context of variants of polypeptides comprised in bicyclic peptide ligands, such as one or more amino acid modifications.

[0793] In some embodiments, the HLE moiety is an FN3 domain, e.g. as disclosed in 11 ,203,630 or WO 2017 / 210425, which are each hereby incorporated by reference. In some embodiments the HLE moiety is an albumin-binding peptide, e.g. an albumin-binding peptide disclosed in any one of WO 2009 / 016043, WO 2012 / 004384, WO 2013 / 177398, WO 2014 / 048977 or WO 2015 / 091957, which are each hereby incorporated by reference. In some embodiments, the HLE moiety is an ankyrin repeat domain, e.g. as disclosed in WO 2016 / 156596, which is hereby incorporated by reference. In some embodiments, the HLE moiety is a VHH domain of an albumin-binding antibody, as disclosed in WO 2017 / 201488 or WO2018 / 104444, which are each hereby incorporated by reference. In some embodiments the HLE moiety is a VHH domain of an albumin-binding antibody and has an amino acid sequence EVQLVESGGGLVQPGNSLRLSCAASGFTFSRFGMSWVRQAPGKGLEWV SSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIgGSLSRSSQGTLV TVSS or a variant thereof. In some embodiments, the HLE is an ScFv, e.g. as disclosed in WO20 12 / 162068, which is hereby incorporated by reference. In some embodiments, the HLE moiety is a nanofitin, e.g. as disclosed in WO 2022 / 171852, which is hereby incorporated 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 is Fclll (DCtAWHLGELVWCtT) or a variant thereof; or is PAM (GK(K(YTR)YTR)K(YTR)YTR) or a variant thereof; or is 15-IGP (Ac- DCtAYHKGELVWCtTFH-NH2) or a variant thereof. In some embodiments the HLE moiety is a FcRn binding moiety. In some embodiments the FcRn moiety is Mezo_1 (QRFCtTGHFGGLYPCtNGP) or a variant thereof; Mezo_283 (C2H4-[(C(O)NH-RF[Pen]*- TGHFT-[Sar]-[NMeL]-YPCt]2) or a variant thereof; BM07918 (EQDAAAHEIRWLPNLTFDQRVAFIHKLAD) or a variant thereof; ZFcRn_2 (VDAKYAKEQDAAAHEIRWLPNLTFDQRVAFIHKLADDPSQSSELLSEAKKLNDSQAPK) or a variant thereof; FcBP(Y286H) (QRFCtTGHFGGLHPCtNG) or a variant thereof; or SYN1327 (Ac-RF-[Pen] t-TGHFG-[Sar]-[NMeL]-YPCt) or a variant thereof; wherein indicates that two amino acids are joined by a disulfide bond.

[0794] In some embodiments the FcRn binding moiety comprises a peptide (e.g. a cyclic peptide), e.g. as disclosed in WO 2007 / 098420, WO 2014 / 205072 or WO 2014 / 140366, each of which are hereby incorporated by reference. In some embodiments a variant may comprise one or more modifications as described herein in the context of variants of polypeptides comprised in bicyclic peptide ligands, such as one or more amino acid modifications.

[0795] In some embodiments the HLE moiety increases the half-life (e.g. the plasma half-life) of the bicycle peptide ligand by a factor or at least 2, 3, 4, 5, 10, 20, 50, 100, 200 or 500 or more relative to the half-life of the bicycle peptide ligand in the absence of the HLE moiety. It will be appreciated that the HLE moiety may be directly bonded to the bicyclic peptide ligand(s), i.e. via a covalent bond or there may be a spacer or linker present between the bicyclic peptide ligand(s) and the HLE moiety. The design and construction of said spacer or linker will be readily apparent to one skilled in the art.

[0796] Pharmaceutical Compositions

[0797] According to a further aspect of the invention, there is provided a pharmaceutical composition comprising the a bicyclic peptide ligand or bicyclic peptide ligand complex as defined herein in combination with one or more pharmaceutically acceptable excipients.

[0798] Generally, the present peptide ligands and complexes will be utilised in purified form together with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

[0799] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

[0800] The peptide ligands and complexes of the present invention may be used as separately administered compositions or in conjunction with other agents. These can include antibodies, antibody fragments and various immunotherapeutic drugs, such as cyclosporine, methotrexate, adriamycin or cisplatinum and immunotoxins. Further examples of other agents which may be administered separately or in conjunction with the peptide ligands of the invention include cytokines, lymphokines, other hematopoietic factors, thrombolytic and anti-thrombotic factors. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in conjunction with the protein ligands of the present invention, or even combinations of selected polypeptides according to the present invention having different specificities, such as polypeptides selected using different target ligands, whether or not they are pooled prior to administration. The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, the peptide ligands of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. Preferably, the pharmaceutical compositions according to the invention will be administered intravenously. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.

[0801] The peptide ligands and complexes of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that levels may have to be adjusted upward to compensate.

[0802] The compositions containing the present peptide ligands and complexes or a cocktail thereof can be administered for prophylactic and / or therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically-effective dose". Amounts needed to achieve this dosage will depend upon the severity of the disease, but generally range from 0.005 to 5.0 mg of selected peptide ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg / kg / dose being more commonly used. For prophylactic applications, compositions containing the present peptide ligands or cocktails thereof may also be administered in similar or slightly lower dosages.

[0803] A composition containing a peptide ligand or complexes according to the present invention may be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal. In addition, the peptide ligands described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells. Blood from a mammal may be combined extracorporeally with the selected peptide ligands whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques. Payloads

[0804] In one embodiment, the bicyclic peptide ligand complex additionally comprises a payload which is other than an antisense oligonucleotide (ASO).

[0805] Thus, according to a further aspect of the invention there is provided a bicyclic peptide ligand complex which comprises:

[0806] (a) one or more bicyclic peptide ligands specific for transferrin receptor 1 (TfR1) comprising a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold;

[0807] (b) a half-life extending (HLE) moiety; and

[0808] (c) a payload which is other than an antisense oligonucleotide (ASO).

[0809] It will be appreciated that the payload can be any suitable agent which is desired to be delivered across the blood-brain barrier and into the brain. For example, the bicyclic peptide ligand complex of the invention can be considered as being a vehicle to deliver the attached payload across the blood-brain barrier via the transcytotic pathway involving Tfr1. Therefore, the payload can be any agent which is required to be presented to the brain. It can be envisaged that such purposes can include, inter alia, therapy, diagnosis or imaging. For example, in one embodiment the payload comprises a therapeutic agent which is other than an antisense oligonucleotide (ASO). In an alternative embodiment, the payload comprises a diagnostic agent which is other than an antisense oligonucleotide (ASO). In an alternative embodiment, the payload comprises an imaging agent which is other than an antisense oligonucleotide (ASO). In some embodiments the payload is an effector moiety as described herein. In some embodiments the effector moiety is selected from one or more cytotoxic agents, radiochelators, or chromophores.

[0810] As with the HLE moiety, it will be appreciated that the payload may be directly bonded to the bicyclic peptide ligand(s) / HLE moiety complex, i.e. via a covalent bond or there may be a spacer or linker present between the bicyclic peptide ligand(s) / HLE moiety complex and the payload. The design and construction of said spacer or linker will be readily apparent to one skilled in the art.

[0811] Therapy It will be appreciated that when the payload comprises a therapeutic agent it may comprise any suitable agent (other than an antisense oligonucleotide (ASO)) capable of preventing, suppressing or treating a disease or disorder through TfR1 mediated delivery of a therapeutic agent. Examples of suitable therapeutic agents include: small molecules (inhibitors, agonists and antagonists, ion channel modulators), peptides and bicyclic peptide ligands, antibodies and fragments thereof (such Fv, Fab and Fc), proteins and lipid nanobodies.

[0812] Provided herein is therefore the a bicyclic peptide ligand as described herein for use in delivery of a payload to a subject in need thereof. Also provided is a complex comprising a bicyclic peptide ligand as described herein and a half-life extending (HLE) moiety as described herein for use in delivery of a payload to a subject in need thereof. In some embodiments the payload is an effector moiety as described in more detail herein.

[0813] Thus, according to a further aspect of the invention, there is provided a bicyclic peptide ligand complex which comprises:

[0814] (a) one or more bicyclic peptide ligands specific for transferrin receptor 1 (TfR 1 ) comprising a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold;

[0815] (b) a half-life extending (HLE) moiety; and

[0816] (c) a payload which is a therapeutic agent other than an antisense oligonucleotide (ASO), for use in preventing, suppressing or treating a disease or disorder through TfR1 mediated delivery of a therapeutic agent.

[0817] Also provided is the use of a complex comprising (a) one or more bicyclic peptide ligands as described herein; (b) a half-life extending (HLE) moiety as described herein and (c) a payload as described herein; in the manufacture of a medicament for preventing, suppressing or treating a disease or disorder through TfR1 mediated delivery of a therapeutic agent. Also provided is a method of delivering a therapeutic agent to a subject in need thereof, 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 halflife extending (HLE) moiety as described herein and (c) a payload as described herein. An example of a disease or disorder which may be prevented, suppressed or treated through TfR1 mediated delivery of a therapeutic agent includes a neurological disorder.

[0818] Examples of such neurological disorders include but are not limited to: a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioural disorder, and CNS inflammation.

[0819] In one embodiment, the neurological disorder is in a human subject. It will be appreciated that the dose amount and / or frequency of administration is modulated to reduce the concentration of peptide ligand to which the red blood cells are exposed. In a further embodiment, the treatment further comprises the step of monitoring the human subject for depletion of red blood cells.

[0820] References herein to the term "prevention" involves administration of the protective composition prior to the induction of the disease. "Suppression" refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease. "Treatment" involves administration of the protective composition after disease symptoms become manifest.

[0821] Animal model systems which can be used to screen the effectiveness of the peptide ligands in protecting against or treating the disease are available. The use of animal model systems is facilitated by the present invention, which allows the development of polypeptide ligands which can cross react with human and animal targets, to allow the use of animal models.

[0822] Diagnosis

[0823] It will be appreciated that when the payload comprises a diagnostic agent it may comprise any suitable agent (other than an antisense oligonucleotide (ASO)) capable of providing information regarding the status of the individual, i.e. diagnosis.

[0824] Thus, according to a further aspect of the invention, there is provided a bicyclic peptide ligand complex which comprises:

[0825] (a) one or more bicyclic peptide ligands specific for transferrin receptor 1 (TfR1) comprising a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold; (b) a half-life extending (HLE) moiety; and

[0826] (c) a payload which is a diagnostic agent other than an antisense oligonucleotide (ASO), for use in diagnosis.

[0827] Also provided is the use of a complex comprising (a) one or more bicyclic peptide ligands as described herein; (b) a half-life extending (HLE) moiety as described herein and (c) a payload as described herein; in the manufacture of an agent for diagnosing a disease or disorder in a subject in need thereof. In some embodiments the payload is an effector moiety as described herein. Also provided is a method of diagnosing a disease or disorder in a subject, 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 halflife extending (HLE) moiety as described herein and (c) a payload as described herein. In some embodiments the diagnosing step comprises taking one or more measurements characteristic of the subject, e.g. characteristic of TfR1 function in the subject.

[0828] One particular example of a diagnostic agent includes a detectable moiety which typically comprises a dye, radiolabel or chromophore. Such an agent demonstrates the advantage of providing diagnostic information from within the brain, which may also comprise imaging information.

[0829] As used herein, the term “detectable moiety” is used interchangeably with the term "label" and relates to any moiety capable of being detected, e.g., primary labels and secondary labels. Primary labels, such as radioisotopes (e.g., tritium,225Ac,227Ac,241Am,72As,74As,211At,198Au,11B,7Be,212Bi,213Bi,75Br,77Br,11C,14C,48Ca,109Cd,139Ce,141Ce,252Cf,55Co,57Co,60Co,51Cr,130Cs,131Cs,137Cs,61Cu,62Cu,64Cu,67Cu,165Dy,152Eu,155Eu,18F,55Fe,59Fe,64Ga,67Ga,68Ga,153Gd,68Ge,122l,123l,124l,125l,131l,132l,111ln,115mln,191mlr,192lr,81mKr,177Lu,51Mn,52Mn,99Mo,13N,95Nb,15O,191Os,1940s,32P,33P,203Pb,212Pb,103Pd,109Pd,238Pu,223Ra,226Ra,82Rb,186Re,188Re,105Rh,97Ru,103Ru,35S,46Sc,47Sc,72Se,75Se,28Si,145Sm,153Sm,117mSn,85Sr,89Sr,90Sr,178Ta,179Ta,182Ta,149Tb,96Tc,99mTc,228Th,229Th,201TI,170Tm,171Tm,188W,127Xe,133Xe,88Y,90Y 9iy169Yb,62Zn,65Zn,89Zr or95Zr, wherein a superscripted m denotes a meta-state), masstags, and fluorescent labels are signal generating reporter groups which can be detected without further modifications. Detectable moieties also include luminescent and phosphorescent groups.

[0830] The term “secondary label” as used herein refers to moieties such as biotin and various protein antigens that require the presence of a second intermediate for production of a detectable signal. For biotin, the secondary intermediate may include streptavidin-enzyme conjugates. For antigen labels, secondary intermediates may include antibody-enzyme conjugates. Some fluorescent groups act as secondary labels because they transfer energy to another group in the process of nonradiative fluorescent resonance energy transfer (FRET), and the second group produces the detected signal.

[0831] The terms “fluorescent label”, “fluorescent dye”, and “fluorophore” as used herein refer to moieties that absorb light energy at a defined excitation wavelength and emit light energy at a different wavelength. 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, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591 , BODIPY 630 / 650, BODIPY 650 / 665), Carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5, Cy7, Cy7.5), Dansyl, Dapoxyl, Dialkylaminocoumarin, 4',5'-Dichloro-2',7'-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin, Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800), JOE, Lissamine 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'-Tetra- bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR), Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X.

[0832] The term “mass-tag” as used herein refers to any moiety that is capable of being uniquely detected by virtue of its mass using mass spectrometry (MS) detection techniques. Examples of mass-tags include electrophore release tags such as N-[3-[4’-[(p- Methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceronyl]isonipecotic Acid, 4’-[2, 3,5,6- Tetrafluoro-4-(pentafluorophenoxyl)]methyl acetophenone, and their derivatives. The synthesis and utility of these mass-tags is described in United States 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 mass-tags include, but are not limited to, nucleotides, dideoxynucleotides, oligonucleotides of varying length and base composition, oligopeptides, oligosaccharides, and other synthetic polymers of varying length and monomer composition. A large variety of organic molecules, both neutral and charged (biomolecules or synthetic compounds) of an appropriate mass range (100-2000 Daltons) may also be used as mass-tags.

[0833] The term “quantum dot” as used herein refers to any moiety that is a highly luminescent semiconductor nanocrystal (e.g. zincsulfide-capped cadmium selenide). The synthesis and utility of these quantum dots is described in United States Patents 6,326,144, 6,468,808, 7,192,785, 7,151,047, and in the scientific literature (see: Chan and Nie (1998) Science 281(5385) 2016-2018).

[0834] Imaging

[0835] It will be appreciated that when the payload comprises imaging agent it may comprise any suitable agent (other than an antisense oligonucleotide (ASO)) capable of providing imaging information relating to the individual.

[0836] Thus, according to a further aspect of the invention, there is provided a bicyclic peptide ligand complex which comprises:

[0837] (a) one or more bicyclic peptide ligands specific for transferrin receptor 1 (TfR1) comprising a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold;

[0838] (b) a half-life extending (HLE) moiety; and

[0839] (c) a payload which is an imaging agent other than an antisense oligonucleotide (ASO), for use in diagnosis.

[0840] One particular example of an imaging agent includes a detectable moiety which typically comprises a dye, radiolabel or chromophore. Such an agent demonstrates the advantage of providing imaging information from within the brain.

[0841] The invention is further described below with reference to the following examples.

[0842] EXAMPLES

[0843] Preparation of HLE Containing Tfr1 Bicyclic Peptide Ligand Complexes

[0844] Example 1: Preparation of B192

[0845]

[0846] Step 1

[0847] The title compound was synthesized using standard Fmoc chemistry. Rink amide MBHA resin (0.5mmol, 1 eq, 0.3mmol / g, 1.67g resin) in DMF was agitated with N2 for 2 hours at 20°C then filtered to collect the resin. For each step of the peptide synthesis, sequential deprotection, followed by coupling was performed.

[0848] Deprotection: 20% piperidine in DMF (50ml) was added to the resin and agitated with N2 for 15 mins. The resin was washed with DMF (50ml x 5) and filtered to isolate the resin. Coupling: A solution of Fmoc-Lys(Dde)-OH (1.5 eq), diisopropylethylamine (DIEA) (3.0 eq) and HATLI (1.425 eq) in DMF (15.0 mL) was added to the resin and agitated with N2 for 30 mins at 20 °C. The resin was then washed with DMF (50ml x 5).

[0849] The deprotection I coupling sequence was repeated with the following amino acids: 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 (3eq) and DIEA (4eq) were added, and the reaction was stirred for 15 mins. This was washed with DMF (50ml x 5).

[0850] Deprotection: 3% hydrazine in DMF (50ml) was added and the resin was agitated with N2 for another 15 mins. The resin was washed with DMF (50ml x 5) and filtered to obtain the resin.

[0851] Coupling: Fmoc-PEG2-OH (1.5 eq), DIEA (3.0 eq) and HATLI (1.425 eq) in DMF was added to the resin and agitated with N2 for 30 mins at 20 °C. The resin was then washed with DMF (50m x 5).

[0852] Deprotection: 20% piperidine in DMF (50ml) was added to the resin and agitated with N2 for 15 mins.

[0853] Coupling: Fmoc-PEG2-OH (1.5 eq), DIEA (3.0 eq) and HATLI (1.425 eq) in DMF was added to the resin and agitated with N2 for 30 mins at 20 °C. The resin was then washed with DMF (50ml x 5).

[0854] Deprotection: 20% piperidine in DMF (50ml) was added to the resin and agitated with N2 for 15 mins.

[0855] Coupling: A solution of Fmoc-g-Glu-OH (3 eq), DIEA (6.0 eq) and HATLI (1.425 eq) in DMF (15.0 ml) was added to the resin and agitated with N2 for 30 mins at 20 °C. The resin was then washed with DMF (50ml x 5).

[0856] Deprotection: 20% piperidine in DMF (50ml) was added to the resin and agitated with N2 for 15 mins.

[0857] Coupling: A solution of 18-(tert-butoxy)-18-oxooctadecanoic acid (1.5 eq), DIEA (6.0 eq) and HATLI (1.425 eq) in DMF (15.0 ml) was added to the resin and agitated with N2 for 30 mins at 20 °C. The resin was then washed with DMF (50ml x 5).

[0858] Cleavage solution (45ml, 85% TFA / 2.50% TIS / 2.50% H2O / 10% 3-mercaptopropionic acid) was added to the flask containing resin at room temperature and stirred for 2 hrs. The peptide was precipitated with cold isopropyl ether (230ml). Filter and collect the filter cake. The filter cake was washed with isopropyl ether (230ml x2). The crude peptide was dried under vacuum for 2 hrs to get a crude solid (900.0 mg).

[0859] Step 2

[0860] To a solution of linear peptide (230 mg) and TATB (36.5 mg, 0.95 eq) in H2O (160.0 mL) and acetonitrile (70mL) CS2CO3 (aq. 1 M) was slowly added to adjust the reaction mixture pH to 8-9. The mixture was stirred at 25°C for 90 mins. 1 M HCI (aq.) was added dropwise until the solution reached pH = 6-7. The mixture was lyophilized to give the crude bicyclic peptide. The crude peptide was purified by preparative HPLC (TFA conditions: A: 0.075 % TFA in H2O, B: MeCN) to give the final product B192 (64.1 mg, 97.61 % purity, TFA salt) as a white solid and confirmed by LCMS (Rt = 1.266 min, MS cal.: 2899.35, MS observed: [M+2H]2+= 1450.17)

[0861] Example 2: Preparation of B193

[0862] B193 can also be depicted as follows: Step 1: Preparation ofB194

[0863] 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 following the method of B192 (Step 1).

[0864] Step 2: Preparation of B 195

[0865] Linear sequence: [Ac][K(N3)]GRLIEDICLPRWGCLWEDD (SEQ ID NO: 119).

[0866] The linear peptide was prepared by standard Fmoc chemistry following the method of B192 (Step 1).

[0867] The crude peptide (1 eq) is dissolved in 2M urea (35ml) and the resulting pH adjusted to 8.5 by the addition of 1M ammonium bicarbonate. Hydrogen peroxide (30% w / w in H2O, 25pl) was added and the reaction was agitated for 30 minutes. The reaction was monitored by LCMS to determine the presence of any linear peptide, if so an additional aliquot of hydrogen peroxide was added and agitation continued. The reaction mixture was evaporated, and the residue purified by HPLC (MeCN I H2O + 0.1% TFA, 20 to 50% of 10 minutes, 20ml / min flow rate) to afford the title compound.

[0868] Step 3

[0869] A mixture of B194 (40.0 mg, 17.6 pmol, 1.00 eq), B195 (43.8 mg, 17.6 pmol, 1.10 eq) and THPTA (7.68 mg, 17.6 pmol, 1.00 eq) was dissolved in t-BuOH (0.30 ml) and H2O (0.30 ml) (pre-degassed and purged with N2 for 3 times). Then aqueous solution of CuSC>4 (0.40 M, 44.1 pl, 1.00 eq) and VcNa (7.00 mg, 35.3 pmol, 2.00 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.20 M NH4HCO3 (in 1 :1 t- BUOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 20 °C for 1 h under N2 atmosphere. LCMS showed B194 was consumed completely and one main peak with desired m / z (MW: 4746.37, observed m / z 1583.2 ([M / 3+H]+), 1187.7 ([M / 4+H]+), 950.4 ([M / 5+H]+)) was detected. The reaction mixture was filtered to remove the undissolved residue. The crude product was purified by prep-HPLC (TFA condition), B193 (48.8 mg, 9.66 pmol, 54.6% yield, 94.0% purity) was obtained as a white solid and confirmed by LCMS (Rt = 1.266 min, MS cal.: 4745.4, MS observed: [M+H]+= 4746.52).

[0870] Example 3: In Vivo Half Life Analysis of HLE Containing Tfr1 Bicyclic Peptide Ligand Complexes

[0871] B191 is a bicyclic peptide which has specificity for Tfrl As a control experiment (to demonstrate in vivo clearance in the absence of an HLE), B191 was dosed intravenously to mice (following the methodology described below) to determine the plasma concentration time profile and associated pharmacokinetic parameters which are displayed in Figure 1 and Table 1 , respectively.

[0872] Table 1 : Average PK parameters for B191 after dosing 2.69 mg / kg by iv infusion for 15 min From the results shown in Figure 1 and Table 1, it can be seen that an in vivo clearance (CL) of 31 ml / min / kg and half-life (V / 2) of 0.28 hours was observed. The fraction unbound in plasma of B191 was 0.772.

[0873] B192 was prepared by conjugation of B191 to a C-18-diacid-gGlu-PEG2-PEG2 albumin binder as described in Example 1. The resultant HLE containing complex B192 was dosed intravenously to mice (following the methodology described below) to determine the plasma concentration time profile and associated pharmacokinetic parameters which are displayed in Figure 2 and Table 2, respectively.

[0874] Table 2: Average PK parameters for B192 after dosing 3.45 mg / kg by iv infusion for 15 min

[0875] From the results shown in Figure 2 and Table 2, it can be seen that B192 surprisingly demonstrated a significant increase in plasma protein binding (fraction unbound in plasma of 0.005). Consequently, a reduction in CL to 0.25 ml / min / kg and volume of distribution (Vd) to 0.16 L / kg led to an extended half-life (V / 2) of 7.8 hours.

[0876] B193 was prepared by conjugation of B191 to albumin binding peptide SA21 (J Biol Chem 2002, 277(38), 35035-43). The resultant HLE containing complex B193 was dosed intravenously to mice (following the methodology described below) to determine the plasma concentration time profile and associated pharmacokinetic parameters which are displayed in Figure 3 and Table 3, respectively.

[0877] Table 3: Average PK parameters for B193 after dosing 0.28 mg / kg by iv infusion for 15 min

[0878] From the results shown in Figure 2 and Table 2, it can be seen that B193 also surprisingly resulted in a significant increase in plasma protein binding (fraction unbound in plasma of <0.015). Consequently, a reduction in CL to 0.10 ml / min / kg and Vd to 0.18 L / kg led to an extended half-life (t1X) of 22.6 hours.

[0879] The results shown in Figures 2 and 3 and Tables 2 and 3 (compared with the results shown in Figure 1 and Table 1) clearly demonstrate that the addition of albumin binding moieties to the parent molecule B191 significantly increase the plasma protein binding. This reduces the plasma CL by preventing glomerular filtration, reduces the volume of distribution by preventing distribution outside of the systemic circulation and consequently increases the half-life.

[0880] Methodology for Pharmacokinetic Studies and Bioanalysis

[0881] All studies and bioanalysis were carried out by WuXi AppTec (HongKong) Limited. The plasma protein binding of all compounds was assessed by ultracentrifugation. In vivo studies were conducted on CD-1 (ICR) male mice of 7-9 weeks of age. Animals were housed in groups of up to four in polysulfone cages with aspen bedding. Accommodation was maintained at 20-26°C with a humidity of 40-70% and a 12 hour light / dark cycle.

[0882] Animals were given free access to autoclaved drinking water and certified rodent diet. Before being placed on study, animals were acclimatised to the test facility for at least 3 days.

[0883] 3 animals per group were dosed with either 2.69 mg / kg B191 or 3.45 mg / kg B192 in 25 mM Histidine*HCI, 10% sucrose pH 7 neutralised with NaOH, at a dosing concentration of 1 mg / ml or 0.28 mg / kg B193 in 25 mM Histidine*HCI, 10% sucrose pH 7 neutralised with NaOH, at a dosing concentration of 0.1 mg / ml. All dosing solutions were sterile-filtered before administration by intravenous infusion over 15 minutes via a jugular vein indwelling cannula. Blood samples were taken from the saphenous vein of animals at 0.25, 0.33, 0.5, 1, 2, 4, 6, 8, 10 and 24 hours post start of the infusion for B191 and B192 and 0.25, 0.33, 0.5, 1 , 2, 4, 6, 8, 10, 24 and 48 hours post start of the infusion for B193 into low-binding polypropylene tubes. All blood samples were immediately transferred into prechilled low- binding microcentrifuge tubes containing 2 pL K2-EDTA (0.5M) as anti-coagulant and placed on wet ice until processed for plasma by centrifuging at 4°C, 3200 g for 10 minutes. Resulting plasma was transferred into 1.5 mL low-binding tubes, quick frozen over dry ice and stored at -60°C or below until extraction prior to LC-MS / MS analysis.

[0884] Samples were extracted by quenching either 15 pL (B192) or 30 pL (B191) of sample (either unknown, blank, calibration or QC) with 150 pL of internal standard containing 100 ng / ml each of labetalol, tolbutamide, verapamil, dexamethasone, glyburide and celecoxib in methanol containing 0.5% Triton X-100. For B193, samples were extracted by adding 15 pL of 4% H3PO4 in water to 15 pl of sample (either unknown, blank, calibration or QC), mixed by vortexing and then quenched with 300 pl of internal standard containing 100 ng / ml each of labetalol, tolbutamide, verapamil, dexamethasone, glyburide and celecoxib in methanol. Following quenching, all samples were vortexed and centrifuged for 15 minutes at 12000 g, 4°C. Supernatant was either undiluted, or diluted 10- or 20-fold in supernatant from blank samples and vortexed. Aliquots were transferred to a 96-well plate and centrifuged for 5 minutes at 3220 g, 4°C, then the supernatant was directly injected for LC-MS / MS analysis. LC-MS / MS analysis was carried out using single reaction monitoring on a Sciex Triple Quad 6500 (B191) or 6500+ (B192 and B193) equipped with an electrospray ionization source. Chromatographic separation was achieved using the columns and conditions described in Table 4. Eight point calibration curves were used for quantitation of unknown samples, four quality control samples within this range were also included. The internal standards selected for quantitation and calibration ranges are also given in Table 4.

[0885] Table 4: LC conditions for bioanalysis and quantification of compounds 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).

[0886] The following are numbered aspects of the disclosure:

[0887] 1. A bicyclic peptide ligand complex which comprises:

[0888] (a) one or more bicyclic peptide ligands specific for transferrin receptor 1 (TfR1) comprising a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold; and

[0889] (b) a half-life extending (HLE) moiety.

[0890] 2. The bicyclic peptide ligand complex according to aspect 1, wherein the bicyclic peptide ligand specific for transferrin receptor 1 (TfR1) is selected from any of B1 to B191 or B196 to B198.

[0891] 3. The bicyclic peptide ligand complex according to aspectl or aspect2, wherein the HLE moiety is a moiety capable of binding to albumin, such as serum albumin, in particular human serum albumin (HSA).

[0892] 4. The bicyclic peptide ligand complex according to aspects, wherein the moiety capable of binding to albumin is selected from:

[0893] (i) Short chain fatty acids (i.e. those with five or fewer carbon atoms) such as butyric (C4) acid;

[0894] (ii) Medium chain fatty acids (i.e. those with 6 to 12 carbon atoms) such as caproic (C6) acid, caprylic (C8) acid, capric (C10) acid or lauric (C12) acid;

[0895] (iii) Long chain fatty acids (i.e. those with 13 to 21 carbon atoms) such as myristic (C14) acid, palmitic (C16) acid, stearic (C18) acid, arachidic (C20) acid;

[0896] (iv) Very long chain fatty acids (i.e. those with 22 or more carbon atoms) such as behenic (C22) acid, lignoceric (C24) and cerotic (C26) acid;

[0897] (v) Genentech’s small molecule FVIIa peptide-albumin binding inhibitors (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) Fmoc protecting groups which have been shown to demonstrate a significant reduction in hydrolysis of compounds in the presence of HSA (Int J Pep Res Ther, 2007, 13(1-2), 12);

[0898] (vii) Coumarin derivatives, such as warfarin type coumarin derivatives which have been shown to improve the in vivo half-life in rats;

[0899] (viii) Evans Blue which binds to the Sudlow site I of HSA with low micromolar affinity (2.5pM) and has a long half-life in blood (Theranostics, 2016, 6(2), 243-53);

[0900] (ix) Diflunisal which binds to sites I and II of HSA with affinities of ~ 3pM (J Med Chem

[0901] 2017, 60(17), 7434-46);

[0902] (x) Indomethacin which binds mainly to site I of HSA with an affinity of 0.7pM (J Med Chem 2017, 60(17), 7434-46);

[0903] (xi) Lithocholic acid (Bile acid) which is an example of an endogenous molecule and when conjugated to insulin was capable of controlling glucose levels for more than 24 hours (Pharm Res 2006, 23(1), 49-55);

[0904] (xii) 4-(p-lodophenyl)butyric acid (Albutag) and related phenylbutyric acid examples which have been found to be stable in mouse and human serum albumin (Angew Chem Int Ed, 2008, 47(17), 3196-3201);

[0905] (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

[0906] 2018, 15, 2297-306), a NODAGA analogue (Cu) (Mol Pharmaceutics 2018, 15, 5556-64) or those described in EP 3 778 592;

[0907] (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);

[0908] (xv) Liraglutide (Diabetes Care 2002; 25:1398-1404);

[0909] (xvi) Semaglutide (Med Chem; 2015, 58, 7370-7380);

[0910] (xvii) Knob Domain Antibody Fragments;

[0911] (xviii) Pegylated compounds such as those described in WO 2020 / 205948;

[0912] (xix) Small molecule aromatic containing compounds such as those described in WO 2008 / 053360; and

[0913] (xx) PPB03 (Bioconjugate Chem 2017, 28(9), 2372-2383).

[0914] 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, in particular a C18 diacid, especially octadecanoic acid. 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).

[0915] 7. The bicyclic peptide ligand complex according to any of aspects 1 to 6, which additionally comprises a spacer or linker between the bicyclic peptide ligand(s) and the HLE moiety.

[0916] 8. The peptide ligand complex according to any of aspects 1 to 7, which additionally comprises a payload which is other than an antisense oligonucleotide (ASO).

[0917] 9. The peptide ligand complex according to aspect 8, which additionally comprises a spacer or linker between the bicyclic peptide ligand(s) / HLE moiety complex and the payload.

[0918] 10. The peptide ligand complex according to aspect 4, wherein the payload is selected from a therapeutic agent, a diagnostic agent and an imaging agent.

[0919] 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 (such as Fv, Fab and Fc), proteins and lipid nanobodies.

[0920] 12. The peptide ligand complex according to aspect 10, wherein the diagnostic agent or imaging agent is a detectable moiety, such as a dye, radiolabel or chromophore.

[0921] 13. A bicyclic peptide ligand specific for transferrin receptor 1 (TfR1), wherein the peptide ligand is B191 or B196 to B198.

[0922] 14. A pharmaceutical composition comprising the bicyclic peptide ligand complex according to any of aspects 1 to 12 or the bicyclic peptide ligand according to aspect 13, in combination with one or more pharmaceutically acceptable excipients.

[0923] 15. A peptide ligand complex according to any of aspects 1 to 11, or the bicyclic peptide ligand according to aspect 13, or pharmaceutical composition according to aspect 14 for use in preventing, suppressing or treating a disease or disorder through TfR1 mediated delivery of a therapeutic agent. 16. The peptide ligand complex, bicyclic peptide ligand or pharmaceutical composition for use of aspect 15, wherein the disease or disorder through TfR1 mediated delivery of a therapeutic agent is selected from neurological disorders, such as a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioural disorder, and CNS inflammation.

Claims

CLAIMS1 . A bicyclic peptide ligand complex which comprises:(a) one or more bicyclic peptide ligands specific for transferrin receptor 1 (TfR1) comprising a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold; and(b) a half-life extending (HLE) moiety.

2. The bicyclic peptide ligand complex according to claim 1 , wherein the polypeptide comprises a first loop sequence comprising 7 amino acids and a second loop sequence comprising 3 amino acids; wherein the first loop sequence has an amino acid sequence- AA 1 - AA2- AA3- AA4- AA5-AA6-AA7 ; wherein:Ai is selected from S, P, A, [K(N3)], [HyP], [Oxa] and [Cis-HyP];A2is 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 [Gia];A4 is selected from D, A, S, [Aib], [Abu] and [K(N3)];As is selected from W, Y, H, A, [DOPA], [hTyr], [pCaPhe] and [pCoPhe];Ae is selected from L, Q, [tBuAla], [Cba], [Abu], [Aib], A, S, T, D, E and N; andA7is selected from G, [dA], A, [dS], [dT], [dD], [dE], [dN], [dQ], [dY], S, D, Y and N; and wherein the second loop sequence has an amino acid sequence- AA 1 - A A2- AA3- ; wherein:AA1 is selected from [tBuGly], [EPA], [Chg], [tBuAla], [C5g], [Cbg], [Cpg], [B-Melle], I, A, Y and [3HyV];AA2is selected from S, A, [HSer], T, D, E, N, and Q; andAA3is selected from W, Y, [2Nal], [3tBuTyr], [1 Nal], [4Pal], A, [DOPA], [pCaPhe], [pCoPhe] and [hTyr]; or a variant thereof, or a pharmaceutically acceptable salt thereof.

3. The bicyclic peptide complex according to claim 2, wherein the first loop sequence has an amino acid sequence- AA 1 - AA2- AA3- AA4- AA5-AA6-AA7 ;wherein:Ai is selected from S, P, A, [K(N3)], [HyP], [Oxa] and [Cis-HyP]; preferably wherein Ai is selected from S and P;A2is selected from A, S, P, Q, G, [HyP], [Aib], N, I, [Aze], [dA] ,[K(N3)], [Oxa], and [Cis- HyP]; preferably wherein A2is selected from P and [HyP];A3 is D;A4 is A;As is selected from H and Y;Ae is L; andA? is G; and wherein the second loop sequence has an amino acid sequence- AA 1 - A A2- AA3- ; wherein:AA1 is selected from [tBuGly] and I;AA2is S; andAA3is Y; or a pharmaceutically acceptable salt thereof.

4. The bicyclic peptide ligand complex according to any one of claims 1 to 3, wherein the first loop 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]DAYLGP[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]DAYLSP[HyP]DAYLDP[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; andYLPDW[tBuAla]; and variants thereof; and pharmaceutically acceptable salts thereof; and / or wherein the second loop sequence is selected fromISW;I SY;[tBuGly]SY;[EPA]SY;[Chg]SY;IS[2Nal];IS[3tBuTyr];IS[1Nal];IS[4Pal];[tBuAla]SY;[C5g]SY;[Cbg]SY;[Cpg]SY;[B-Melle]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; andGDEY; and variants thereof; and pharmaceutically acceptable salts thereof.

5. The bicyclic peptide ligand complex according to any one of claims 1 to 4, wherein the polypeptide comprises one or more N- 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,T,1"-(1,3,5-triazinane-1,3,5-triyl)triprop-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-bromoethanone) (TATB).

7. The bicyclic peptide ligand complex according to any one of claims 1 to 6, wherein the bicyclic peptide ligand specific for transferrin receptor 1 (TfR1) is selected from any of B1 to B191 or B196 to B198.

8. The bicyclic peptide ligand complex according any one of claims 1 to 7, wherein the HLE moiety is a moiety capable of binding to albumin, such as serum albumin, in particular human serum albumin (HSA).

9. The bicyclic peptide ligand complex according to claim 8, wherein the moiety capable of binding to albumin is selected from:(i) Short chain fatty acids (i.e. those with five or fewer carbon atoms) such as butyric (04) acid;(ii) Medium chain fatty acids (i.e. those with 6 to 12 carbon atoms) such as caproic (06) acid, caprylic (08) acid, capric (010) acid or lauric (012) acid;(iii) Long chain fatty acids (i.e. those with 13 to 21 carbon atoms) such as myristic (014) acid, palmitic (016) acid, stearic (018) acid, arachidic (020) acid;(iv) Very long chain fatty acids (i.e. those with 22 or more carbon atoms) such as behenic (C22) acid, lignoceric (C24) and cerotic (C26) acid;(v) Genentech’s small molecule FVIIa peptide-albumin binding inhibitors (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) Fmoc protecting groups which have been shown to demonstrate a significant reduction in hydrolysis of compounds in the presence of HSA (I nt J Pep Res Ther, 2007, 13(1-2), 12);(vii) Coumarin derivatives, such as warfarin type coumarin derivatives which have been shown to improve the in vivo half-life in rats;(viii) Evans Blue which binds to the Sudlow site I of HSA with low micromolar affinity (2.5pM) 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 affinities of ~ 3pM (J Med Chem2017, 60(17), 7434-46);(x) Indomethacin which binds mainly to site I of HSA with an affinity of 0.7pM (J Med Chem 2017, 60(17), 7434-46);(xi) Lithocholic acid (Bile acid) which is an example of an endogenous molecule and when conjugated to insulin was capable of controlling glucose levels for more than 24 hours (Pharm Res 2006, 23(1), 49-55);(xii) 4-(p-lodophenyl)butyric acid (Albutag) and related phenylbutyric acid examples which 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 (Mol Pharmaceutics2018, 15, 2297-306), a NODAGA analogue (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) Semaglutide (Med Chem; 2015, 58, 7370-7380);(xvii) Knob Domain Antibody Fragments;(xviii) Pegylated compounds such as those described in WO 2020 / 205948;(xix) Small molecule aromatic containing compounds such as those described in WO 2008 / 053360; and(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 moiety is a fatty acid, such as a long chain fatty acid, in particular 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 an albumin-binding derivative thereof12. The bicyclic peptide ligand complex according to claim 11, wherein the HLE moiety is ([Ac][K(N3)]GRLIEDICLPRWGCLWEDD (SEQ ID NO: 119).

13. The bicyclic peptide ligand complex according to any of claims 1 to 12, which additionally comprises a spacer or linker between the bicyclic peptide ligand(s) and the HLE moiety.

14. The peptide ligand complex according to any of claims 1 to 13, which additionally comprises a payload which is other than an antisense oligonucleotide (ASO).

15. The peptide ligand complex according to claim 14, which additionally comprises a spacer or linker between the bicyclic peptide ligand(s) / HLE moiety complex and the payload.

16. The peptide ligand complex according to claim 14 or 15, wherein the payload is selected from a therapeutic agent, a diagnostic agent and an imaging agent.

17. The peptide ligand complex according to 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 (such as Fv, Fab and Fc), proteins and lipid nanobodies.

18. The peptide ligand complex according to claim 16, wherein the diagnostic agent or imaging agent is a detectable moiety, such as a dye, radiolabel or chromophore.

19. A bicyclic peptide ligand specific for transferring receptor 1 (TfR1), wherein the peptide ligand comprises a polypeptide as defined in claim 2 or 3.

20. A bicyclic peptide ligand specific for transferrin receptor 1 (TfR1), wherein the peptideligand is B191 or B196 to B198.

21. A pharmaceutical composition comprising the bicyclic peptide ligand complex according to any of claims 1 to 18 or the bicyclic peptide ligand according to claim 19 or 20, in combination with one or more pharmaceutically acceptable excipients.

22. A peptide ligand complex according to any of claims 1 to 18, or a bicyclic peptide ligand according to claim 19 or 20, or a pharmaceutical composition according to claim 21 for use in preventing, suppressing or treating a disease or disorder through TfR1 mediated delivery of a therapeutic agent.

23. The peptide ligand complex, bicyclic peptide ligand or pharmaceutical composition for use of claim 22, wherein the disease or disorder is selected from neurological disorders, such as a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioural disorder, and CNS inflammation.

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