WO2022079445A1

WO2022079445A1 - Bicyclic peptide ligand drug conjugates

Info

Publication number: WO2022079445A1
Application number: PCT/GB2021/052677
Authority: WO
Inventors: Paul Beswick; Gemma Mudd; Daniel Teufel
Original assignee: Bicyclerd Limited
Priority date: 2020-10-15
Filing date: 2021-10-15
Publication date: 2022-04-21
Also published as: GB202016331D0

Abstract

The present invention relates to drug conjugates comprising at least two polypeptides which are each covalently bound to aromatic molecular scaffolds such that two or more peptide loops are subtended between attachment points to the scaffold. The invention also relates to pharmaceutical compositions comprising said drug conjugates and to the use of said drug conjugates in preventing, suppressing or treating diseases, such as those which may be alleviated by cell death, in particular diseases characterised by defective cell types, proliferative disorders such as cancer and autoimmune disorders such as rheumatoid arthritis.

Description

BICYCLIC PEPTIDE LIGAND DRUG CONJUGATES FIELD OF THE INVENTION The present invention relates to drug conjugates comprising at least two polypeptides which are each covalently bound to aromatic molecular scaffolds such that two or more peptide loops are subtended between attachment points to the scaffold. The invention also relates to pharmaceutical compositions comprising said drug conjugates and to the use of said drug conjugates in preventing, suppressing or treating diseases, such as those which may be alleviated by cell death, in particular diseases characterised by defective cell types, proliferative disorders such as cancer and autoimmune disorders such as rheumatoid arthritis. BACKGROUND OF THE INVENTION Cyclic peptides are able to bind with high affinity and target specificity to protein targets and hence are an attractive molecule class for the development of therapeutics. In fact, several cyclic peptides are already successfully used in the clinic, as for example the antibacterial peptide vancomycin, the immunosuppressant drug cyclosporine or the anti-cancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). Good binding properties result from a relatively large interaction surface formed between the peptide and the target as well as the reduced conformational flexibility of the cyclic structures. Typically, macrocycles bind to surfaces of several hundred square angstrom, as for example the cyclic peptide CXCR4 antagonist CVX15 (400 Å²; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with the Arg-Gly-Asp motif binding to integrin αVb3 (355 Å²) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1 binding to urokinase-type plasminogen activator (603 Å²; Zhao et al. (2007), J Struct Biol 160 (1), 1-10). Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, leading to a smaller loss of entropy upon binding to targets and resulting in a higher binding affinity. The reduced flexibility also leads to locking target-specific conformations, increasing binding specificity compared to linear peptides. This effect has been exemplified by a potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8) which lost its selectivity over other MMPs when its ring was opened (Cherney et al. (1998), J Med Chem 41 (11), 1749- 51). The favorable binding properties achieved through macrocyclization are even more pronounced in multicyclic peptides having more than one peptide ring as for example in vancomycin, nisin and actinomycin. Different research teams have previously tethered polypeptides with cysteine residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and co-workers had used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclisation of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for the generation of candidate drug compounds wherein said compounds are generated by linking cysteine containing polypeptides to a molecular scaffold as for example tris(bromomethyl)benzene are disclosed in WO 2004/077062 and WO 2006/078161. Further suitable examples of molecular scaffolds include the non- aromatic scaffolds described in Heinis et al (2014) Angewandte Chemie, International Edition 53(6) 1602-1606. Phage display-based combinatorial approaches have been developed to generate and screen large libraries of bicyclic peptides to targets of interest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO 2009/098450). Briefly, combinatorial libraries of linear peptides containing three cysteine residues and two regions of six random amino acids (Cys-(Xaa)₆-Cys-(Xaa)₆-Cys) were displayed on phage and cyclised by covalently linking the cysteine side chains to a small molecule (tris-(bromomethyl)benzene). SUMMARY OF THE INVENTION According to a first aspect of the invention, there is provided a drug conjugate comprising at least two peptide ligands, which may be the same or different, each of which comprises a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and an aromatic 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. According to a second aspect of the invention, there is provided a drug conjugate comprising one or more cytotoxic agents conjugated to at least two peptide ligands, which may be the same or different, each comprising a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and an aromatic 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. According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a drug conjugate as defined herein in combination with one or more pharmaceutically acceptable excipients. According to a further aspect of the invention, there is provided a drug conjugate as defined herein for use in preventing, suppressing or treating diseases, such as those which may be alleviated by cell death, in particular diseases characterised by defective cell types, proliferative disorders such as cancer and autoimmune disorders such as rheumatoid arthritis. BRIEF DESCRIPTION OF THE FIGURES Figure 1: Relative affinity/kinetics of binding of BDC Reference BT17BDC18 (grey lines) and the tandem version BT17BDC-35 (Compound A) to MT1-MMP PEX protein. Figure 2: Body weight changes and tumor volume trace after administering BT66BDC-2 (Compound D) and BDC Reference BT66BDC-1 to female CB17-SCID mice bearing MOLP-8 xenograft. Data points represent group mean body weight. Error bars represent standard error of the mean (SEM). Figure 3: Body weight changes and Tumor volume trace after administering BT66BDC-3 (Compound E) and BDC Reference BT66BDC-1 to female CB17-SCID mice bearing MOLP-8 xenograft. Data points represent group mean body weight. Error bars represent standard error of the mean (SEM). Figure 4: Body weight changes and tumor volume traces after administering BCY8252 to female Balb/c nude mice bearing NCI-H292 xenograft. Data points represent group mean body weight. Error bars represent standard error of the mean (SEM). DETAILED DESCRIPTION OF THE INVENTION According to a first aspect of the invention, there is provided a drug conjugate comprising at least two peptide ligands, which may be the same or different, each of which comprises a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and an aromatic 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. It will be appreciated that as well as the drug conjugate containing a plurality of peptide ligands having potentially differing sequences, said peptide ligands may be specific for the same or different targets. The arrangement wherein the drug conjugate comprises one peptide ligand specific for one target and one or more further peptide ligands specific for a different target is known as bi-paratopic binding. In one embodiment, at least one of said peptide ligands is specific for an epitope present on a cancer cell. In one embodiment, at least one of said peptide ligands is specific for MT1-MMP. MT1-MMP is a transmembrane metalloprotease that plays a major role in the extracellular matrix remodeling, directly by degrading several of its components and indirectly by activating pro- MMP2. MT1-MMP is crucial for tumor angiogenesis (Sounni et al (2002) FASEB J.16(6), 555-564) and is over-expressed on a variety of solid tumours, therefore the MT1-MMP – binding bicycle peptides of the present invention have particular utility in the targeted treatment of cancer, in particular solid tumours such as non-small cell lung carcinomas. In one embodiment, at least one of said bicyclic peptides of the invention is specific for human MT1-MMP. In a further embodiment, at least one of said bicyclic peptides of the invention is specific for mouse MT1-MMP. In a yet further embodiment, at least one of said bicyclic peptides of the invention is specific for human and mouse MT1-MMP. In a yet further embodiment, at least one of said bicyclic peptides of the invention is specific for human, mouse and dog MT1-MMP. Examples of suitable MT1-MMP specific peptide ligands are described in WO 2016/067035 and PCT/GB2017/051250, the bicyclic peptide ligands of which are herein incorporated by reference. In the embodiment where at least one of said peptide ligands is specific for MT1-MMP, said loop sequences comprise 5 or 6 amino acid acids. In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences one of which consists of 5 amino acids and the other of which consists of 6 amino acids. In one embodiment, the at least one of said peptide ligand specific for MT1-MMP has a core sequence of: CiYNEFGCiiEDFYDICiii (SEQ ID NO: 1) (referred to as 17-69-07 and SEQ ID NO: 2 in WO 2016/067035). In a further embodiment, the at least one of said peptide ligand specific for MT1-MMP has the full sequence of: βAla-Sar10-A-C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)C (SEQ ID NO: 2) (referred to as 17-69-07-N241 and SEQ ID NO: 5 in WO 2016/067035). In one embodiment, at least one of said peptide ligands is specific for CD38. CD38 is a 45 kD type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. The CD38 protein is a bifunctional ectoenzyme that can catalyze the conversion of NAD+ into cyclic ADP-ribose (cADPR) and also hydrolyze cADPR into ADP-ribose. During ontogeny, CD38 appears on CD34+ committed stem cells and lineage-committed progenitors of lymphoid, erythroid and myeloid cells. CD38 expression persists mostly in the lymphoid lineage with varying expression levels at different stages of T and B cell development. CD38 is upregulated in many hematopoeitic malignancies and in cell lines derived from various hematopoietic malignancies, including non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's Lymphoma (HL), and chronic myeloid leukemia (CML). On the other hand, most primitive pluripotent stem cells of the hematopoietic system are CD38−. CD38 expression in hematopoietic malignancies and its correlation with disease progression makes CD38 an attractive target for antibody therapy. CD38 has been reported to be involved in Ca²⁺ mobilization (Morra et al. (1998) FASEB J. 12; 581-592; Zilber et al. (2000) Proc Natl Acad Sci USA 97, 2840-2845) and in the signal transduction through tyrosine phosphorylation of numerous signaling molecules, including phospholipase C-γ, ZAP-70, syk, and c-cbl, in lymphoid and myeloid cells or cell lines (Funaro et al. (1993) Eur J Immunol 23, 2407-2411; Morra et al. (1998), supra; Funaro et al. (1990) J Immunol 145, 2390-2396; Zubiaur et al. (1997) J Immunol 159, 193-205; Deaglio et al. (2003) Blood 102, 2146-2155; Todisco et al. (2000) Blood 95, 535-542; Konopleva et al. (1998) J Immunol 161, 4702-4708; Zilber et al. (2000) Proc Natl Acad Sci USA 97, 2840- 2845; Kitanaka et al. (1997) J Immunol 159, 184-192; Kitanaka et al. (1999) J Immunol 162, 1952-1958; Mallone et al. (2001) Int Immunol 13, 397-409). On the basis of these observations, CD38 was proposed to be an important signaling molecule in the maturation and activation of lymphoid and myeloid cells during their normal development. The exact role of CD38 in signal transduction and hematopoiesis is still not clear, especially since most of these signal transduction studies have used cell lines ectopically overexpressing CD38 and anti-CD38 monoclonal antibodies, which are non-physiological ligands. Because the CD38 protein has an enzymatic activity that produces cADPR, a molecule that can induce Ca²⁺ mobilization (Lee et al. (1989) J Biol Chem 264, 1608-1615; Lee and Aarhus (1991) Cell Regul 2, 203-209), it has been proposed that CD38 ligation by monoclonal antibodies triggers Ca²⁺ mobilization and signal transduction in lymphocytes by increasing production of cADPR (Lee et al. (1997) Adv Exp Med Biol 419, 411-419). Contrary to this hypothesis, the truncation and point-mutation analysis of CD38 protein showed that neither its cytoplasmic tail nor its enzymatic activity is necessary for the signaling mediated by anti-CD38 antibodies (Kitanaka et al. (1999) J Immunol 162, 1952- 1958; Lund et al. (1999) J Immunol 162, 2693-2702; Hoshino et al. (1997) J Immunol 158, 741-747). The best evidence for the function of CD38 comes from CD38−/− knockout mice, which have a defect in their innate immunity and a reduced T-cell dependent humoral response due to a defect in dendritic cell migration (Partida-Sanchez et al. (2004) Immunity 20, 279-291; Partida-Sanchez et al. (2001) Nat Med 7, 1209-1216). Nevertheless, it is not clear if the CD38 function in mice is identical to that in humans since the CD38 expression pattern during hematopoiesis differs greatly between human and mouse: a) unlike immature progenitor stem cells in humans, similar progenitor stem cells in mice express a high level of CD38 (Randall et al. (1996) Blood 87, 4057-4067; Dagher et al. (1998) Biol Blood Marrow Transplant 4, 69-74), b) while during the human B cell development, high levels of CD38 expression are found in germinal center B cells and plasma cells (Uckun (1990) Blood 76, 1908-1923; Kumagai et al. (1995) J Exp Med 181, 1101-1110), in the mouse, the CD38 expression levels in the corresponding cells are low (Oliver et al. (1997) J Immunol 158, 1108-1115; Ridderstad and Tarlinton (1998) J Immunol 160, 4688-4695). Several anti-human CD38 antibodies with different proliferative properties on various tumor cells and cell lines have been described in the literature. For example, a chimeric OKT10 antibody with mouse Fab and human IgG1 Fc mediates antibody-dependent cell-mediated cytotoxicity (ADCC) very efficiently against lymphoma cells in the presence of peripheral blood mononuclear effector cells from either MM patients or normal individuals (Stevenson et al. (1991) Blood 77, 1071-1079). A CDR-grafted humanized version of the anti-CD38 antibody AT13/5 has been shown to have potent ADCC activity against CD38-positive cell lines (U.S. Patent Application No.09/797,941). Human monoclonal anti-CD38 antibodies have been shown to mediate the in vitro killing of CD38-positive cell lines by ADCC and/or complement-dependent cytotoxicity (CDC), and to delay the tumor growth in SCID mice bearing MM cell line RPMI-8226 (WO 2005/103083). On the other hand, several anti-CD38 antibodies, IB4, SUN-4B7, and OKT10, but not IB6, AT1, or AT2, induced the proliferation of peripheral blood mononuclear cells (PBMC) from normal individuals (Ausiello et al. (2000) Tissue Antigens 56, 539-547). Some of the antibodies of the prior art have been shown to be able to trigger apoptosis in CD38+ B cells. However, they can only do so in the presence of stroma cells or stroma- derived cytokines. An agonistic anti-CD38 antibody (IB4) has been reported to prevent apoptosis of human germinal center (GC) B cells (Zupo et al. (1994) Eur J Immunol 24, 1218-1222), and to induce proliferation of KG-1 and HL-60 AML cells (Konopleva et al. (1998) J Immunol 161, 4702-4708), but induces apoptosis in Jurkat T lymphoblastic cells (Morra et al. (1998) FASEB J 12, 581-592). Another anti-CD38 antibody T16 induced apoptosis of immature lymphoid cells and leukemic lymphoblast cells from an ALL patient (Kumagai et al. (1995) J Exp Med 181, 1101-1110), and of leukemic myeloblast cells from AML patients (Todisco et al. (2000) Blood 95, 535-542), but T16 induced apoptosis only in the presence of stroma cells or stroma-derived cytokines (IL-7, IL-3, stem cell factor). In one embodiment, the CD38 is mammalian CD38. In a further embodiment, the mammalian CD38 is human CD38 (hCD38). Examples of suitable CD38 specific peptide ligands are described in GB 1701834.2 and GB 1705013.9, the bicyclic peptide ligands of which are herein incorporated by reference. In the embodiment where at least one of said peptide ligands is specific for CD38, said loop sequences comprise 2 or 7 amino acid acids. In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences one of which consists of 2 amino acids and the other of which consists of 7 amino acids. In one embodiment, the at least one peptide ligand specific for CD38 has a core sequence of: CiVPCiiADFPIWYCiii (SEQ ID NO: 3) (referred to as SEQ ID NO: 5 in GB 1701834.2 and GB 1705013.9). In one embodiment, the at least one peptide ligand specific for CD38 has the full sequence of: (β-Ala)-Sar₁₀-A-CVPCADFPIWYC (SEQ ID NO: 4) (referred to as 66-03-00-N006 in GB 1701834.2 and GB 1705013.9). In one embodiment, at least one of said peptide ligands is specific for Nectin, such as Nectin- 4. Nectin-4 is a surface molecule that belongs to the nectin family of proteins, which comprises 4 members. Nectins are cell adhesion molecules that play a key role in various biological processes such as polarity, proliferation, differentiation and migration, for epithelial, endothelial, immune and neuronal cells, during development and adult life. They are involved in several pathological processes in humans. They are the main receptors for poliovirus, herpes simplex virus and measles virus. Mutations in the genes encoding Nectin-1 (PVRL1) or Nectin-4 (PVRL4) cause ectodermal dysplasia syndromes associated with other abnormalities. Nectin-4 is expressed during foetal development. In adult tissues its expression is more restricted than that of other members of the family. Nectin-4 is a tumour-associated antigen in 50%, 49% and 86% of breast, ovarian and lung carcinomas, respectively, mostly on tumours of bad prognosis. Its expression is not detected in the corresponding normal tissues. In breast tumours, Nectin-4 is expressed mainly in triple-negative and ERBB2+ carcinomas. In the serum of patients with these cancers, the detection of soluble forms of Nectin-4 is associated with a poor prognosis. Levels of serum Nectin-4 increase during metastatic progression and decrease after treatment. These results suggest that Nectin-4 could be a reliable target for the treatment of cancer. Accordingly, several anti-Nectin-4 antibodies have been described in the prior art. In particular, Enfortumab Vedotin (ASG-22ME) is an antibody-drug conjugate (ADC) targeting Nectin-4 and is currently clinically investigated for the treatment of patients suffering from solid tumours. Examples of suitable Nectin-4 specific peptide ligands are described in GB 1810250.9 and GB 1815684.4, the bicyclic peptide ligands of which are herein incorporated by reference. In the embodiment where at least one of said peptide ligands is specific for Nectin-4, said loop sequences comprise 3 or 9 amino acid acids. In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences one of which consists of 3 amino acids and the other of which consists of 9 amino acids. In one embodiment, the at least one peptide ligand specific for Nectin-4 has a core sequence of: CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: 5) (referred to as SEQ ID NO: 169 in GB 1810250.9 and GB 1815684.4). In a further embodiment, the at least one peptide ligand specific for Nectin-4 has the full sequence of Ac-(SEQ ID NO: 5) (hereinafter referred to as BCY8126). In an alternative embodiment, the at least one peptide ligand specific for Nectin-4 has the full sequence of: (β-Ala)-Sar₁₀-CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: 6) (referred to as BCY8234 in GB 1810250.9 and GB 1815684.4). 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) 4^th ed., John Wiley & Sons, Inc.), which are incorporated herein by reference. In one embodiment, said drug conjugate comprises two peptide ligands, both of which are specific for the same target. In a further embodiment, said drug conjugate comprises two peptide ligands, both of which are specific for MT1-MMP. In a yet further embodiment, said drug conjugate comprises two peptide ligands, both of which are specific for MT1-MMP and both of which comprise the same peptide sequence. In a further embodiment, said drug conjugate comprises two peptide ligands, both of which are specific for CD38. In a yet further embodiment, said drug conjugate comprises two peptide ligands, both of which are specific for CD38 and both of which comprise the same peptide sequence. In an alternative embodiment, said drug conjugate comprises two peptide ligands, one of which is specific for a first target and the other of which is specific for a second target. In a further embodiment, said first target is MT1-MMP and said second target is Nectin-4. Nomenclature Numbering When referring to amino acid residue positions within the bicyclic peptides of the invention, cysteine residues (C_i, C_ii and C_iii) are omitted from the numbering as they are invariant, therefore, the numbering of amino acid residues within a selected bicyclic peptide of the invention is referred to as below: -C_i-Y₁-N₂-E₃-F₄-G₅-C_ii-E₆-D₇-F₈-Y₉-D₁₀-I₁₁-C_iii (SEQ ID NO: 1). For the purpose of this description, all bicyclic peptides are assumed to be cyclised with TBMB (1,3,5-tris(bromomethyl)benzene) yielding a tri-substituted 1,3,5-trismethylbenzene structure. Cyclisation with TBMB occurs on C_i, C_ii, and C_iii. Molecular Format 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 βAla-Sar₁₀-Ala tail would be denoted as: βAla-Sar10-A-(SEQ ID NO: X). Inversed Peptide Sequences 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 becomes C-terminus and vice versa) and their stereochemistry is likewise also reversed (i.e. D-amino acids become L-amino acids and vice versa). Peptide Ligands 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 residues) 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 comprise at least three cysteine residues (referred to herein as Ci, Cii and Ciii), and form at least two loops on the scaffold. Advantages of the Peptide Ligands 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; - Protease stability. Bicyclic peptide ligands should ideally 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 bicycle lead candidate can be developed in animal models as well as administered with confidence to humans; - 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; - 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 for short exposure to develop a bicyclic peptide with enhanced retention in the circulation, and is therefore optimal for the management of more chronic disease states. Other factors driving the desirable plasma half-life are requirements of sustained exposure for maximal therapeutic efficiency versus the accompanying toxicology due to sustained exposure of the agent; and - Selectivity. Certain peptide ligands of the invention demonstrate good selectivity over other receptor subtypes. For example, when the bicyclic peptide is specific for MT1-MMP, said bicyclic peptide will be ideally selective for MT1-MMP over other metalloproteases. In addition, when the bicyclic peptide is specific for CD38, said bicyclic peptide will be ideally selective for CD38 over other CDs. Furthermore, when the bicyclic peptide is specific for nectin-4, said bicyclic peptide will be ideally selective for nectin-4 over other nectins. Pharmaceutically Acceptable Salts 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. 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. 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), α-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. 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. 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., NH3R⁺, NH2R2⁺, NHR3⁺, 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 ⁺. Where the compounds 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 compounds of the invention. Modified Derivatives It will be appreciated that modified derivatives of the peptide ligands as defined herein are within the scope of the present invention. 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 (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. 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. 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. 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. 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, non-natural 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. 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, C ^- disubstituted derivatives (for example, aminoisobutyric acid, Aib), and cyclo amino acids, a simple derivative being amino-cyclopropylcarboxylic acid. 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 (Ciii). 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. 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). 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 ^-turn conformations (Tugyi et al (2005) PNAS, 102(2), 413–418). 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). 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: - Incorporating hydrophobic moieties that exploit the hydrophobic effect and lead to lower off rates, such that higher affinities are achieved; - 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 - 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. (for reviews see Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem (2009), 16, 4399-418). Isotopic variations The present invention includes all pharmaceutically acceptable (radio)isotope-labeled 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. Examples of isotopes suitable for inclusion in the peptide ligands of the invention comprise isotopes of hydrogen, such as ²H (D) and ³H (T), carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, such as ¹⁸F, iodine, such as ¹²³I, ¹²⁵I and ¹³¹I, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, sulfur, such as ³⁵S, copper, such as ⁶⁴Cu, gallium, such as ⁶⁷Ga or ⁶⁸Ga, yttrium, such as ⁹⁰Y and lutetium, such as ¹⁷⁷Lu, and Bismuth, such as ²¹³Bi. 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 EphA2 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. 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. Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy. Isotopically-labeled 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-labeled reagent in place of the non-labeled reagent previously employed. Reactive Groups The molecular scaffold of the invention may be bonded to the polypeptide via functional or reactive groups on the polypeptide. These are typically formed from the side chains of particular amino acids found in the polypeptide polymer. The reactive groups are groups capable of forming a covalent bond with the molecular scaffold. Typically, the reactive groups are present on amino acid side chains on the peptide. Examples are lysine, arginine, histidine and sulfur containing groups such as cysteine, methionine as well as analogues such as selenocysteine. In one embodiment, said reactive groups comprise cysteine. Examples of reactive groups of natural amino acids are the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartate or glutamate, the guanidinium group of arginine, the phenolic group of tyrosine or the hydroxyl group of serine. Non-natural amino acids can provide a wide range of reactive groups including an azide, a keto-carbonyl, an alkyne, a vinyl, or an aryl halide group. The amino and carboxyl group of the termini of the polypeptide can also serve as reactive groups to form covalent bonds to a molecular scaffold/molecular core. The polypeptides of the invention contain at least three reactive groups. Said polypeptides can also contain four or more reactive groups. The more reactive groups are used, the more loops can be formed in the molecular scaffold. In a preferred embodiment, polypeptides with three reactive groups are generated. Reaction of said polypeptides with a molecular scaffold/molecular core having a three-fold rotational symmetry generates a single product isomer. The generation of a single product isomer is favourable for several reasons. The nucleic acids of the compound libraries encode only the primary sequences of the polypeptide but not the isomeric state of the molecules that are formed upon reaction of the polypeptide with the molecular core. If only one product isomer can be formed, the assignment of the nucleic acid to the product isomer is clearly defined. If multiple product isomers are formed, the nucleic acid cannot give information about the nature of the product isomer that was isolated in a screening or selection process. The formation of a single product isomer is also advantageous if a specific member of a library of the invention is synthesized. In this case, the chemical reaction of the polypeptide with the molecular scaffold yields a single product isomer rather than a mixture of isomers. In another embodiment of the invention, polypeptides with four reactive groups are generated. Reaction of said polypeptides with a molecular scaffold/molecular core having a tetrahedral symmetry generates two product isomers. Even though the two different product isomers are encoded by one and the same nucleic acid, the isomeric nature of the isolated isomer can be determined by chemically synthesizing both isomers, separating the two isomers and testing both isomers for binding to a target ligand. In one embodiment of the invention, at least one of the reactive groups of the polypeptides is orthogonal to the remaining reactive groups. The use of orthogonal reactive groups allows the directing of said orthogonal reactive groups to specific sites of the molecular core. Linking strategies involving orthogonal reactive groups may be used to limit the number of product isomers formed. In other words, by choosing distinct or different reactive groups for one or more of the at least three bonds to those chosen for the remainder of the at least three bonds, a particular order of bonding or directing of specific reactive groups of the polypeptide to specific positions on the molecular scaffold may be usefully achieved. In another embodiment, the reactive groups of the polypeptide of the invention are reacted with molecular linkers wherein said linkers are capable to react with a molecular scaffold so that the linker will intervene between the molecular scaffold and the polypeptide in the final bonded state. Alternatives to thiol-mediated conjugations can be used to attach the molecular scaffold to the peptide via covalent interactions. Alternatively these techniques may be used in modification or attachment of further moieties (such as small molecules of interest which are distinct from the molecular scaffold) to the polypeptide after they have been selected or isolated according to the present invention – in this embodiment then clearly the attachment need not be covalent and may embrace non-covalent attachment. These methods may be used instead of (or in combination with) the thiol mediated methods by producing phage that display proteins and peptides bearing unnatural amino acids with the requisite chemical reactive groups, in combination small molecules that bear the complementary reactive group, or by incorporating the unnatural amino acids into a chemically or recombinantly synthesised polypeptide when the molecule is being made after the selection/isolation phase. Further details can be found in WO 2009/098450 or Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7. It will be appreciated that the looped bicyclic peptide structure is further attached to the molecular scaffold via at least one thioether linkage. The thioether linkage provides an anchor during formation of the bicyclic peptides. In one embodiment, there is only one such thioether linkage. In further embodiments, there is one such thioether linkage and two amino linkages. In further embodiments, there is one such thioether linkage and two alkylamino linkages. Suitably, the thioether linkage is a central linkage of the bicyclic or polycyclic peptide conjugate, i.e. in the peptide sequence two residues (e.g. diaminopropionic acid residues) forming the amino linkages in the peptide are spaced from and located on either side of the amino acid residue (e.g. lysine) forming the thioether linkage. Suitably, the looped peptide structure is therefore a bicyclic peptide conjugate having a central thioether linkage and two peripheral amino linkages. In some embodiments, placement of the thioether bond can be N- terminal or C-terminal to two N-alkylamino linkages. In one embodiment, the reactive groups comprise one cysteine residue and two L-2,3- diaminopropionic acid (Dap) or N-beta-C1-4 alkyl-L-2, 3-diaminopropionic acid (N-AlkDap) residues. Aromatic Molecular scaffold References herein to the term “aromatic molecular scaffold” refer to any molecular scaffold as defined herein which contains an aromatic carbocyclic or heterocyclic ring system. It will be appreciated that the aromatic molecular scaffold may comprise an aromatic moiety. Examples of suitable aromatic moieties within the aromatic scaffold include biphenylene, terphenylene, naphthalene or anthracene. It will also be appreciated that the aromatic molecular scaffold may comprise a heteroaromatic moiety. Examples of suitable heteroaromatic moieties within the aromatic scaffold include pyridine, pyrimidine, pyrrole, furan and thiophene. It will also be appreciated that the aromatic molecular scaffold may comprise a halomethylarene moiety, such as a bis(bromomethyl)benzene, a tris(bromomethyl)benzene, a tetra(bromomethyl)benzene or derivatives thereof. Non-limiting examples of aromatic molecular scaffolds include: bis-, tris-, or tetra(halomethyl)benzene; bis-, tris-, or tetra(halomethyl)pyridine; bis-, tris-, or tetra(halomethyl)pyridazine; bis-, tris-, or tetra(halomethyl)pyrimidine; bis-, tris-, or tetra(halomethyl)pyrazine; bis-, tris-, or tetra(halomethyl)-1,2,3-triazine; bis-, tris-, or tetra- halomethyl)-1,2,4-triazine; bis-, tris-, or tetra(halomethyl)pyrrole, -furan, -thiophene; bis-, tris- , or tetra(halomethyl)imidazole, -oxazole, -thiazol; bis-, tris-, or tetra(halomethyl)-3H- pyrazole, -isooxazole, -isothiazol; bis-, tris-, or tetra(halomethyl)biphenylene; bis-, tris-, or tetra(halomethyl)terphenylene; 1,8-bis(halomethyl)naphthalene; bis-, tris-, or tetra(halomethyl)anthracene; and bis-, tris-, or tetra(2-halomethylphenyl)methane. More specific examples of aromatic molecular scaffolds include: 1,2- bis(halomethyl)benzene; 3,4-bis(halomethyl)pyridine; 3,4-bis(halomethyl)pyridazine; 4,5- bis(halomethyl)pyrimidine; 4,5-bis(halomethyl)pyrazine; 4,5-bis(halomethyl)-1,2,3-triazine; 5,6-bis(halomethyl)-1,2,4-triazine; 3,4-bis(halomethyl)pyrrole, -furan, -thiophene and other regioisomers; 4,5-bis(halomethyl)imidazole, -oxazole, -thiazol; 4,5-bis(halomethyl)-3H- pyrazole, -isooxazole, -isothiazol; 2,2′-bis(halomethyl)biphenylene; 2,2″- bis(halomethyl)terphenylene; 1,8-bis(halomethyl)naphthalene; 1,10- bis(halomethyl)anthracene; bis(2-halomethylphenyl)methane; 1,2,3-tris(halomethyl)benzene; 2,3,4-tris(halomethyl)pyridine; 2,3,4-tris(halomethyl)pyridazine; 3,4,5- tris(halomethyl)pyrimidine; 4,5,6-tris(halomethyl)-1,2,3-triazine; 2,3,4-tris(halomethyl)pyrrole, -furan, -thiophene; 2,4,5-bis(halomethyl)imidazole, -oxazole, -thiazol; 3,4,5-bis(halomethyl)- 1H-pyrazole, -isooxazole, -isothiazol; 2,4,2′-tris(halomethyl)biphenylene; 2,3′,2″- tris(halomethyl)terphenylene; 1,3,8-tris(halomethyl)naphthalene; 1,3,10- tris(halomethyl)anthracene; bis(2-halomethylphenyl)methane; 1,2,4,5- tetra(halomethyl)benzene; 1,2,4,5-tetra(halomethyl)pyridine; 2,4,5,6- tetra(halomethyl)pyrimidine; 2,3,4,5-tetra(halomethyl)pyrrole, -furan, -thiophene; 2,2′,6,6′- tetra(halomethyl)biphenylene; 2,2″,6,6″-tetra(halomethyl) terphenylene; 2,3,5,6- tetra(halomethyl)naphthalene and 2,3,7,8-tetra(halomethyl)anthracene; and bis(2,4- bis(halomethyl)phenyl)methane. As noted in the foregoing documents, the molecular scaffold may be a small molecule, such as a small organic molecule. 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. 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. 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. In one embodiment, the molecular scaffold may comprise or may consist of tris(bromomethyl)benzene, especially 1,3,5-tris(bromomethyl)benzene (‘TBMB’), or a derivative thereof. In one embodiment, the molecular scaffold is 2,4,6-tris(bromomethyl)mesitylene. This molecule is similar to 1,3,5-tris(bromomethyl)benzene but contains three additional methyl groups attached to the benzene ring. This has the advantage that the additional methyl groups may form further contacts with the polypeptide and hence add additional structural constraint. The molecular scaffold of the invention contains chemical groups that allow functional groups of the polypeptide of the encoded library of the invention to form covalent links with the molecular scaffold. Said chemical groups are selected from a wide range of functionalities including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides and acyl halides. Scaffold reactive groups that could be used on the molecular scaffold to react with thiol groups of cysteines are alkyl halides (or also named halogenoalkanes or haloalkanes). Examples include bromomethylbenzene (the scaffold reactive group exemplified by TBMB) or iodoacetamide. Other scaffold reactive groups that are used to selectively couple compounds to cysteines in proteins are maleimides, ^ ^ unsaturated carbonyl containing compounds and ^ ^halomethylcarbonyl containing compounds. Examples of maleimides which may be used as molecular scaffolds in the invention include: tris-(2- maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, tris-(maleimido)benzene. An example of an ^ ^halomethylcarbonyl containing compound is N,N',N''-(benzene-1,3,5- triyl)tris(2-bromoacetamide). Selenocysteine is also a natural amino acid which has a similar reactivity to cysteine and can be used for the same reactions. Thus, wherever cysteine is mentioned, it is typically acceptable to substitute selenocysteine unless the context suggests otherwise. Additional Agents In one embodiment, said drug conjugate is additionally conjugated to one or more active agents. Examples of suitable “active” agents include any suitable agent capable of performing a cellular activity upon binding of the bicyclic peptide complex to its target. Such agents include small molecules, inhibitors, agonists, antagonists, partial agonists and antagonists, inverse agonists and antagonists and cytotoxic agents. In a further embodiment, said drug conjugate is additionally conjugated to one or more cytotoxic agents. Thus, according to a second aspect of the invention, there is provided a drug conjugate comprising one or more cytotoxic agents conjugated to at least two peptide ligands, which may be the same or different, each comprising a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and an aromatic 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. Suitable examples of cytotoxic agents include: alkylating agents such as cisplatin and carboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; Anti-metabolites including purine analogs azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids including vinca alkaloids such as Vincristine, Vinblastine, Vinorelbine and Vindesine; Podophyllotoxin and its derivatives etoposide and teniposide; Taxanes, including paclitaxel, originally known as Taxol; topoisomerase inhibitors including camptothecins: irinotecan and topotecan, and type II inhibitors including amsacrine, etoposide, etoposide phosphate, and teniposide. Further agents can include antitumour antibiotics which include the immunosuppressant dactinomycin (which is used in kidney transplantations), doxorubicin, epirubicin, bleomycin, calicheamycins, and others. In one embodiment of the invention, the cytotoxic agent is selected from maytansinoids (such as DM1) or monomethyl auristatins (such as MMAE). DM1 is a cytotoxic agent which is a thiol-containing derivative of maytansine and has the following structure:

Monomethyl auristatin E (MMAE) is a synthetic antineoplastic agent and has the following structure:

In one embodiment, the cytotoxic agent is linked to the bicyclic peptide by a cleavable bond, such as a disulphide bond or a protease sensitive bond. In a further embodiment, the groups adjacent to the disulphide bond are modified to control the hindrance of the disulphide bond, and by this the rate of cleavage and concomitant release of cytotoxic agent. Published work established the potential for modifying the susceptibility of the disulphide bond to reduction by introducing steric hindrance on either side of the disulphide bond (Kellogg et al (2011) Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrance reduces the rate of reduction by intracellular glutathione and also extracellular (systemic) reducing agents, consequentially reducing the ease by which toxin is released, both inside and outside the cell. Thus, selection of the optimum in disulphide stability in the circulation (which minimises undesirable side effects of the toxin) versus efficient release in the intracellular milieu (which maximises the therapeutic effect) can be achieved by careful selection of the degree of hindrance on either side of the disulphide bond. The hindrance on either side of the disulphide bond is modulated through introducing one or more methyl groups on either the targeting entity (here, the bicyclic peptide) or toxin side of the molecular construct. In one embodiment, the cytotoxic agent and linker is selected from any combinations of those described in WO 2016/067035 (the cytotoxic agents and linkers thereof are herein incorporated by reference). In one embodiment, the linker between said cytotoxic agent and said bicyclic peptide comprises a cleavable disulfide linker or a -PABC-Cit-Val- moiety, wherein PABC is p- aminobenzylcarbamate. In a further embodiment, the linker between said cytotoxic agent and said bicyclic peptide additionally comprises an inert spacer portion. Examples of suitable inert spacer portions are shown in the compounds of formula (A) to (E) herein. In an alternative embodiment, the linker between said cytotoxic agent and said bicyclic peptide comprises a -PABC-Val-Cit-Glu- linker, wherein said bicyclic peptides are joined via a branch in said linker between said Glu and the bicyclic peptide (i.e. the resultant bicyclic peptide drug conjugate comprises a MMAE-PABC-Val-Cit-Glu-X-(Bicyclic peptide)2 moiety, wherein X represents a branching linker). Examples of such a linker is shown in the compound of formula (F) herein. In one embodiment, said drug conjugate comprises two bicyclic peptides, both of which are specific for MT1-MMP, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (A):

βAla-Sar10-A-C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)C (SEQ ID NO: 2) (referred to as 17-69-07-N241 and SEQ ID NO: 5 in WO 2016/067035). The BDC of formula (A) is known herein as BT17BDC-35. Data is presented herein in Table A which shows that BT17BDC-35 demonstrated 9 fold greater binding affinity than the single bicyclic peptide containing drug conjugate BT17BDC-18. Data is also presented herein in Figure 1 which demonstrates that reference tandem molecule BT17BDC-35 demonstrated an extended residence time compared with the corresponding single bicyclic peptide containing drug conjugate BT17BDC-18. In an alternative embodiment, said drug conjugate comprises two bicyclic peptides, both of which are specific for MT1-MMP, the cytotoxic agent is MMAE and the drug conjugate comprises a compound of formula (B):

wherein R is selected from: βAla-Sar10-A-C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)C (SEQ ID NO: 2) (referred to as 17-69-07-N241 and SEQ ID NO: 5 in WO 2016/067035). The BDC of formula (B) is known herein as BT17BDC-43. Data is presented herein in Table A which shows that BT17BDC-43 demonstrated almost 5 fold greater binding affinity than the single bicyclic peptide containing drug conjugate BT17BDC-18. In an alternative embodiment, said drug conjugate comprises two bicyclic peptides, both of which are specific for MT1-MMP, the cytotoxic agent is DM1 and the drug conjugate

βAla-Sar10-A-C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)C (SEQ ID NO: 2) (referred to as 17-69-07-N241 and SEQ ID NO: 5 in WO 2016/067035). The BDC of formula (C) is known herein as BT17BDC-44. Data is presented herein in Table A which shows that BT17BDC-44 demonstrated over 3 fold greater binding affinity than the single bicyclic peptide containing drug conjugate BT17BDC-18. In an alternative embodiment, said drug conjugate comprises two bicyclic peptides, both of which are specific for CD38, the cytotoxic agent is DM1 and the drug conjugate comprises a

wherein R is selected from: (β-Ala)-Sar10-A-CVPCADFPIWYC (SEQ ID NO: 4) (referred to as 66-03-00-N006 in GB 1701834.2 and GB 1705013.9). The BDC of formula (D) is known herein as BT66BDC-2. Data is presented in Table B where it can be seen that BT66BDC-2 demonstrated 13 fold greater binding affinity than the reference BDC (BT66BDC-1). Data is also presented herein which demonstrates in Figure 2 and Tables 1 to 4 that BT66BDC-2 produced dose-dependent antitumor activity. In an alternative embodiment, said drug conjugate comprises two bicyclic peptides, both of which are specific for CD38, the cytotoxic agent is DM1 and the drug conjugate comprises a

wherein R is selected from: (β-Ala)-Sar10-A-CVPCADFPIWYC (SEQ ID NO: 4) (referred to as 66-03-00-N006 in GB 1701834.2 and GB 1705013.9). The BDC of formula (E) is known herein as BT66BDC-3. Data is presented in Table B where it can be seen that BT66BDC-3 demonstrated 8 fold greater binding affinity than the reference BDC (BT66BDC- 1). Data is also presented herein which demonstrates in Figure 3 and Tables 1 to 4 that BT66BDC-3 produced dose-dependent antitumor activity. In an alternative embodiment, said conjugate comprises two bicyclic peptides, one of which is specific for MT1-MMP and the other of which is specific for Nectin-4 (i.e. an MT1- MMP/Nectin-4 hetero-tandem), the cytotoxic agent is MMAE and the drug conjugate comprises a compound of formula (F):

The BDC of formula (F) is known herein as BCY8252. Data is presented herein in Table 5 which showed that BCY8252 demonstrated good levels of binding in the SPR binding assay. In particular, the MT1-MMP/Nectin-4 hetero-tandem BCY8252 demonstrated 3.9 fold less binding activity in the SPR binding assay than the monomeric Nectin-4 bicyclic peptide BCY8126. Without being bound by theory, it is believed that although Nectin-4 binding is less potent for the hetero-tandem BCY8252, the binding levels are still at an acceptable level. Furthermore, the heterotandem BCY8252 provides the advantage of demonstrating binding affinity to differing epitopes present on cancer cells (i.e. MT1-MMP and Nectin-4), thereby providing a more effective anti-tumour agent which is capable of targeting specific cancer sub-types in a broader patient population. This benefit therefore compensates for the slight reduction in potency. Synthesis 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). Thus, the invention also relates to 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. Optionally amino acid residues in the polypeptide of interest may be substituted when manufacturing a conjugate or complex. Peptides can also be extended, to incorporate for example another loop and therefore introduce multiple specificities. 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 U S A.1994 Dec 20; 91(26):12544-8 or in Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003). 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. TBMB) 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 disulfide –linked bicyclic peptide-peptide conjugate. Similar techniques apply equally to the synthesis/coupling of two bicyclic and bispecific macrocycles, potentially creating a tetraspecific molecule. 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. Pharmaceutical Compositions According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand or a drug conjugate as defined herein in combination with one or more pharmaceutically acceptable excipients. Generally, the present peptide ligands 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. 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). The peptide ligands 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 cylcosporine, methotrexate, adriamycin or cisplatinum and immunotoxins. 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 by inhalation. 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. The peptide ligands 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. The compositions containing the present peptide ligands 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 and the general state of the patient's own immune system, 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. A composition containing a peptide ligand 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. Therapeutic Uses By virtue of the presence of the cytotoxic agent, the drug conjugates of the invention have specific utility in the treatment of diseases which may be alleviated by cell death. Examples of suitable diseases include diseases characterised by defective cell types, proliferative disorders such as cancer and autoimmune disorders such as rheumatoid arthritis. By virtue of the presence of the cytotoxic agent coupled to a cancer cell binding bicyclic peptide, the bicyclic peptides of the invention have specific utility in the treatment of cancer. Thus, according to a further aspect of the invention, there is provided a drug conjugate as defined herein for use in preventing, suppressing or treating cancer (such as a tumour). According to a further aspect of the invention, there is provided a method of preventing, suppressing or treating cancer (such as a tumour), which comprises administering to a patient in need thereof a drug conjugate as defined herein. Examples of cancers (and their benign counterparts) which may be treated (or inhibited) include, but are not limited to tumours of epithelial origin (adenomas and carcinomas of various types including adenocarcinomas, squamous carcinomas, transitional cell carcinomas and other carcinomas) such as carcinomas of the bladder and urinary tract, breast, gastrointestinal tract (including the esophagus, stomach (gastric), small intestine, colon, rectum and anus), liver (hepatocellular carcinoma), gall bladder and biliary system, exocrine pancreas, kidney, lung (for example adenocarcinomas, small cell lung carcinomas, non-small cell lung carcinomas, bronchioalveolar carcinomas and mesotheliomas), head and neck (for example cancers of the tongue, buccal cavity, larynx, pharynx, nasopharynx, tonsil, salivary glands, nasal cavity and paranasal sinuses), ovary, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (for example thyroid follicular carcinoma), adrenal, prostate, skin and adnexae (for example melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic naevus); haematological malignancies (i.e. leukemias, lymphomas) and premalignant haematological disorders and disorders of borderline malignancy including haematological malignancies and related conditions of lymphoid lineage (for example acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt’s lymphoma, mantle cell lymphoma, T-cell lymphomas and leukaemias, natural killer [NK] cell lymphomas, Hodgkin’s lymphomas, hairy cell leukaemia, monoclonal gammopathy of uncertain significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), and haematological malignancies and related conditions of myeloid lineage (for example acute myelogenousleukemia [AML], chronic myelogenousleukemia [CML], chronic myelomonocyticleukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythaemia vera, essential thrombocythaemia and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocyticleukemia); tumours of mesenchymal origin, for example sarcomas of soft tissue, bone or cartilage such as osteosarcomas, fibrosarcomas, chondrosarcomas, rhabdomyosarcomas,leiomyosarcomas, liposarcomas, angiosarcomas, Kaposi’s sarcoma, Ewing’s sarcoma, synovial sarcomas, epithelioid sarcomas, gastrointestinal stromal tumours, benign and malignant histiocytomas, and dermatofibrosarcomaprotuberans; tumours of the central or peripheral nervous system (for example astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumours and schwannomas); endocrine tumours (for example pituitary tumours, adrenal tumours, islet cell tumours, parathyroid tumours, carcinoid tumours and medullary carcinoma of the thyroid); ocular and adnexal tumours (for example retinoblastoma); germ cell and trophoblastic tumours (for example teratomas, seminomas, dysgerminomas, hydatidiform moles and choriocarcinomas); and paediatric and embryonal tumours (for example medulloblastoma, neuroblastoma, Wilms tumour, and primitive neuroectodermal tumours); or syndromes, congenital or otherwise, which leave the patient susceptible to malignancy (for example Xeroderma Pigmentosum). In a further embodiment, the cancer is selected from: breast cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, liver cancer, glioblastoma and angiogenesis. 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. 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. The invention is further described below with reference to the following examples. Examples Abbreviations

Materials and Methods Peptide Synthesis Peptide synthesis was based on Fmoc chemistry, using a Symphony and SymphonyX peptide synthesiser manufactured by Peptide Instruments and a Syro II synthesiser by MultiSynTech. Standard Fmoc-amino acids were employed (Sigma, Merck), with appropriate side chain protecting groups: where applicable standard coupling conditions were used in each case, followed by deprotection using standard methodology. Peptides were purified by HPLC and following isolation they were modified with 1,3,5-tris(bromomethyl)benzene (TBMB, Sigma). For this, linear peptide was diluted with H2O up to ~35 mL, ~500 µL of 100 mM TBMB in acetonitrile was added, and the reaction was initiated with ~5 mL of 1 M NH₄HCO₃ in H₂O. The reaction was allowed to proceed for ~30 -60 min at RT, and quenched with ~ 500µl of the 1M Cysteine hydrochloride (Sigma) once the reaction had completed (judged by MALDI). Following lyophilisation, the modified peptide was purified in a Gemini C18 column (Phenomenex) using water/acetonitrile with 0.1% trifluoroacetic acid as mobile phase. Pure fractions containing the correct cyclised material were pooled, lyophilised and kept at -20ºC for storage. All amino acids, unless noted otherwise, were used in the L- configurations. Preparation of Reference Bicyclic Peptide Drug Conjugate BT17BDC-18 BT17BDC-18 was prepared as described in WO 2016/067035. Preparation of Reference Bicyclic Peptide Drug Conjugate BT66BDC-1

66-03-00-N041(56mg) was dissolved in DMF (0.8ml). A solution of DM1 (19mg) in DMF (0.78ml) was added followed by diisopropylethylamine (26µL) and the mixture stirred at room temperature for 2 hour. Water (17.5ml) was added and the mixture filtered before being loaded onto a Luna C18(3) prep HPLC column (250mm X 20mm). The product was obtained by eluting with a gradient of acetonitrile (with 1% trfluoroacetic acid) and water (with 1% trfluoroacetic acid) 16%-55% over 50 minutes. Lyophilisation of the pure fractions gave 37.5mg product LC/MS (ES⁺) calc for MH+ 3234.4; found 3234.4. Preparation of Bicyclic Peptide Drug Conjugates (A) to (E) Preparation of amine precursors

The diacid (Boc-AHDA) and HATU were each weighed out and dissolved in 1mL DMSO. The solutions were then combined and mixed well, then DIPEA added. The resulting yellow solution was mixed for 5 minutes, then added to a solution of amine in 2.5 mL DMSO. The mixture was stirred overnight and analysed by LC-MS. Purification was carried out by diluting the reaction mixture up to 45 mL with water and 1 mL acetic acid. The solution was split into 3 equal parts and each one was run on a preparative C8 column using 0.1% TFA in H2O as eluent A and 0.1% TFA in MeCN as eluent B using gradient 207030-10. Due to the low absorption of the compounds at 220 nm, a wavelength of 190 nm was used for detection. Fractions containing the desired material for each of the 3 runs (determined by LC-MS) were combined and lyophilised for 2 days in pre-weighed vessels which had been lyophilised overnight to remove any volatiles. The dried products were weighed and yields calculated.

Add 6 equivalents (with respect to scaffold) of copper sulfate solution to 20 equivalents (with respect to scaffold) ascorbic acid solution and mix thoroughly. Mix the AHDA scaffold solution with 3 equivalents of alkyne functionalised bicycle solution (to give 1.5 equivalents of alkyne with respect to total azide groups – see table below for clarity). Add the catalyst solution to the azide/alkyne mixture, mix the contents then spin down to retrieve droplets. Shake/stir and monitor the reaction by LCMS/HPLC by diluting sample in 50 mM EDTA solution (to chelate excess copper). Typically the reaction is complete within one hour. When complete, dilute sample in 50 mM EDTA solution and purify using preparative RP-HPLC on a C8/18 column using TFA as a counter ion. The dilution should be such that the DMSO content of the solution to be loaded on the HPLC is less than 10%, preferably less than 5 %, so as to ensure that the Peptide-conjugate sticks efficiently to the column

The portions of Boc-protected scaffold compounds were dissolved in 2 mL acetonitrile and 1 mL TFA was added to each solution. The solutions were incubated for 16 hours then analysed by LC-MS, which showed no trace of starting material, only the desired amine products.35 mL water was added to each the mixtures and the resultant mixture frozen and lyophilised. However, repeated melting was observed which was overcome by addition of water to the mixtures each time and re-freezing. Conversion of amine precursors to disulphide linked BDCs

Conjugation of amine precursors with cytotoxic agents via a disulphide linker

Full Procedure Prepare 20 mM solution of the Bicycle peptide in dry DMSO or DMA . Prepare 100 mM solution of N-Succinimidyl 2-pyridyldithio-carboxylates esters in dry DMSO or DMA. N- Succinimidyl esters are moisture sensitive and are kept under nitrogen in a desiccator in - 20°C freezer. Add the N-Succinimidyl 2-pyridyldithio-carboxylate solution to the peptide solution in order to have a 1.25 fold excess of the former over the peptide. Mix well the resulting reaction mix and spin down any droplets on the wall. Add neat DIPEA to the mixture to have a 20 fold excess of the DIPEA over the peptide. Mix well the resulting solution and spin down any droplets on the wall. Thus, the approximate initial concentrations of the reactants are: o Bicycle peptide 15 mM o N-Succinimidyl 2-pyridyldithio-carboxylate 19 mM o DIPEA 300 mM Stir or shake the reaction mix at room temperature and follow the progression of the reaction using LC/MS or MALDI-TOF. When the reaction is complete, precipitate MTBE cold. If the reaction is performed in DMA, add directly cold MTBE 12 times the volume of the reaction. If the reaction is in DMSO, add 2 volume of acetonitrile and 10 volume of cold MTBE. Centrifuge the reaction mixture for 10 minutes at 4000 rpm and then remove the supernatant. Solubilize the precipitated up to 20 mL with 6 M guanidine hydrochloride and purify the mixture by RP-HPLC (see SP058) or up to 50 mL with 6 M guanidine hydrochloride and purify the mixture by FPLC. n of final BDCs

The general procedure below was used to prepare the disulphide linked BDCs and is analogous to that used to prepare the Reference Bicyclic Peptide Drug Conjugate BT66BDC-1 described hereinbefore: Pyridyl disulfide was dissolved in DMF (1 eq) . A solution of DM1 (1.2 eq) in DMF was added followed by diisopropylethylamine (7 eq) and the mixture stirred at room temperature for 2 hour. Water was added and the mixture filtered before being loaded onto a Luna C18(3) prep HPLC column (250mm X 20mm). The product was obtained by eluting with a gradient of acetonitrile (with 1% trfluoroacetic acid) and water (with 1% trfluoroacetic acid) over 50 minutes. Lyophilisation of the pure fractions gave the product. Preparation of Compound of formula (D) (BT66BDC-2) R = H, R1 = Me

The compound was prepared using the general procedure described above. Input material – Pyridyl disulphide prescursor (47mg), DM1 (7.6mg), diisopropylethylamine (10µL) and DMF (1.4ml). Following purification the reaction gave 47mg of the desired product (57%) LC/MS (ES+) calc for M+ 6116.9; found 6116.9 Preparation of Compound of formula (E) (BT66BDC-3) R, R1 = Me

The compound was prepared using the general procedure described above. Input material – Pyridyl disulphide precursor (49mg), DM1 (9.4mg), diisopropylethylamine (11µL) and DMF (1.2ml). Following purification the reaction gave 35mg of the desired product (63%) LC/MS (ES+) calc for M+ 6130.9; found 6130.9. Preparation of Compound of formula (A) (BT17BDC-35)

R = H, R1 = Me The compound was prepared using the general procedure described above. Input material – Pyridyl disulphide precursor (53mg), DM1 (9.1mg), diisopropylethylamine (11µL) and DMF (1.2ml). Following purification the reaction gave 42mg of the desired product (63%) LC/MS (ES+) calc for M+ 5932.9; found 5932.9. Preparation of Compound of formula (C) (BT17BDC-44)

R , R1 = Me The compound was prepared using the general procedure described above. Input material – Pyridyl disulphide precursor (41mg), DM1 (8.9 mg), diisopropylethylamine (11µL) and DMF (1.2ml). Following purification the reaction gave 21mg of the desired product (23%) LC/MS (ES+) calc for M+ 5946.9; found 5946.9 Preparation of Compound of formula (B) (BT17BDC-43) Conversion of amine precursors to Val-Cit linked BDCs

Step 1 - Synthesis of MMAE-PABC-Val-Cit Glutarate

A 7 mL screw top vial containing MMAE-PABC-Val-Cit (250 mg) was purged using a nitrogen balloon. 1.72 mL of anhydrous dimethylacetamide was added with stirring and the solution was cooled to 0 °C in an ice water bath. DIPEA (52.24 mg, 0.40 mmol, 2.0 eqv.) was then added as a stock solution in anhydrous dimethyl acetamide (1.05 mL at 50 mg/ mL) and the reaction was stirred at 0 °C for 10 minutes. Glutaric anhydride (46.12 mg, 0.40 mmol, 2.0 eqv.) was then added as a solution in anhydrous dimethyl acetamide (922.4 µL at 50 mg/mL). The ice bath was then removed and the reaction was stirred to r.t. over the course of 1 h. The progress of the reaction was monitored by LCMS and on completion the reaction was quenched with saturated aqueous ammonium sulphate (4 mL) ensuring that the mixture was at a neutral pH before continuing. Pure water (20 mL) was added causing a precipitate which formed on neutralisation to re- dissolve. The aqueous mixture was then transferred into a 100 mL separating funnel rinsing the reaction vessel into the funnel with dichloromethane (25 mL). The layers were separated and the aqueous phase was extracted twice more with fresh dichloromethane (2 x 25 mL) before drying the combined organic phases by passing through a biotage® phase separation cartridge into a clean 250 mL round-bottom flask. The solvent was removed by rotary evaporation with a maximum water bath temperature of 30 °C to give the crude material as a sticky yellow oil which was taken up into 1: 5 dimethylacetamide: acetonitrile (4 mL) and loaded as liquid onto a 60 g C18 biotage cartridge which had been equilibrated into 1 % acetonitrile in water with 0.05 % TFA buffer. The cartridge was eluted with 1 – 99 % acetonitrile in water with 0.05 % TFA over 40 minutes. Pure product containing fractions were identified using LCMS, combined and lyophilised to dryness. The final yield was 91%, with a purity of >90%.

Step 2 - Synthesis of NHS Activated MMAE-PABC-Val-Cit Glutarate

A 50 mL round bottom flask which contained MMAE-PABC-Val-Cit Glutarate (136.6 mg, 0.11 mmol) was purged using a nitrogen balloon. Anhydrous dimethylacetamide (4.20 mL) and anhydrous dichloromethane (1.5 mL) were then added with stirring before adding N- hydroxysuccinimide (38.4 mg, 0.033 mmol, 3.0 eqv.) was then added as a solid and the solution was cooled to 0 °C in an ice water bath. EDCI (63.51 mg, 0.33 mmol, 3.0 eqv.) was then added as a solid before removing the ice water bath and allowing the reaction to warm to r.t. overnight. Monitoring of the reaction using LCMS (method 1) showed that the reaction had proceeded to completion. Dichloromethane was removed by gentle rotary evaporation before injecting the resulting dimethyl acetamide solution onto a 30 g biotage® cartridge which had been equilibrated into 5 % acetonitrile in water (0.05 % TFA buffer) before the cartridge was eluted with 5 – 99 % acetonitrile in water (0.1 % TFA) over 40 minutes. Pure product containing fractions were identified using LCMS, combined and lyophilised to dryness. The final yield was 61%, with a purity of >90%. Step 3 - Synthesis of MMAE-PABC-Val-Cit Glutarate-Bicycle

A 7 mL screw top vial which contained bicycle peptide solution (i.e.32 mM, 1.34 mL in DMA, 42.9 µmol, 114 mg) was purged using a nitrogen balloon. DIPEA (37.4 µL, 27.1 mg, 0.22 mmol, 5 eqv.) was then added and the reaction was stirred at r.t. for 10 minutes. NHS Activated MMAE-PABC-Val-Cit Glutarate (85.9 mg, 64.4 µmol, 1.5 eqv.) in DMA (1.85 mL, 34.8 mM solution) was then added and the reaction was stirred under a positive nitrogen atmosphere overnight at r.t. The crude reaction was purified by reverse phase preparative chromatography in 0.1% TFA as mobile phase. Pure product containing fractions were identified using LCMS, combined and lyophilised to dryness. The final yield was 80%, with a purity of >95%. Free toxin was not detectable (or <1%) versus the reference by HPLC. LC/MS (ES+) calc for M+ 6213.9; found 6213.9 Preparation of BCY8252

Reactions were monitored using LC-MS (Acquity UPLC CSH C18 column, 1.7µm, 2.1 x 30mm; acetonitrile/water/HCOOH containing buffers and 15 to 60% acetonitrile gradient elution over 10min). Products from the reactions were purified using RP-HPLC (Gemini C18 - semi prep column, 5µm, 110 Å, 250 x 10mm; acetonitrile/water/TFA containing buffers and 20 to 80% acetonitrile gradient elution over 20min). General procedure for preparation of Compound 2

A solution of compound 1 (1.20 g, 1.68 mmol) in DMF (10 mL) was added DIEA (902 mg, 6.98 mmol, 1.22 mL) under nitrogen atmosphere, the solution was stirred at 0 °C for 10 min, then HOBt (226 mg, 1.68 mmol, 1.2 eq) and MMAE (1.00 g, 1.40 mmol) were added thereto, the mixture was degassed and purged with N2 for three times, and then the mixture was stirred at 35 °C for 16 hr under N2 atmosphere. LC-MS (ES8396-28-P1A1) showed compound 1 was consumed completely and one main peak with desired mass was detected. The resulting reaction mixture was taken in 200 mL of water with stirring for 1 hr, a precipitate was formed and filtered to give a yellow solid. The yellow solid was triturated with EtOAc overnight to give compound 2 (2.60 g, 2.12 mmol, 76.1% yield) as a white solid. LCMS m/z 1223.7 = [M+H - Boc]⁺ , RT = 1.18 min. General procedure for preparation of Compound 3

To a solution of compound 2 (2.60 g, 2.12 mmol) in DCM (28 mL) was added TFA (12.3 g, 108 mmol, 8.00 mL) with stirring at 25 °C for 3 hr. LCMS (ES8396-30-P1B2) showed the Boc- group was removed completely and desired product (MS = 1123) was formed with a byproduct (MS = 1220). Then the mixture was concentrated under pressure to remove the TFA to give a residue. The residue was dissolved in THF (20 mL) and K2CO3 (1.47 g, 10.6 mmol) was added. The mixture was stirred at 25 °C for 12 hr. LC-MS (ES8396-30-P1B3) showed one main peak with desired mass was detected. The resulting reaction mixture was filtered and the filtrate was concentrated to give a residue which was purified by prep-HPLC (TFA condition) to give compound 3 (1.40 g, 1.13 mmol, 53.2% yield, TFA) as a white solid. LCMS m/z 610.8 = [M+H]²⁺ , RT = 0.82 min. General procedure for preparation of Compound 4

A 50 mL of round flask containing compound 3 (1.80 g, 1.45 mmol, TFA) was purged using a nitrogen balloon. Anhydrous DMA (10 mL) was added with stirring and the solution was cooled to 0 °C in an ice water bath. DIEA (376 mg, 2.91 mmol, 507 µL) was then added and the reaction was stirred at 0 °C for 10 min. Tetrahydropyran-2,6-dione (332 mg, 2.91 mmol) was added. The ice bath was then removed and the reaction was stirred at 25 °C for 1 hr. LC-MS (ES8396-31-P1A1) showed one main peak with desired mass was detected. The resulting reaction mixture was purified by prep-HPLC (TFA condition) to give compound 4 (1.70 g, 1.26 mmol, 86.5% yield, TFA) as a white solid. LCMS m/z 619.8 = [M-H]2- , RT = 1.12 min. General procedure for preparation of Compound 5

To a solution of compound 4 (200 mg, 162 µmol) in DMA (4.5 mL) and DCM (1.5 mL) was added 1-hydroxypyrrolidine-2,5-dione (55.8 mg, 485 µmol) under nitrogen atmosphere with stirring for 10 min at 0 °C using an ice bath, then EDCI (92.9 mg, 485 µmol) was added to the mixture with further stirring at 25 °C for 16 hr. LC-MS (ES8396-32-P1A) showed compound 4 was consumed completely and one main peak with desired mass was detected.1 mL of water was added to the mixture. The mixture was purified by prep-HPLC (neutral condition) to give compound 5 (170 mg, 117 µmol, 72.6% yield, TFA) as a white solid. LCMS m/z 667.7= [M+H]²⁺ , RT = 1.1 min. General procedure for preparation of Compound 6

To a solution of format 5 (60.0 mg, 19.3 µmol) in DMA (3 mL) was added DIEA (8.09 mg, 62.6 µmol, 10.9 µL) and stirred under N2 atmosphere. Then compound 5 (25.8 mg, 19.3 µmol) was added and stirred at 25 °C for 16 hr. LC-MS (ES8396-35-P1A4) showed compound 5 was consumed completely and desired mass was detected.2 mL water was added to the reaction mixture. The mixture was purified by prep-HPLC (TFA condition) to give compound 6 (30.0 mg, 6.93 µmol, 35.9% yield) as a white solid. LCMS m/z 1258.5 = [M+H]²⁺ , RT = 1.1 min.

To a solution of compound 6 (26.0 mg, 6.01 µmol), (17-69-07-N241)-PYA (16.5 mg, 6.01 µmol) in anhydrous DMF (2 mL) were added CuI (5.72 mg, 30.0 µmol) and DIEA (15.5 mg, 120 µmol, 20.9 µL). The mixture was stirred at 25 °C under nitrogen atmosphere for 1 hr. LC-MS (ES8396-41-P1A1) showed desired mass was detected. HPLC (ES8396-41-P1H1) showed the purity of reaction mixture.2 mL water was added to the reaction mixture which was purified by prep-HPLC (TFA condition) to give compound BCY8252 (11.4 mg, 1.61 µmol, 26.86% yield) as a white solid. LCMS m/z 1766.2 = [M+H]³⁺ , RT = 1.1 min. HPLC RT = 12.5 min. DATA 1. MT1 Competition Binding Assay MT1 Competition Binding Assay Affinity of the peptides of the invention for human MT1- MMP (K_i) was determined using a fluorescence polarisation assay, using the method reported in WO 2016/067035. The results of the binding affinity are shown in Table A where it can be seen that the tandem versions (BT17BDC-35, BT17BDC-43 and BT17BDC-44) demonstrated at least 3 fold greater binding affinity than the reference BDC (BT17BDC-18): Table A

2. CD38 Competition Binding Assay CD38 Competition Binding Assay Affinity of the peptides of the invention for human CD38 (Ki) was determined using a fluorescence polarisation assay, using the method reported by Lea et al (Expert Opin Drug Discov.20116(1): 17–3) and using the following fluorescently labelled peptide: ACTPCADFPIWGCA-Sar6-K(Fl) The results of the binding affinity are shown in Table B where it can be seen that the tandem versions (BT66BDC-2 and BT66BDC-3) demonstrated at least 8 fold greater binding affinity than the reference BDC (BT66BDC-1): Table B

3. Biacore binding analysis of Reference BDC (BT17BDC-18) and tandem version (BT17BDC-35; Compound (A)) to MT1-MMP PEX protein 3.1 Aim Use Biacore system to compare the affinity/kinetics of binding of the tandem version of BT17BDC-18 to MT1-MMP PEX protein. By immobilising different densities of MT1-MMP protein on the chip surface and flowing the peptide over, it is hoped we can compare the affinity/kinetics under conditions where the tandem binding nature of the tandem molecule is evident and the expected extended residence time is evident. 3.2 Materials • Biacore T200 system, from Technology Development Laboratory • CM5 Biacore chip, Series S Sensor Chip CM5, #BR-1005-30 • General reagents from GE, provided by TDL lab • Human MT1-MMP PEX protein, 79.1 μM (T-017-019) • BT17BDC-18 = Lonza P4731-3 • BT17BDC-35 = batch #1.2 • Running buffer – 10 mM HEPES, 150 mM NaCl, 1 mM CaCl2, 0.025% Tween20 (GE) (pH 7.4); made up from our own lab stocks and filtered 3.3 Method & Results The work flow for using the Biacore system can be split into: (a) Docking & preparation of chip (b) Immobilisation of protein on chip (“ligand”) (c) Initial confirmation of binding of a peptide (“analyte”) (d) Scouting for regeneration conditions (e) Confirming surface performance (f) Kinetic analyses of peptides of interest Prior to use the Biacore T200 had been cleaned with the Desorb procedure and was in Standby mode using water with the Maintenance chip inserted. (a) Docking & preparation of chip • Running buffer – 10 mM HEPES, 150 mM NaCl, 1 mM CaCl2, 0.025% Tween20, pH 7.4. • Undock maintenance chip and store protected from dust. • Switch to my running buffer (keep some in 50 ml tube for diluting samples). • Dock my new CM5 Biacore chip. • Prime in running buffer. • Run Normalise to normalise the signal across the chip. (b) Immobilisation of protein on chip • Wanted to immobilise a low, medium and high amount on the chip to explore densities that might give more opportunity for clustering and tandem effects. • Use the formula from Biacore to estimate how much ligand to immobilise to produce a given maximum binding response (Rmax): Rmax = analyte MW x RL (immobilisation level of ligand) x stoichiometry ligand MW e.g. for an Rmax of ~25 then: 25 = ~2500 x RL x 1 ~24500 RL = ~250 i.e. if I immobilise ~250 RUs of MT1-MMP PEX protein then the maximum theoretical binding response with a peptide of 2500 MW will be ~25 RUs (Biacore recommend keeping Rmax below 100, and the lower the better, for good kinetics). • MT1-MMP protein was diluted and immobilised as below using the Aim to immobilise wizard using the standard amine coupling procedure: o Fc1 = blank o Fc2 = Aim for 150 RU of PEX using 10 ug/ml diluted in NaAc pH 5.5 o Fc3 = Aim for 1500 RU of PEX using 10 ug/ml diluted in NaAc pH 5.5 o Fc4 = Aim for 15000 RU of PEX using 20 ug/ml diluted in NaAc pH 5.5 • The immobilisation procedure worked well o Fc2 = immobilised 146 RU o Fc3 = immobilised 1486 RU o Fc4 = immobilised 5055 RU (c) Initial confirmation of binding of a peptide • Test binding was conducted with 100 nM BT17BDC-18 o Binding response of ~8 RU • Test binding was conducted with 100 nM tandem BT17BDC-35 o Binding response of ~15 RU (d) Scouting for regeneration conditions • Regeneration with 10 mM NaOH was confirmed as being optimal (as used before) (e) Confirming surface performance • Not performed as previously confirmed. (f) Kinetic analyses of peptides of interest • Used the Wizard o 50 µl/min o 300 sec association o 1800 sec dissociation o 30 sec regeneration with 10 mM NaOH o 30 sec stabilisation o Peptides diluted from 100 nM down to 0.195 or 0.048 nM, with buffer blank before and after. The data from the lowest immobilisation density for this experiment is shown in Figure 1 wherein it can be seen that reference tandem molecule BT17BDC-35 demonstrated an extended residence time compared with the corresponding single bicyclic peptide containing drug conjugate BT17BDC-18. 4. In vivo efficacy test of Compounds of Formula (D) and (E) in treatment of MOLP-8 xenograft in CB17-SCID mice 4.1 Study Objective The objective of this study was to evaluate the in vivo anti-tumor efficacy of Compounds of Formula (D) and (E) in comparison with single bicyclic peptide containing drug conjugate BT66BDC-1 in the treatment of the subcutaneous MOLP-8 xenograft model in CB17-SCID mice. 4.2 Experimental Design Table 1

Note: n: animal number; Dosing volume: adjust dosing volume based on body weight 10 ^l/g 4.3 Materials 4.3.1Animals and Housing Condition 4.3.1.1 Animals Species: Mus Musculus Strain: CB17-SCID Age: 6-8 weeks Sex: female Body weight: 18-22 g Number of animals: 24 mice plus spare Animal supplier: Shanghai SLAC Laboratory Animal Co., LTD. 4.3.1.2 Housing condition The mice were kept in individual ventilation cages at constant temperature and humidity with 3 animals in each cage. ^ Temperature: 20~26^oC. ^ Humidity 40-70%. Cages: Made of polycarbonate. Size: 300 mm x 180 mm x 150 mm. The bedding material was corn cob, which was changed twice per week. Diet: Animals had free access to irradiation sterilized dry granule food during the entire study period. Water: Animals had free access to sterile drinking water. Cage identification: The identification labels for each cage contained the following information: number of animals, sex, strain, the date received, treatment, study number, group number and the starting date of the treatment. Animal identification: Animals were marked by ear coding. 4.3.2Test and Positive Control Articles Product identification: BT66BDC1 Physical description: DMSO stock MW: 3234.4, Purity: > 95% Concentration: 20 mg/ml Package and storage condition: stored at -80^oC Product identification: BT66BDC2 Manufacturer: Bicycle Therapeutics Lot number: N/A Physical description: Lyophilized powder Molecular weight: 6117.1 Specification: 5mg/bottle Package and storage condition: stored at -80^oC Product identification: BT66BDC3 Manufacturer: Bicycle Therapeutics Lot number: N/A Physical description: Lyophilized powder Molecular weight: 6130.3 Specification: 5mg/bottle Package and storage condition: stored at -80^oC 4.4 Experimental Methods and Procedures 4.4.1Cell Culture The MOLP-8 tumor cells were maintained in RMPI-1640 supplemented with 20% heat inactivated fetal bovine serum at 37ºC in an atmosphere of 5% CO2 in air. The tumor cells were routinely subcultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation. 4.4.2Tumor Inoculation Each mouse was inoculated subcutaneously at the right flank with MOLP-8 tumor cells (10 x 10⁶) in 0.2 ml PBS with 50% matrigel for tumor development. The animals were randomized and treatment was started when the average tumor volume reached approximately 173 mm³ for the efficacy study. The test article administration and the animal numbers in each group were shown in Table 2: Table 2: Testing Article Formulation Preparation

4.4.3Observations All the procedures related to animal handling, care and the treatment in the study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec, following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). At the time of routine monitoring, the animals were daily checked for any effects of tumor growth and treatments on normal behavior such as mobility, food and water consumption (by looking only), body weight gain/loss (body weights were measured twice weekly), eye/hair matting and any other abnormal effect as stated in the protocol. Death and observed clinical signs were recorded on the basis of the numbers of animals within each subset. 4.4.4Tumor Measurements and the Endpoints The major endpoint was to see if the tumor growth could be delayed or mice could be cured. Tumor size was measured three times weekly in two dimensions using a caliper, and the volume was expressed in mm³ using the formula: V = 0.5 a x b² where a and b are the long and short diameters of the tumor, respectively. The tumor size was then used for calculations of T/C value. The T/C value (in percent) is an indication of antitumor effectiveness; T and C are the mean volumes of the treated and control groups, respectively, on a given day. TGI was calculated for each group using the formula: TGI (%) = [1-(Ti-T0)/ (Vi-V0)] ×100; Ti is the average tumor volume of a treatment group on a given day, T0 is the average tumor volume of the treatment group on the day of treatment start, Vi is the average tumor volume of the vehicle control group on the same day with Ti, and V0 is the average tumor volume of the vehicle group on the day of treatment start. 4.4.5Sample Collection On PG-D14, mice of group 2-8 were re-dosed and plasma was collected at 5min, 15min, 30min, 60min and 120min after the dosing for PK analysis and tumors were fixed for FFPE. 4.4.6Statistical Analysis Summary statistics, including mean and the standard error of the mean (SEM), are provided for the tumor volume of each group at each time point. Statistical analysis of difference in tumor volume among the groups was conducted on the data obtained at the best therapeutic time point after the final dose. A one-way ANOVA was performed to compare tumor volume among groups, and when a significant F-statistics (a ratio of treatment variance to the error variance) was obtained, multiple comparison procedures will be applied after ANOVA. The potential synergistic effect between treatments will be analyzed by two-way ANOVA. All data will be analyzed using Prism 5.0. p < 0.05 is considered to be statistically significant. 4.5 Results 4.5.1Body Weight change and Tumor Growth Curve Body weight and tumor growth are shown in Figures 2 and 3. 4.5.2Tumor Volume Trace Mean tumor volume over time in female CB17-SCID mice bearing MOLP-8 xenograft is shown in Table 3. Table 3: Tumor volume trace over time Days after the start of treatment Group Treatment 0 2 4 7 9 11 14 1 Vehicle , 17 335±85 584±15 1149±1 1858±2 2423±3 3034±3 biw 3± 5 72 01 32 77 BT66BDC-2 17 (Compound 525±16 1073±2 1371±3 1709±4 2335±4 2 2± 296±85 D) 6 66 27 20 20 28 0.3 mpk, tiw BT66BDC-2 17 (Compound 3 5± 289±73 362±38 410±28 352±32 330±32 363±81 D) 1 mpk, 20 tiw BT66BDC-2

(Com 232±10 4 pound 4± 139±45 71±22 71±19 54±23 38±17 D) 3 53 3 mpk, tiw BT66BDC-3 17 (Compound 567±12 954±12 1169±9 1725±1 2297±1 5 4± 320±95 E) 2 6 9 47 67 33 0.3 mpk, tiw BT66BDC-3

(Compound 923±17 1190±1 1669±2 2246±1 6 6± 331±35 518±78 E) 7 61 40 52 17 1 mpk, tiw BT66BDC-3 16 (C 317±12 505±14 608±15 648±13 791±18 7 ompound 9± 390±96 E) 2 1 6 9 9 55 3 mpk, tiw BT66BDC- 17 8 1, 3± 204±48 200±59 228±99 156±66 94±34 68±15 3 mpk, tiw 21 4.5.3Tumor Growth Inhibition Analysis Tumor growth inhibition rate for BT66BDC1, BT66BDC2 and BT66BDC3 in the MOLP-8 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment. Table 4: Tumor growth inhibition analysis (T/C and TGI) Tumor b P value P value T/C TGI Gr Treatment Volume ( vs ( vs BDC1) (%) (%) (mm3)a vehicle) 1 Vehicle, tiw 3034±377 -- -- BT66BDC-2 (Compound -- 2 2335±420 p>0.05 (D) 0.3 mpk, tiw 77.0 24.4 BT66BDC-2 (Compound -- 3 363±81 p<0.001 (D) 1 mpk, tiw 12.0 93.4 BT66BDC-2 (Compound p>0.05 4 38±17 p<0.001 (D) 3 mpk, tiw 1.2 104.8 BT66BDC-3 (Compound -- 5 2297±167 p>0.05 (E) 0.3 mpk, tiw 75.7 25.8 BT66BDC-3 (Compound -- 6 2246±152 p>0.05 (E) 1 mpk, tiw 74.0 27.6 BT66BDC-3 (Compound p<0.01 7 791±189 p<0.001 (E) 3 mpk, tiw 26.1 78.3 BT66BDC-1 (Compound -- 8 68±15 p<0.001 (E) 3 mpk, tiw 2.2 103.7 a. Mean ± SEM. b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C). 4.6 Results Summary and Discussion In this study, the therapeutic efficacy of Reference BDC BT66BDC-1 and tandem BDCs BT66BDC-2 (Compound D) and BT66BDC-3 (Compound E) in the MOLP-8 xenograft model was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the Figures 2 and 3 and Tables 1 to 4. The mean tumor size of vehicle treated mice reached 3034 mm³ on day 14. BT66BDC-2 (Compound D) at 0.3 mg/kg (TV=2335 mm³, TGI=24.4%, p>0.05),1 mg/kg (TV=363 mm³, TGI=93.4%, p<0.001), 3 mg/kg (TV=38 mm³, TGI=104.8%, p<0.001) and BT66BDC-3 (Compound E) at 0.3 mg/kg (TV=2297 mm³, TGI=25.8%, p>0.05), 1 mg/kg (TV=2246 mm³, TGI=27.6%, p>0.05), 3 mg/kg (TV=791 mm³, TGI=78.3%, p<0.001) produced dose- dependent antitumor activity. BT66BDC-2 (Compound D) at 3 mg/kg produced comparable anti-tumor effect as compared with Reference monomer BDC BT66BDC-1. BT66BDC-3 (Compound E) at 3 mg/kg showed less anti-tumor activity. Body weight was monitored regularly as an indirect measure of toxicity. In this study, all mice maintained the bodyweight well. 5. Nectin-4 Biacore SPR Binding Assay Biacore experiments were performed to determine ka (M^-1s^-1), kd (s^-1), KD (nM) values of monomeric peptides binding to human Necin-4 protein (obtained from Charles River). Human Nectin-4 (residues Gly32-Ser349; NCBI RefSeq: NP_112178.2) with a gp67 signal sequence and C-terminal FLAG tag was cloned into pFastbac-1 and baculovirus made using standard Bac-to-Bac™ protocols (Life Technologies). Sf21 cells at 1 x 10⁶ml^-1 in Excell-420 medium (Sigma) at 27°C were infected at an MOI of 2 with a P1 virus stock and the supernatant harvested at 72 hours. The supernatant was batch bound for 1 hour at 4°C with Anti-FLAG M2 affinity agarose resin (Sigma) washed in PBS and the resin subsequently transferred to a column and washed extensively with PBS. The protein was eluted with 100µg/ml FLAG peptide. The eluted protein was concentrated to 2ml and loaded onto an S- 200 Superdex column (GE Healthcare) in PBS at 1ml/min. 2ml fractions were collected and the fractions containing Nectin-4 protein were concentrated to 16mg/ml. The protein was randomly biotinylated in PBS using EZ-Link™ Sulfo-NHS-LC-LC-Biotin reagent (Thermo Fisher) as per the manufacturer’s suggested protocol. The protein was extensively desalted to remove uncoupled biotin using spin columns into PBS. For analysis of peptide binding, a Biacore 3000 instrument was used utilising a CM5 chip (GE Healthcare). Streptavidin was immobilized on the chip using standard amine-coupling chemistry at 25°C with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated with a 7 minute injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1 M N- hydroxy succinimide (NHS) at a flow rate of 10 μl/min. For capture of streptavidin, the protein was diluted to 0.2 mg/ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120µl of streptavidin onto the activated chip surface. Residual activated groups were blocked with a 7 minute injection of 1 M ethanolamine (pH 8.5) and biotinylated Nectin-4 captured to a level of 1,200-1,800 RU. Buffer was changed to PBS/0.05% Tween 20 and a dilution series of the peptides was prepared in this buffer with a final DMSO concentration of 0.5%. The top peptide concentration was 100nM with 6 further 2-fold dilutions. The SPR analysis was run at 25°C at a flow rate of 50µl/min with 60 seconds association and dissociation between 400 and 1,200 seconds depending upon the individual peptide. Data were corrected for DMSO excluded volume effects. All data were double-referenced for blank injections and reference surface using standard processing procedures and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Data were fitted using simple 1:1 binding model allowing for mass transport effects where appropriate. BCY8252 (as well as its constituent monomeric Nectin-4 bicyclic peptide, BCY8126) were both tested in the above mentioned Nectin-4 binding assays and the results are shown in Table 5: Table 5

6. In vivo efficacy study of BCY8252 in treatment of NCI-H292 xenograft in Balb/c nude mice 6.1 Study Objective The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY8252 in treatment of NCI-H292 xenograft in Balb/c nude mice. 6.2 Experimental Design Table 6

6.3 Materials 6.3.1 Animals and Housing Condition 6.3.1.1. Animals Species: Mus Musculus Strain: Balb/c nude Age: 6-8 weeks Sex: female Body weight: 18-22 g Number of animals: 43 mice plus spare Animal supplier: Shanghai Lingchang Biotechnology Experimental Animal Co. Ltd 6.3.1.2. Housing condition The mice were kept in individual ventilation cages at constant temperature and humidity with 3 or 4 animals in each cage. ^ Temperature: 20~26 ^oC. ^ Humidity 40-70%. Cages: Made of polycarbonate. The size is 300 mm x 180 mm x 150 mm. The bedding material is corn cob, which is changed twice per week. Diet: Animals had free access to irradiation sterilized dry granule food during the entire study period. Water: Animals had free access to sterile drinking water. Cage identification: The identification labels for each cage contained the following information: number of animals, sex, strain, the date received, treatment, study number, group number and the starting date of the treatment. Animal identification: Animals were marked by ear coding. 6.3.2 Test and Positive Control Articles Product identification: BCY8252 Manufacturer: Bicycle Therapeutics Lot number: 1 Physical description: Lyophilised powder Molecular weight: 7067.19 Purity: 95.90% Package and storage condition: stored at -80^oC 6.4 Experimental Methods and Procedures 6.4.1 Cell Culture The NCI-H292 tumor cells were maintained in vitro as a monolayer culture in RPMI-1640 medium supplemented with 10% heat inactivated fetal bovine serum at 37ºC in an atmosphere of 5% CO2 in air. The tumor cells were routinely subcultured twice weekly by trypsin-EDTA treatment. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation. 6.4.2 Tumor Inoculation Each mouse was inoculated subcutaneously at the right flank with NCI-H292 tumor cells (10 x 10⁶) in 0.2 ml of PBS for tumor development.43 animals were randomized when the average tumor volume reached 168 mm³. The test article administration and the animal numbers in each group were shown in the experimental design table. 6.4.3 Testing Article Formulation Preparation Table 7

6.4.4 Observations All the procedures related to animal handling, care and the treatment in the study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec, following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). At the time of routine monitoring, the animals were checked for any effects of tumor growth and treatments on normal behavior such as mobility, food and water consumption (by looking only), body weight gain/loss, eye/hair matting and any other abnormal effect as stated in the protocol. Death and observed clinical signs were recorded on the basis of the numbers of animals within each subset. 6.4.5 Tumor Measurements and the Endpoints The major endpoint was to see if the tumor growth could be delayed or mice could be cured. Tumor volume was measured three times weekly in two dimensions using a caliper, and the volume was expressed in mm³ using the formula: V = 0.5 a x b² where a and b are the long and short diameters of the tumor, respectively. The tumor size was then used for calculations of T/C value. The T/C value (in percent) is an indication of antitumor effectiveness; T and C are the mean volumes of the treated and control groups, respectively, on a given day. TGI was calculated for each group using the formula: TGI (%) = [1-(T_i-T₀)/ (V_i-V₀)] ×100; T_i is the average tumor volume of a treatment group on a given day, T₀ is the average tumor volume of the treatment group on the day of treatment start, Vi is the average tumor volume of the vehicle control group on the same day with Ti, and V0 is the average tumor volume of the vehicle group on the day of treatment start. 6.4.6 Sample Collection At the end of study, the plasma of group 2, 5, 9, 10, 11, 12 mice was collected at 5 min, 15 min, 30 min, 1 h and 2 h post the last dosing. The tumors of group 1, 5, 6, 12 mice were collected for FFPE at 2 h post the last dosing. 6.4.7 Statistical Analysis Summary statistics, including mean and the standard error of the mean (SEM), were provided for the tumor volume of each group at each time point. Statistical analysis of difference in tumor volume among the groups was conducted on the data obtained at the best therapeutic time point after the final dose. A t-test was performed to compare tumor volume among groups, and when a significant .All data were analyzed using GraphPad Prism 5.0. P< 0.05 was considered to be statistically significant. 6.5 Results 6.5.1 Body Weight change and Tumor Growth Curve Body weight and tumor growth curve are shown in Figure 4. 6.5.2 Tumor Volume Trace Mean tumor volume over time in female Balb/c nude mice bearing NCI-H292 xenograft is shown in Table 8. Table 8: Tumor volume trace over time Gr Days after the start of treatment Treatment . 0 2 4 7 9 11 14 168±1 297±4 362±5 697±10 843±15 1 Vehicle, qw 460±62 548±69 6 8 8 2 2 BCY8252 (Compound 168±3 207±4 230±5 313±10 337±12 344±12 410±15 F), 1 8 2 7 2 6 7 3 mpk, qw 6.5.3 Tumor Growth Inhibition Analysis Tumor growth inhibition rate for test articles in the NCI-H292 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment. Table 9: Tumor growth inhibition analysis Tumor T/Cb TGI Gr Treatment Volume P value (mm 1 Vehicle, qw 843±1

BCY8252 2 (Compound F), 410±157 48.7 64.1 p>0.05 3 mpk, qw a. Mean ± SEM. b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C). 6.6 Results Summary and Discussion In this study, the therapeutic efficacy of BCY8252 in the NCI-H292 xenograft model was evaluated. The measured body weight and tumor volume of all treatment groups at various time points are shown in the Figure 4 and Tables 8 and 9. The mean tumor size of vehicle treated mice reached 843 mm³ on day 14. BCY8252 at 3 mg/kg didn’t show obvious antitumor activity. In this study, all mice maintained the bodyweight well.

Claims

CLAIMS 1. A drug conjugate comprising at least two peptide ligands, which may be the same or different, each of which comprises a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and an aromatic 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.

2. The drug conjugate as defined in claim 1, wherein said peptide ligands are specific for the same or different targets.

3. The drug conjugate as defined in claim 1 or claim 2, wherein at least one of said peptide ligands is specific for an epitope present on a cancer cell.

4. The drug conjugate as defined in any one of claims 1 to 3, which comprises two peptide ligands, both of which are specific for the same target.

5. The drug conjugate as defined in any one of claims 1 to 3, wherein at least one of said peptide ligands is specific for MT1-MMP, such as said drug conjugate comprises two peptide ligands, both of which are specific for MT1-MMP, in particular said drug conjugate comprises two peptide ligands, both of which are specific for MT1-MMP and both of which comprise the same peptide sequence.

6. The drug conjugate as defined in claim 4 or claim 5, wherein said loop sequences comprise 5 or 6 amino acid acids.

7. The drug conjugate as defined in claim 5 or claim 6, wherein said loop sequences comprise three cysteine residues separated by two loop sequences one of which consists of 5 amino acids and the other of which consists of 6 amino acids.

8. The drug conjugate as defined in any one of claims 5 to 7, wherein the at least one of said peptide ligand specific for MT1-MMP has a core sequence of: CiYNEFGCiiEDFYDICiii (17-69-07; SEQ ID NO: 1).

9. The drug conjugate as defined in any one of claims 5 to 8, wherein the at least one of said peptide ligand specific for MT1-MMP has the full sequence of: βAla-Sar10-A-C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)C (17-69-07-N241; SEQ ID NO: 2).

10. The drug conjugate as defined in any one of claims 1 to 9, wherein at least one of said peptide ligands is specific for CD38, such as said drug conjugate comprises two peptide ligands, both of which are specific for CD38, in particular said drug conjugate comprises two peptide ligands, both of which are specific for CD38 and both of which comprise the same peptide sequence.

11. The drug conjugate as defined in claim 10, wherein said loop sequences comprise 2 or 7 amino acid acids.

12. The drug conjugate as defined in claim 10 or claim 11, wherein said loop sequences comprise three cysteine residues separated by two loop sequences one of which consists of 2 amino acids and the other of which consists of 7 amino acids.

13. The drug conjugate as defined in any one of claims 10 to 12, wherein the at least one of said peptide ligand specific for CD38 has a core sequence of: CiVPCiiADFPIWYCiii (SEQ ID NO: 3).

14. The drug conjugate as defined in any one of claims 10 to 13, wherein the at least one of said peptide ligand specific for CD38 has the full sequence of: (β-Ala)-Sar10-A-CVPCADFPIWYC (66-03-00-N006; SEQ ID NO: 4).

15. The drug conjugate as defined in any one of claims 1 to 14, wherein at least one of said peptide ligands is specific for Nectin-4.

16. The drug conjugate as defined in any one of claims 1 to 3, which comprises two peptide ligands, one of which is specific for a first target (such as MT1-MMP) and the other of which is specific for a second target (such as Nectin-4).

17. The drug conjugate as defined in claim 15 or claim 16, wherein said loop sequences comprise 3 or 9 amino acid acids.

18. The drug conjugate as defined in any one of claims 15 to 17, wherein said loop sequences comprise three cysteine residues separated by two loop sequences one of which consists of 3 amino acids and the other of which consists of 9 amino acids.

19. The drug conjugate as defined in any one of claims 15 to 18, wherein the at least one of said peptide ligand specific for Nectin-4 has a core sequence of: CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: 5).

20. The drug conjugate as defined in any one of claims 15 to 19, wherein the at least one of said peptide ligand specific for Nectin-4 has the full sequence of: (β-Ala)-Sar₁₀-CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: 6).

21. The drug conjugate as defined in any one of claims 1 to 20, wherein said reactive groups comprise cysteine.

22. The drug conjugate as defined in any one of claims 1 to 21, wherein the aromatic molecular scaffold is selected from TBMB (1,3,5-tris(bromomethyl)benzene).

23. The drug conjugate as defined in any one of claims 1 to 22, which is conjugated to one or more active agents, such as small molecules, inhibitors, agonists, antagonists, partial agonists and antagonists, inverse agonists and antagonists and cytotoxic agents.

24. The drug conjugate as defined in any one of claims 1 to 23, which is conjugated to one or more cytotoxic agents.

25. The drug conjugate as defined in claim 24, wherein the cytotoxic agent is selected from maytansinoids, such as DM1 which has the following structure:

or monomethyl auristatins, such as MMAE which has the following structure:

26. The drug conjugate as defined in claim 24 or claim 25, which is selected from a compound of formula (A) to (F):

wherein R is selected from: βAla-Sar10-A-C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)C (SEQ ID NO: 2);

(β-Ala)-Sar₁₀-A-CVPCADFPIWYC (SEQ ID NO: 4);

27. A pharmaceutical composition which comprises the drug conjugate of any one of claims 1 to 26, in combination with one or more pharmaceutically acceptable excipients.

28. The drug conjugate as defined in any one of claims 1 to 26, for use in preventing, suppressing or treating diseases.

29. The drug conjugate for use as defined in claim 28, wherein said disease is one which may be alleviated by cell death 30. The drug conjugate for use as defined in claim 29, wherein said disease is selected from diseases characterised by defective cell types, proliferative disorders such as cancer and autoimmune disorders such as rheumatoid arthritis.