Heterotandem bicyclic peptide complexes

Abstract

HETEROTANDEM BICYCLIC PEPTIDE COMPLEXES The present invention relates to heterotandem bicyclic peptide complexes which comprise a first peptide ligand, which binds to a component present on an immune cell, conjugated via a linker to a second peptide ligand, which binds to a component present on a cancer cell. The invention also relates to the use of said heterotandem bicyclic peptide complexes in preventing, suppressing or treating cancer. 20 24 20 51 05 25 J ul 2 02 4 A B S T R A C T H E T E R O T A N D E M B I C Y C L I C P E P T I D E C O M P L E X E S 2 0 2 4 2 0 5 1 0 5 2 5 J u l 2 0 2 4

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a divisional of Australian Patent Application No 2019247795 which is the 5 Australian national phase entry of PCT / GB2019 / 050951, which claims priority to GB provisional patent application Nos GB1805492.4 filed 4 April 2018 and GB1820981.7 filed 21 December 2018. Each of these applications is herein incorporated by reference in their entireties. 10 FIELD OF THE INVENTION The present invention relates to heterotandem bicyclic peptide complexes which comprise a first peptide ligand, which binds to a component present on an immune cell, conjugated via a linker to a second peptide ligand, which binds to a component present on a cancer cell. The invention also relates to the use of said heterotandem bicyclic peptide complexes in 15 preventing, suppressing or treating cancer. 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 20 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 25 bind to surfaces of several hundred square angstrom, as for example the cyclic peptide CXCR4 antagonist CVX15 (400 A2; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with the Arg-Gly-Asp motif binding to integrin aVb3 (355 A2) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1 binding to urokinase-type plasminogen activator (603 A2; Zhao et al. (2007), J Struct Biol 160 (1), 1-10). 30 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 35 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 2024205105   08 Sep 2026 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 5 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 0 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. 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 15   (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). 20 For the avoidance of doubt, in this specification, the terms ‘comprises’, ‘comprising’, ‘includes’, ‘including’, or similar terms are intended to mean a non-exclusive inclusion, such that a method, system or apparatus that comprises a list of elements does not include those elements solely, but may well include other elements not listed. 25 Any reference to or discussion of any document, act or item of knowledge in this specification is included solely for the purpose of providing a context for the present invention. It is not suggested or represented that any of these matters or any combination thereof formed at the priority date part of the common general knowledge, or was known to be relevant to an attempt to solve any problem with which this specification is concerned. 30 SUMMARY OF THE INVENTION In a first aspect, the invention relates to a heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof comprising: (a) a first peptide ligand which binds to a component present on an immune cell, wherein 35 said component present on an immune cell is CD137, and wherein the CD137 binding bicyclic peptide ligand comprises an amino acid sequence selected from: CiIEEGQYCiiFADPY[Nle]Ciii (SEQ ID NO: 1); 2024205105   08 Sep 2026 Ci[tBuAla]PE[D-Ala]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 3); CiIEEGQYCiiF[D-Ala]DPY[Nle]Ciii (SEQ ID NO: 4); Ci[tBuAla]PK[D-Ala]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 5); Ci[tBuAla]PE[D-Lys]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 6); 5 Ci[tBuAla]P[K(PYA)][D-Ala]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 7); Ci[tBuAla]PE[D-Lys(PYA)]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 8); CiIEE[D-Lys(PYA)]QYCiiFADPY(Nle)Ciii (SEQ ID NO: 9); [dCi][dI][dE][dE][K(PYA)][dQ][dY][dCii][dF][dA][dD][dP][dY][dNle][dCiii] (SEQ ID NO: 10); conjugated via a linker to 0   (b) a second peptide ligand which binds to a component present on a cancer cell, wherein said component present on a cancer cell is EphA2, PD-L1, or Nectin-4, wherein (b)(i) the EphA2 binding bicyclic peptide ligand comprises an amino acid sequence selected from: Ci[HyP]LVNPLCiiLHP[dD]W[HArg]Ciii (SEQ ID NO: 2); and 15          CiLWDPTPCiiANLHL[HArg]Ciii (SEQ ID NO: 11); or a modified derivative thereof, (b)(ii) the PD-L1 binding bicyclic peptide ligand comprises an amino acid sequence selected from: Ci[HArg]DWCiiHWTFSHGHPCiii (SEQ ID NO: 12); 20         CiSAGWLTMCiiQKLHLCiii (SEQ ID NO: 13); and CiSAGWLTMCiiQ[K(PYA)]LHLCiii (SEQ ID NO: 14); or a modified derivative thereof, or (b)(iii) the Nectin-4 binding bicyclic peptide ligand comprises an amino acid sequence selected from: 25          CiP[1Nal][dD]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 15; hereinafter referred to as BCY8116); CiP[1Nal][dD]CiiM[HArg]D[dW]STP[HyP][dW]Ciii (SEQ ID NO: 16; hereinafter referred to as BCY11415); and CiP[1Nal][dK](Sar10-(B-Ala))CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 17); 30          CiPFGCiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 18; hereinafter referred to as BCY11414); or a modified derivative thereof; wherein Ci, Cii and Ciii represent first, second and third cysteine residues, respectively, Nle represents norleucine, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid, 35 HyP represents hydroxyproline, dD represents aspartic acid in D-configuration and HArg 2024205105   08 Sep 2026 represents homoarginine, 1Nal represents 1-naphthylalanine, Sar10 represents 10 sarcosine units, B-Ala represents beta-alanine, or a pharmaceutically acceptable salt thereof; wherein each of said peptide ligands comprises a polypeptide comprising at least three cysteine residues, separated by at least two loop sequences, and a molecular scaffold which 5 forms covalent bonds with the cysteine residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold. In a second aspect, the invention relates to a pharmaceutical composition which comprises the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof of 0 the first aspect in combination with one or more pharmaceutically acceptable excipients. In a third aspect, the invention relates to a method of preventing, suppressing or treating cancer comprising administering to a subject in need thereof an effective amount of the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as 15 defined in the first aspect, or the pharmaceutical composition as defined in the second aspect, wherein: (i) the second peptide ligand binds to EphA2 and the cancer is an EphA2 associated cancer; (ii) the second peptide ligand binds to PD-L1 and the cancer is a PD-L1 associated cancer; or (iii) the second peptide ligand binds to Nectin-4 and the cancer is a Nectin-4 associated 20 cancer. In a fourth aspect, the invention relates to use of the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in the first aspect, or the pharmaceutical composition as defined in the second aspect, in the manufacture of a 25 medicament for preventing, suppressing or treating cancer, wherein: (i) the second peptide ligand binds to EphA2 and the cancer is an EphA2 associated cancer; (ii) the second peptide ligand binds to PD-L1 and the cancer is a PD-L1 associated cancer; or (iii) the second peptide ligand binds to Nectin-4 and the cancer is a Nectin-4 associated cancer. 30 According to a further aspect of the invention, there is provided a heterotandem bicyclic peptide complex comprising: (a) a first peptide ligand which binds to a component present on an immune cell; conjugated via a linker to 2024205105   08 Sep 2026 (b) a second peptide ligand which binds to a component present on a cancer cell; wherein each of said peptide ligands comprise a polypeptide comprising at least three cysteine residues, separated by at least two loop sequences, and a molecular scaffold which forms 5 covalent bonds with the cysteine residues 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 heterotandem bicyclic peptide complex as defined herein in combination with 0 one or more pharmaceutically acceptable excipients. According to a further aspect of the invention, there is provided a heterotandem bicyclic peptide complex as defined herein for use in preventing, suppressing or treating cancer. 15 BRIEF DESCRIPTION OF THE FIGURES Figure 1:     Schematic representation of a heterotandem bicyclic peptide complex comprising an EphA2 and CD137 peptide ligand binding to both an immune cell and a cancer cell. Figure 2:     Structure and composition of the EphA2-CD137 heterotandem bicyclic 20 peptide complex BCY79 2024205105  24 Nov 2025 Figure 3: Analysis of the EphA2-CD137 heterotandem bicyclic peptide complex BCY7985 in the Promega CD137 luciferase reporter assay (CS196008) in the presence of EphA2-expressing HT1080 cells. Figure 4: EphA2 / CD137 heterotandems are active in CD137 reporter assay and the 5 fold induction of activation is dependent on tumour target expression level on the cell line used in co-culture. Figure 5: EphA2 / CD137 heterotandems induce tumour cell killing in primary human T-cell and cancer cell co-culture assay. Tumour cell killing is evaluated by counting viable Nuclight red positive tumour cells over time. A Caspase 3 / 7 dye is used to identify apoptotic . 0 tumour cells. Figure 6: Nectin-4 / CD137 heterotandems are active in CD137 reporter assay and the fold induction of activation is dependent on tumour target expression level on the cell line (HT1376:Nectin-4 high and NCI-H292: Nectin-4 Medium) used in co-culture. Figure 7: Nectin-4 / CD137 heterotandems induce IL-2 and IFN-y cytokine secretion in . 5 a PBMC-4T1 co-culture assay. BCY9350 and BCY9351 are non-binding controls for Nectin-4 and CD137 respectively. Figure 8: Nectin-4 / CD137 heterotandems induce target dependent cytokine release in ex-vivo cultures of primary patient-derived lung tumours. (A) Ex vivo patient derived tumour cells form 3D spheroids within 4h in culture, 10X image under light microscope. (B) Flow 10 analysis of Nectin-4 expression in patient derived tumour samples from 3 donors. (C) Table indicates %CD137+ T cells and Nectin-4+ cells in 3 donor samples. (D) Heatmap indicating % change in immune markers (normalized to vehicle) in response to treatment with control / test compounds. (E) %CD8 +ki67+ T cells in response to treatment with control / test compounds (vehicle indicated with dotted line). 25          Figure 9: PD-L1 / CD137 heterotandems are active in CD137 reporter assay in presence of PD-L1 expressing cell line RKO. Figure 10: Pharmacokinetics of heterotandems in SD Rats: BCY10572 and BCY10000 were dosed IV at 2 mg / kg (n =3). 30 DETAILED DESCRIPTION OF THE INVENTION According to an aspect of the invention, there is provided a heterotandem bicyclic peptide complex comprising: (a) a first peptide ligand which binds to a component present on an immune cell; conjugated via a linker to 35          (b) a second peptide ligand which binds to a component present on a cancer cell; wherein each of said peptide ligands comprise a polypeptide comprising at least three cysteine residues, separated by at least two loop sequences, and a molecular scaffold which forms 2024205105   25 Jul 2024 covalent bonds with the cysteine residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold. First Peptide Ligands 5 References herein to the term “immune cell” includes any cell within the immune system. Suitable examples include white blood cells, such as lymphocytes (e.g. T lymphocytes or T cells, B cells or natural killer cells). In one embodiment, the T cell is CD8 or CD4. In a further embodiment, the T cell is CD8. Other examples of immune cells include dendritic cells, follicular dendritic cells and granulocytes. 10 In one embodiment, the component present on an immune cell is CD137. CD137 is a member of the tumour necrosis factor (TNF) receptor family. Its alternative names are tumour necrosis factor receptor superfamily member 9 (TNFRSF9), 4- IBB and induced 15 by lymphocyte activation (ILA). CD137 can be expressed by activated T cells, but to a larger extent on CD8+ than on CD4+ T cells. In addition, CD137 expression is found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes and cells of blood vessel walls at sites of inflammation. One characterized activity of CD137 is its costimulatory activity for activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival 20 and cytolytic activity. Further, it can enhance immune activity to eliminate tumours in mice. CD137 is a T-cell costimulatory receptor induced on TCR activation (Nam et al., Curr. Cancer Drug Targets, 5:357-363 (2005); Waits et al., Annu. Rev, Immunol., 23:23-68 (2005)). In addition to its expression on activated CD4+ and CD8+ T cells, CD137 is also expressed on 25   CD4+CD25+ regulatory T cells, natural killer (NK) and NK-T cells, monocytes, neutrophils, and dendritic cells. Its natural ligand, CD137L, has been described on antigen- presenting cells including B cells, monocyte / macrophages, and dendritic cells (Watts et al. Annu. Rev. Immunol, 23:23-68 (2005)). On interaction with its ligand, CD137 leads to increased TCR-induced T-cell proliferation, cytokine production, functional maturation, and prolonged CD8+ 30 T-cell survival (Nam et al, Curr. Cancer Drug Targets, 5:357-363 (2005), Watts et d -1., Annu. Rev. Immunol, 23:23-68 (2005)). Signalling through CD137 by either CD137L or agonistic monoclonal antibodies (mAbs) against CD137 leads to increased TCR-induced T cell proliferation, cytokine production and 35 functional maturation, and prolonged CD8+ T cell survival. These effects result from: (1) the activation of the NF-kB, c-Jun NH2-terminal kinase / stress-activated protein kinase 2024205105   25 Jul 2024 (JNK / SAPK), and p38 mitogen-activated protein kinase (MAPK) signalling pathways, and (2) the control of anti-apoptotic and cell cycle -related gene expression. Experiments performed in both CD137 and CD137L-deficient mice have additionally 5 demonstrated the importance of CD137 costimulation in the generation of a fully competent T cell response. IL-2 and IL-15 activated NK cells express CD137, and ligation of CD137 by agonistic mAbs stimulates NK cell proliferation and IFN-y secretion, but not their cytolytic activity. 10 Furthermore, CD137-stimulated NK cells promote the expansion of activated T cells in vitro. In accordance with their costimulatory function, agonist mAbs against CD137 have been shown to promote rejection of cardiac and skin allografts, eradicate established tumours, 15 broaden primary antiviral CD8+ T cell responses, and increase T cell cytolytic potential. These studies support the view that CD137 signalling promotes T cell function which may enhance immunity against tumours and infection. In one embodiment, the first peptide ligand comprises a CD137 binding bicyclic peptide ligand. 20 Suitable examples of CD137 binding bicyclic peptide ligands are disclosed in GB Patent Application Nos. 1712589.9 and 1802934.8, the peptides of which are incorporated herein by reference. 25 In one embodiment, the CD137 binding bicyclic peptide ligand comprises an amino acid sequence: CilEEGQYCiiFADPY[Nle]Ciii (SEQ ID NO: 1); Ci[tBuAla]PE[D-Ala]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 3); CilEEGQYCiiF[D-Ala]DPY[Nle]Ciii (SEQ ID NO: 4); 30          CittBuAlajPKtD-AlajPYCiiFADPYtNIejCiii (SEQ ID NO: 5); CittBuAlajPEtD-LysjPYCiiFADPYtNIejCiii (SEQ ID NO: 6); CittBuAlajPt^PYAjjtD-AlajPYCiiFADPYtNIejCiii (SEQ ID NO: 7); Ci[tBuAla]PE[D-Lys(PYA)]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 8); CiIEE[D-Lys(PYA)]QYCiiFADPY(Nle)Ciii (SEQ ID NO: 9); and 35           [dCi][dl][dE][dE][K(PYA)][dQ][dY][dCii][dF][dA][dD][dP][dY][dNle][dCiii] (SEQ ID NO: 10); 2024205105   25 Jul 2024 wherein Ci, Cu and CiH represent first, second and third cysteine residues, respectively, Nie represents norleucine, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid, or a pharmaceutically acceptable salt thereof. 5 In one particular embodiment which may be mentioned, the CD137 binding bicyclic peptide ligand comprises an amino acid sequence: CilEEGQYCiiFADPY[Nle]Ciii (SEQ ID NO: 1); wherein Ci, Ch and Cui represent first, second and third cysteine residues, respectively, Nie represents norleucine, or a pharmaceutically acceptable salt thereof. 10 In a further embodiment, the CD137 binding bicyclic peptide ligand comprises N- and C-terminal modifications and comprises: Ac-A-(SEQ ID NO: 1)-Dap (hereinafter referred to as BCY7732); Ac-A-(SEQ ID NO: 1)-Dap(PYA) (hereinafter referred to as BCY7741); 15          Ac-(SEQ ID NO: 3)-Dap (hereinafter referred to as BCY9172); Ac-(SEQ ID NO: 3)-Dap(PYA) (hereinafter referred to as BCY11014); Ac-A-(SEQ ID NO: 4)-Dap (hereinafter referred to as BCY8045); Ac-(SEQ ID NO: 5)-A (hereinafter referred to as BCY8919); Ac-(SEQ ID NO: 6)-A (hereinafter referred to as BCY8920); 20          Ac-(SEQ ID NO: 7)-A (hereinafter referred to as BCY8927); Ac-(SEQ ID NO: 8)-A (hereinafter referred to as BCY8928); Ac-A-(SEQ ID NO: 9)-A (hereinafter referred to as BCY7744); and Ac-[dA]-(SEQ ID NO: 10)-[dA]-NH2 (hereinafter referred to as BCY11506); wherein Ac represents an acetyl group, Dap represents diaminopropionic acid and PYA 25 represents 4-pentynoic acid, or a pharmaceutically acceptable salt thereof. In a further embodiment which may be mentioned, the CD137 binding bicyclic peptide ligand comprises N- and C-terminal modifications and comprises: Ac-A-(SEQ ID NO: 1)-Dap (hereinafter referred to as BCY7732); 30 wherein Ac represents an acetyl group and Dap represents diaminopropionic acid, or a pharmaceutically acceptable salt thereof. Second Peptide Ligands References herein to the term “cancer cell” includes any cell which is known to be involved in 35 cancer. Cancer cells are created when the genes responsible for regulating cell division are damaged. Carcinogenesis is caused by mutation and epimutation of the genetic material of normal cells, which upsets the normal balance between proliferation and cell death. This 2024205105   25 Jul 2024 results in uncontrolled cell division and the evolution of those cells by natural selection in the body. The uncontrolled and often rapid proliferation of cells can lead to benign or malignant tumours (cancer). Benign tumors do not spread to other parts of the body or invade other tissues. Malignant tumors can invade other organs, spread to distant locations (metastasis) 5 and become life-threatening. In one embodiment, the cancer cell is selected from an HT1080, SC-OV-3, PC3, H1376, NCI-H292, LnCap, MC38, 4T1-D02 and RKO tumor cell. 10 In one embodiment, the component present on a cancer cell is EphA2. Eph receptor tyrosine kinases (Ephs) belong to a large group of receptor tyrosine kinases (RTKs), kinases that phosphorylate proteins on tyrosine residues. Ephs and their membrane bound ephrin ligands (ephrins) control cell positioning and tissue organization (Poliakov etal. 15   (2004) Dev Cell 7, 465-80). Functional and biochemical Eph responses occur at higher ligand oligomerization states (Stein etal. (1998) Genes Dev 12, 667-678). Among other patterning functions, various Ephs and ephrins have been shown to play a role in vascular development. Knockout of EphB4 and ephrin-B2 results in a lack of the ability to 20 remodel capillary beds into blood vessels (Poliakov et al., supra) and embryonic lethality. Persistent expression of some Eph receptors and ephrins has also been observed in newly-formed, adult micro-vessels (Brantley-Sieders et al. (2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12). 25 The de-regulated re-emergence of some ephrins and their receptors in adults also has been observed to contribute to tumor invasion, metastasis and neo-angiogenesis (Nakamoto et al. (2002) Microsc Res Tech 59, 58-67; Brantley-Sieders etal., supra). Furthermore, some Eph family members have been found to be over-expressed on tumor cells from a variety of human tumors (Brantley-Sieders etal., supra), Marme (2002) Ann Hematol 81 Suppl 2, S66; 30 Booth etal. (2002) Nat Med 8, 1360-1). EPH receptor A2 (ephrin type-A receptor 2) is a protein that in humans is encoded by the EPHA2 gene. 35 EphA2 is upregulated in multiple cancers in man, often correlating with disease progression, metastasis and poor prognosis e.g.: breast (Zelinski etal (2001) Cancer Res. 61, 23012306; Zhuang et al (2010) Cancer Res. 70, 299-308; Brantley-Sieders et al (2011) PLoS 2024205105   25 Jul 2024 One 6, e24426), lung (Brannan etal (2009) Cancer Prev Res (Phila) 2, 1039-1049; Kinch et al (2003) Clin Cancer Res. 9, 613-618; Guo etal (2013) J Thorac Oncol. 8, 301-308), gastric (Nakamura etal (2005) Cancer Sci. 96, 42-47; Yuan etal (2009) Dig Dis Sci 54, 2410-2417), pancreatic (Mudali etal (2006) Clin Exp Metastasis 23, 357-365), prostate (Walker-Daniels 5 et al (1999) Prostate 41,275-280), liver (Yang et al (2009) Hepatol Res. 39, 1169-1177) and glioblastoma (Wykosky etal (2005) Mol Cancer Res. 3, 541-551; Li etal (2010) Tumour Biol. 31, 477-488). The full role of EphA2 in cancer progression is still not defined although there is evidence for 10 interaction at numerous stages of cancer progression including tumour cell growth, survival, invasion and angiogenesis. Downregulation of EphA2 expression suppresses tumour cancer cell propagation (Binda etal (2012) Cancer Cell 22, 765-780), whilst EphA2 blockade inhibits VEGF induced cell migration (Hess etal (2001) Cancer Res. 61, 3250-3255), sprouting and angiogenesis (Cheng et al (2002) Mol Cancer Res. 1, 2-11; Lin et al (2007) 15 Cancer 109, 332-40) and metastatic progression (Brantley-Sieders et al (2005) FASEB J. 19, 1884-1886). An antibody drug conjugate to EphA2 has been shown to significantly diminish tumour growth in rat and mouse xenograft models (Jackson et al (2008) Cancer Research 68, 936720   9374) and a similar approach has been tried in man although treatment had to be discontinued for treatment related adverse events (Annunziata et al (2013) Invest New drugs 31, 77-84). In one embodiment, the second peptide ligand comprises an EphA2 binding bicyclic peptide 25 ligand. Suitable examples of EphA2 binding bicyclic peptide ligands are disclosed in GB Patent Application Nos. 1721259.8 and 1804102.0, the peptides of which are incorporated herein by reference. 30 In one embodiment, the EphA2 binding bicyclic peptide ligand comprises an amino acid sequence: Ci[HyP]LVNPLCiiLHP[dD]W[HArg]Ciii (SEQ ID NO: 2); and CiLWDPTPCiANLHL[HArg]Ciii (SEQ ID NO: 11); 35 wherein Ci, Ch and Cui represent first, second and third cysteine residues, respectively, HyP represents hydroxyproline, dD represents aspartic acid in D-configuration and HArg represents homoarginine, or a pharmaceutically acceptable salt thereof. 2024205105   25 Jul 2024 In one embodiment which may be mentioned, the EphA2 binding bicyclic peptide ligand comprises an amino acid sequence: Ci[HyP]LVNPLCiiLHP[dD]W[HArg]Ciii (SEQ ID NO: 2); 5 wherein Ci, Ch and Cui represent first, second and third cysteine residues, respectively, HyP represents hydroxyproline, dD represents aspartic acid in D-configuration and HArg represents homoarginine, or a pharmaceutically acceptable salt thereof. In a further embodiment, the EphA2 binding bicyclic peptide ligand comprises N-terminal 10 modifications and comprises: A-HArg-D-(SEQ ID NO: 2) (hereinafter referred to as BCY9594); [B-Ala]-[Sario]-A-[HArg]-D-(SEQ ID NO: 2) (hereinafter referred to as BCY6099); [PYA]-[B-Ala]-[Sario]-A-[HArg]-D-(SEQ ID NO: 2) (hereinafter referred to as BCY6169); and 15          [PYA]-[B-Ala]-[Sario]-VGP-(SEQ ID NO: 11) (hereinafter referred to as BCY8941); wherein HArg represents homoarginine, PYA represents 4-pentynoic acid, Sano represents 10 sarcosine units, B-Ala represents beta-alanine, or a pharmaceutically acceptable salt thereof. 20 In a further embodiment which may be mentioned, the EphA2 binding bicyclic peptide ligand comprises N-terminal modifications and comprises: A-HArg-D-(SEQ ID NO: 2) (hereinafter referred to as BCY9594). wherein HArg represents homoarginine, or a pharmaceutically acceptable salt thereof. 25 In an alternative embodiment, the component present on a cancer cell is PD-L1. Programmed cell death 1 ligand 1 (PD-L1) is a 290 amino acid type I transmembrane protein encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9. PD-L1 expression is involved in evasion of immune responses involved in chronic infection, e.g., 30 chronic viral infection (including, for example, HIV, HBV, HCV and HTLV, among others), chronic bacterial infection (including, for example, Helicobacter pylori, among others), and chronic parasitic infection (including, for example, Schistosoma mansoni). PD-L1 expression has been detected in a number of tissues and cell types including T-cells, B-cells, macrophages, dendritic cells, and nonhaematopoietic cells including endothelial cells, 35 hepatocytes, muscle cells, and placenta. 2024205105   25 Jul 2024 PD-L1 expression is also involved in suppression of anti-tumour immune activity. Tumours express antigens that can be recognised by host T-cells, but immunologic clearance of tumours is rare. Part of this failure is due to immune suppression by the tumour microenvironment. PD-L1 expression on many tumours is a component of this suppressive 5 milieu and acts in concert with other immunosuppressive signals. PD-L1 expression has been shown in situ on a wide variety of solid tumours including breast, lung, colon, ovarian, melanoma, bladder, liver, salivary, stomach, gliomas, thyroid, thymic epithelial, head, and neck (Brown JA et al. 2003 Immunol. 170:1257-66; Dong H etal. 2002 Nat. Med. 8:793-800; Hamanishi J, et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Strome SE et al. 2003 10 Cancer Res. 63:6501-5; Inman BA et al. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi Tetal. 2007 Clin. Cancer Res. 13:2151-57; Thompson RH etal. 2004 Proc. Natl. Acad. Sci. USA101:17174-79; Wu C etal. 2006 Acta Histochem. 108:19-24). In addition, the expression of the receptor for PD-L1, Programmed cell death protein 1 (also known as 15 PD-1 and CD279) is upregulated on tumour infiltrating lymphocytes, and this also contributes to tumour immunosuppression (Blank C etal. 2003 Immunol. 171:4574-81). Most importantly, studies relating PD-L1 expression on tumours to disease outcome show that PD-L1 expression strongly correlates with unfavourable prognosis in kidney, ovarian, bladder, breast, gastric, and pancreatic cancer (Hamanishi J etal. 2007 Proc. Natl. Acad. Sci. USA 104:336020   65; Inman BA et al. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi T et al. 2007 Clin. Cancer Res. 13:2151-57; Thompson RH etal. 2004 Proc. Natl. Acad. Sci. USA 101:17174-79; Wu C et al. 2006 Acta Histochem. 108:19-24). In addition, these studies suggest that higher levels of PD-L1 expression on tumours may facilitate advancement of 25 tumour stage and invasion into deeper tissue structures. The PD-1 pathway can also play a role in haematologic malignancies. PD-L1 is expressed on multiple myeloma cells but not on normal plasma cells (Liu J etal. 2007 Blood 110:296-304). PD-L1 is expressed on some primary T-cell lymphomas, particularly anaplastic large cell T 30 lymphomas (Brown JA et al, 2003 Immunol. 170:1257-66). PD-1 is highly expressed on the T-cells of angioimmunoblastic lymphomas, and PD-L1 is expressed on the associated follicular dendritic cell network (Dorfman DM et al. 2006 Am. J. Surg. Pathol. 30:802-10). In nodular lymphocyte-predominant Hodgkin lymphoma, the T-cells associated with lymphocytic or histiocytic (L&H) cells express PD-1. Microarray analysis using a readout of genes induced 35 by PD-1 ligation suggests that tumour-associated T-cells are responding to PD-1 signals in situ in Hodgkin lymphoma (Chemnitz JM etal. 2007 Blood 110:3226-33). PD-1 and PD-L1 are expressed on CD4 T-cells in HTLV-1 -mediated adult T-cell leukaemia and lymphoma 2024205105   25 Jul 2024 (Shimauchi T etal. 2007 Int. J. Cancer 121: 2585-90). These tumour cells are hyporesponsive to TCR signals. Studies in animal models demonstrate that PD-L1 on tumours inhibits T-cell activation and 5 lysis of tumour cells and in some cases leads to increased tumour-specific T-cell death (Dong H et al. 2002 Nat. Med. 8:793-800; Hirano F et al. 2005 Cancer Res. 65:1089-96). Tumour-associated APCs can also utilise the PD-1:PD-L1 pathway to control antitumour T-cell responses. PD-L1 expression on a population of tumour-associated myeloid DCs is upregulated by tumour environmental factors (Curiel TJ et al. 2003 Nat. Med. 9:562-67). 10 Plasmacytoid dendritic cells (DCs) in the tumour-draining lymph node of B16 melanoma express IDO, which strongly activates the suppressive activity of regulatory T-cells. The suppressive activity of IDO-treated regulatory T-cells required cell contact with IDO-expressing DCs (Sharma MD etal. 2007 Clin. Invest. 117:2570-82). 15 In one embodiment, the second peptide ligand comprises a PD-L1 binding bicyclic peptide ligand. Suitable examples of PD-L1 binding bicyclic peptide ligands are disclosed in GB Patent Application Nos. 1820956.9 and 1820969.2, the peptides of which are incorporated herein 20 by reference. In one embodiment, the PD-L1 binding bicyclic peptide ligand comprises an amino acid sequence selected from: Ci[HArg]DWCiiHWTFSHGHPCiii (SEQ ID NO: 12); 25         CiSAGWLTMCiiQKLHLCiii (SEQ ID NO: 13); and CiSAGWLTMCiiQ[K(PYA)]LHLCiii (SEQ ID NO: 14); wherein Q, Ch and Cui represent first, second and third cysteine residues, respectively, HArg represents homoarginine and PYA represents 4-pentynoic acid, or a pharmaceutically acceptable salt thereof. 30 In a further embodiment, the PD-L1 binding bicyclic peptide ligand comprises N-terminal and / or C-terminal modifications and comprises: [PYA]-[B-Ala]-[Sario]-(SEQ ID NO: 12) (hereinafter referred to as BCY8938); [PYA]-[B-Ala]-[Sario]-SDK-(SEQ ID NO: 13) (hereinafter referred to as BCY10043); 35          NH2-SDK-(SEQ ID NO: 13)-[Sarw]-[K(PYA)] (hereinafter referred to as BCY10044); NH2-SDK-(SEQ ID NO: 14) (hereinafter referred to as BCY10045); and Ac-SDK-(SEQ ID NO: 14)-PSH (hereinafter referred to as BCY10861); 2024205105   25 Jul 2024 wherein PYA represents 4-pentynoic acid, B-Ala represents beta-alanine, Sano represents 10 sarcosine units, or a pharmaceutically acceptable salt thereof. In an alternative embodiment, the component present on a cancer cell is Nectin-4. 5 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 10 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 15 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 20 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. 25 In one embodiment, the second peptide ligand comprises a Nectin-4 binding bicyclic peptide ligand. Suitable examples of Nectin-4 binding bicyclic peptide ligands are disclosed in GB Patent 30 Application Nos 1810250.9, 1815684.4 and 1818499.4, the peptides of which are incorporated herein by reference. In one embodiment, the Nectin-4 binding bicyclic peptide ligand comprises an amino acid sequence selected from: 35          CiP[1Nal][dD]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 15; hereinafter referred to as BCY8116); 2024205105   25 Jul 2024 CiP[1Nal][dD]CiiM[HArg]D[dW]STP[HyP][dW]Ciii (SEQ ID NO: 16; hereinafter referred to as BCY11415); and CiP[1Nal][dK](Sario-(B-Ala))CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 17); CiPFGCiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 18; hereinafter referred to as 5   BCY11414); wherein Ci, Ch and Cm represent first, second and third cysteine residues, respectively, 1 Nai represents 1-naphthylalanine, HArg represents homoarginine, HyP represents hydroxyproline, Sano represents 10 sarcosine units, B-Ala represents beta-alanine, or a pharmaceutically acceptable salt thereof. 10 In a further embodiment, the Nectin-4 binding bicyclic peptide ligand optionally comprises N-terminal modifications and comprises: SEQ ID NO: 15 (hereinafter referred to as BCY8116); [PYA]-[B-Ala]-[Sario]-(SEQ ID NO: 15) (hereinafter referred to as BCY8846); 15          SEQ ID NO: 16 (hereinafter referred to as BCY11415); [PYA]-[B-Ala]-[Sario]-(SEQ ID NO: 16) (hereinafter referred to as BCY11942); Ac-(SEQ ID NO: 17) (hereinafter referred to as BCY8831); and SEQ ID NO: 18 (hereinafter referred to as BCY11414); wherein PYA represents 4-pentynoic acid, B-Ala represents beta-alanine, Sano represents 20   10 sarcosine units, or a pharmaceutically acceptable salt thereof. In an alternative embodiment, the component present on a cancer cell is prostate-specific membrane antigen (PSMA). 25 Prostate-specific membrane antigen (PSMA) (also known as Glutamate carboxypeptidase II (GOPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I) and NAAG peptidase) is an enzyme that in humans is encoded by the FOLH1 (folate hydrolase 1) gene. Human GOPI I contains 750 amino acids and weighs approximately 84 kDa. 30 Human PSMA is highly expressed in the prostate, roughly a hundred times greater than in most other tissues. In some prostate cancers, PSMA is the second-most upregulated gene product, with an 8- to 12-fold increase over levels in noncancerous prostate cells. Because of this high expression, PSMA is being developed as potential biomarker for therapy and imaging of some cancers. In human prostate cancer, the higher expressing tumors are 35 associated with quicker time to progression and a greater percentage of patients suffering relapse. 2024205105   25 Jul 2024 In one embodiment, the second peptide ligand comprises a PSMA binding bicyclic peptide ligand. Suitable examples of PSMA binding bicyclic peptide ligands are disclosed in GB Patent 5 Application Nos 1810318.4, 1810325.9 and 1820325.7, the peptides of which are incorporated herein by reference. Linkers It will be appreciated that the first peptide ligand may be conjugated to the second peptide 10 ligand via any suitable linker. Typically the design of said linker will be such that the two Bicyclic peptides are presented in such a manner that they can bind unencumbered to their respective targets either alone or while simultaneously binding to both target receptors. Additionally, the linker should permit binding to both targets simultaneously while maintaining an appropriate distance between the target cells that would lead to the desired functional 15 outcome. The properties of the linker may be modulated to increase length, rigidity or solubility to optimise the desired functional outcome. The linker may also be designed to permit the attachment of more than one Bicycle to the same target. Increasing the valency of either binding peptide may serve to increase the affinity of the heterotandem for the target cells or may help to induce oligomerisation of one or both of the target receptors. 20 In one embodiment, the linker is selected from the following sequences: -CH2-, -PEG5-, -PEG10-, -PEG12-, -PEG23-, -PEG24-, -PEGis-Sars-, -PEGwSario-, -PEGs^Saris-, -PEGs^Sars-, -B-Ala-Sar2o-, -B-Ala-Sario-PEGio-, -B-Ala-Sars-PEGis- and -B-Ala-Sars-PEGs-. 25 Structural representations of suitable linkers are detailed below: 2024205105   25 Jul 2024 H2N-Peg5-N3 COM00000132 O N3-PEG5-COOH COM00000467 H2N-Peg10-N3 COM00000134 N3-CH2-COOH COM00000468 H2N-Peg23-N3 COM00000135 N3-PEG12-COOH COM00000466 O NHS-PEG5-N3 O NHS-PEG12-N3 2024205105   25 Jul 2024 H2N-PEG15-SAR5-N3 COM00000128 15       5 H2N-PEG5-SAR15-N3 COM00000130 H2N-(B-Ala)-SAR10-PEG10-N3 COM00000471 10        10 H2N-PEG10-SAR10-N3 COM00000129 5          5 H2N-PEG5-SAR5-N3 COM00000131 H2N-(B-Ala)-SAR20-N3 COM00000470 H2N-(B-Ala)-SAR5-PEG15-N3 COM00000472 H2N-(B-Ala)-SAR5-PEG5-N3 COM00000473 Heterotandem Complexes In one specific embodiment, the first peptide ligand comprises a CD137 binding bicyclic 5 peptide ligand attached to a TATA scaffold, the second peptide ligand comprises an EphA2 binding bicyclic peptide ligand attached to a TATA scaffold and said heterotandem complex is selected from: Complex No. EphA2 BCY No. Attachment Point Linker CD137 BCY No. Attachment Point BCY9173 BCY6169 N-terminal PYA -PEG12- BCY9172 C-terminal Dap BCY7985 BCY6169 N-terminal PYA -PEG12- BCY7732 C-terminal Dap 2024205105   25 Jul 2024 BCY8942 BCY6169 N-terminal PYA -PEG12- BCY8045 C-terminal Dap BCY8943 BCY8941 N-terminal PYA -PEG12- BCY7732 C-terminal Dap BCY9647 BCY6099 N-terminus -PEG10- BCY7741 C-terminal Dap(PYA) BCY9648 BCY6099 N-terminus -PEG23- BCY7741 C-terminal Dap(PYA) BCY9655 BCY6099 N-terminus -PEG15- Sar5- BCY7741 C-terminal Dap(PYA) BCY9656 BCY6099 N-terminus -PEG10- Sarw- BCY7741 C-terminal Dap(PYA) BCY9657 BCY6099 N-terminus -PEG5- Saris- BCY7741 C-terminal Dap(PYA) BCY9658 BCY6099 N-terminus -PEG5- Sar5- BCY7741 C-terminal Dap(PYA) BCY9659 BCY6099 N-terminus -PEG5- BCY7741 C-terminal Dap(PYA) BCY9758 BCY6099 N-terminus -PEG24- BCY7732 C-terminal Dap BCY10568 BCY6169 N-terminal PYA -PEG12- BCY8919 Lys3 BCY10570 BCY6169 N-terminal PYA -PEG12- BCY8920 dLys4 BCY10574 BCY9594 N-terminus -PEG5- BCY8927 Lys (PYA)3 BCY10575 BCY9594 N-terminus -PEG5- BCY8928 dLys (PYA)4 BCY10576 BCY9594 N-terminus -PEG5- BCY11014 C-terminal Dap(PYA) BCY10577 BCY6169 N-terminus -ch2- BCY9172 C-terminal Dap The heterotandem bicyclic peptide complex BCY7985 consists of a CD137-specific peptide BCY7859 linked to the N-terminal PYA group of an EphA2-specific peptide BCY6169 via PEG12 (shown pictorially in Figure 2). 5 CD137 is a homotrimeric protein and the natural ligand CD137L exists as a homotrimer either expressed on immune cells or secreted. The biology of CD137 is highly dependent on multimerization to induce CD137 activity in immune cells. One way to generate CD137 multimerization is through cellular cross-linking of the CD137 specific agonist through 10 interaction with a specific receptor present on another cell. EphA2 is highly expressed on tumour cells and oligomerization of this receptor tyrosine kinase by Ephrin-A ligands drives its activation. Without being bound by theory, the inventors believe that a EphA2-CD137 heterotandem consisting of one EphA2-specific peptide coupled to one 15 CD137-specific peptide acts to cross-link CD137. The implication is that CD137 would be 2024205105   25 Jul 2024 multimerized and activated in the presence of EphA2 on cells such as tumour cells. This would drive CD137 immune cell activation in the local tumour environment (Figure 1). This hypothesis was tested in the CD137 cellular activity reporter assay described herein and 5 the results are shown herein in Figure 3 wherein it can be seen that BCY7985 showed strong induction of CD137 cell activity in the Promega CD137 luciferase reporter assay (CS196008) in the presence of EphA2-expressing HT1080 cells. In one alternative specific embodiment, the first peptide ligand comprises a CD137 binding 10 bicyclic peptide ligand attached to a TATA scaffold, the second peptide ligand comprises a Nectin-4 binding bicyclic peptide ligand attached to a TATA scaffold and said heterotandem complex is selected from: Complex No. Nectin-4 BCY No. Attachment Point Linker CD137 BCY No. Attachment Point BCY8854 BCY8846 N-terminal PYA -PEG12- BCY7732 C-terminal Dap BCY9350 BCY11942 N-terminal PYA -PEG12- BCY7732 C-terminal Dap BCY9351 BCY8846 N-terminal PYA -PEG12- BCY8045 C-terminal Dap BCY9399 BCY8116 N-terminus -PEG10- BCY7741 C-terminal Dap(PYA) BCY9400 BCY8116 N-terminus -PEG23- BCY7741 C-terminal Dap(PYA) BCY9401 BCY8116 N-terminus -B-Ala- Sar2o- BCY7741 C-terminal Dap(PYA) BCY9403 BCY8116 N-terminus -B-Ala- Sarw- PEG10- BCY7741 C-terminal Dap(PYA) BCY9405 BCY8116 N-terminus -B-Ala- Sar5- PEG15- BCY7741 C-terminal Dap(PYA) BCY9406 BCY8116 N-terminus -B-Ala- Sar5- PEG5- BCY7741 C-terminal Dap(PYA) BCY9407 BCY8116 N-terminus -PEG15- Sar5- BCY7741 C-terminal Dap(PYA) BCY9408 BCY8116 N-terminus -PEG10- Sarw- BCY7741 C-terminal Dap(PYA) 2024205105   25 Jul 2024 BCY9409 BCY8116 N-terminus -peg5- Saris- BCY7741 C-terminal Dap(PYA) BCY9410 BCY8116 N-terminus -peg5- Sar5- BCY7741 C-terminal Dap(PYA) BCY9411 BCY8116 N-terminus -peg5- BCY7741 C-terminal Dap(PYA) BCY9759 BCY8116 N-terminus -PEG24- BCY7732 C-terminal Dap BCY10000 BCY8846 N-terminal PYA -PEG12- BCY9172 C-terminal Dap BCY10567 BCY8846 N-terminal PYA -PEG12- BCY8919 Lys3 BCY10569 BCY8846 N-terminal PYA -PEG12- BCY8920 dLys4 BCY10571 BCY8116 N-terminus -PEG5- BCY8927 Lys(PYA)3 BCY10572 BCY8116 N-terminus -PEG5- BCY8928 dLys (PYA)4 BCY10573 BCY8116 N-terminus -PEG5- BCY11014 C-terminal Dap(PYA) BCY10578 BCY8846 N-terminal PYA -ch2- BCY9172 C-terminal Dap BCY10917 BCY8831 dLys(Sario)-(B- Ala))4 -PEG12- BCY11014 C-terminal Dap(PYA) BCY11020 BCY8831 dLys(Sario)-(B- Ala))4 -PEG5- BCY11014 C-terminal Dap(PYA) BCY11373 BCY8116 N-terminus -ch2- BCY8927 Lys(PYA)3 BCY11374 BCY8116 N-terminus -ch2- BCY8928 dLys (PYA)4 BCY11375 BCY8116 N-terminus -ch2- BCY11014 C-terminal Dap(PYA) BCY11616 BCY8116 N-terminus -PEG5- BCY7744 dLys (PYA)4 BCY11617 BCY8116 N-terminus -PEG5- BCY11506 Lys(PYA)4 BCY11857 BCY11414 N-terminus -PEG5- BCY7744 dLys (PYA)4 BCY11858 BCY11414 N-terminus -PEG5- BCY8928 dLys (PYA)4 BCY11859 BCY11415 N-terminus -PEG5- BCY8928 dLys (PYA)4 Without being bound by theory, the inventors believe that a Nectin-4-CD137 heterotandem consisting of one Nectin-4-specific peptide coupled to one CD137-specific peptide acts to cross-link CD137 in the same manner as described hereinbefore for EphA2. In one embodiment, the Nectin-4-CD137 heterotandem is other than any one or more of: BCY11857, BCY11858 and / or BCY11859. In one alternative specific embodiment, the first peptide ligand comprises a CD137 binding 10 bicyclic peptide ligand attached to a TATA scaffold, the second peptide ligand comprises a 2024205105   25 Jul 2024 PD-L1 binding bicyclic peptide ligand attached to a TATA scaffold and said heterotandem complex is selected from: Complex No. PD-L1 BCY No. Attachment Point Linker CD137 BCY No. Attachment Point BCY8939 BCY8938 N-terminal PYA -PEG12- BCY7732 C-terminal Dap BCY10580 BCY10043 N-terminal PYA -PEG12- BCY9172 C-terminal Dap BCY10581 BCY10044 C-terminal Lys(PYA) -PEG12- BCY9172 C-terminal Dap BCY10582 BCY10045 Lys(PYA)9 -PEG12- BCY9172 C-terminal Dap BCY11017 BCY10861 Lys(PYA)9 -PEG12- BCY8919 Lys3 BCY11018 BCY10861 Lys(PYA)9 -PEG12- BCY8920 dLys4 BCY11019 BCY10861 Lys(PYA)9 -PEG12- BCY9172 C-terminal Dap BCY11376 BCY10861 Lys(PYA)9 -ch2- BCY8919 Lys3 BCY11377 BCY10861 Lys(PYA)9 -ch2- BCY8920 dLys4 BCY11378 BCY10861 Lys(PYA)9 -ch2- BCY9172 C-terminal Dap BCY11379 BCY10861 Lys(PYA)9 -peg5- BCY8919 Lys3 BCY11380 BCY10861 Lys(PYA)9 -peg5- BCY8920 dLys4 BCY11381 BCY10861 Lys(PYA)9 -peg5- BCY9172 C-terminal Dap 5 Without being bound by theory, the inventors believe that a PD-L1-CD137 heterotandem consisting of one PD-L1-specific peptide coupled to one CD137-specific peptide acts to crosslink CD137 in the same manner as described hereinbefore for EphA2. Unless defined otherwise, all technical and scientific terms used herein have the same 10 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 Sam brook etal., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel etal., Short Protocols in Molecular Biology 15   (1999) 4th ed., John Wiley & Sons, Inc.), which are incorporated herein by reference. Nomenclature Numbering 2024205105   25 Jul 2024 When referring to amino acid residue positions within compounds of the invention, cysteine residues (Ci, Ch and Cui) are omitted from the numbering as they are invariant, therefore, the numbering of amino acid residues within SEQ ID NO: 1 is referred to as below: 5                 Ci-li-E2-E3-G4-Q5-Y6-Cii-F7-A8-D9-Pio-Yii-[Nle]i2-Ciii (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) or 1,T,1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) and yielding a tri-substituted structure. Cyclisation with TBMB and TATA occurs on Ci, 10 Cii, and Cui. 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 pAla-Sar10-Ala tail would 15 be denoted as: PAIa-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 20 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). 25 Peptide Ligands A peptide ligand, as referred to herein, refers to a peptide covalently bound to a molecular scaffold. Typically, such peptides comprise 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 30 forms a loop when the peptide is bound to the scaffold. In the present case, the peptides comprise at least three cysteine residues (referred to herein as Ci, Ch and Cm), and form at least two loops on the scaffold. Pharmaceutically Acceptable Saits 35 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. 2024205105   25 Jul 2024 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 5 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 10 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, (+)-(1 S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, 15 glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic), isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1,5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L-20 pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenicand 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, 25 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. 30 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, 35 ammonium ion (i.e., NH4+) and substituted ammonium ions (e.g., NHsR+, NH2R2+, NHRs+, NR4+). Examples of some suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, 2024205105   25 Jul 2024 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(CHs)4+. 5 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 invention. 10 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 15 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 20 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, 25 glutamate / aspartate and tyrosine with suitable amine, thiol, carboxylic acid and phenolreactive 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. 30 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-35 terminal modification comprises addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator or a chromophore. 2024205105   25 Jul 2024 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 cysteine group (the group referred to herein as Ci) is capped with acetic anhydride or other appropriate reagents during peptide synthesis leading to a molecule 5 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 10 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 15 embodiment, the C-terminal cysteine group (the group referred to herein as Ciii) 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 carboxy peptidase and reduces the potential for proteolytic degradation of the bicyclic peptide. 20 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. 25 Alternatively, non-natural amino acids may be used having constrained amino acid side chains, such that proteolytic hydrolysis of the nearby peptide bond is conformationally and sterically impeded. In particular, these concern proline analogues, bulky sidechains, Ca-disubstituted derivatives (for example, aminoisobutyric acid, Aib), and cyclo amino acids, a simple derivative being amino-cyclopropylcarboxylic acid. 30 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). 35 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 2024205105   25 Jul 2024 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 5 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 10 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 15 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 20 increase proteolytic stability by steric hindrance and by a propensity of D-amino acids to stabilise p-turn conformations (Tugyi etal (2005) PNAS, 102(2), 413-418). In one embodiment, the modified derivative comprises removal of any amino acid residues and substitution with alanines. This embodiment provides the advantage of removing potential 25 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: 30 - 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 35 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 2024205105   25 Jul 2024 - 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. 5 (for reviews see Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor etal, Curr. Medicinal Chern (2009), 16, 4399-418). Isotopic variations 10 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, 15 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, 20 such as 36CI, fluorine, such as 18F, iodine, such as 123l, 125l and 131l, 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. 25 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 Nectin-4 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 30 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 35 of incorporation and ready means of detection. 2024205105   25 Jul 2024 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. 5 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 10 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. Molecular scaffold 15 Molecular scaffolds are described in, for example, WO 2009 / 098450 and references cited therein, particularly WO 2004 / 077062 and WO 2006 / 078161. As noted in the foregoing documents, the molecular scaffold may be a small molecule, such as a small organic molecule. 20 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. 25 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, 30 alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides. In one embodiment, the molecular scaffold may comprise or may consist of hexahydro-1,3,5-triazine, especially 1,3,5-Triacryloylhexahydro-1,3,5-triazine (‘TATA’), or a derivative thereof. 35 In one embodiment, the molecular scaffold is 2,4,6-tris(bromomethyl)mesitylene. This molecule is similar to 1,3,5-tris(bromomethyl)benzene (TBMB) but contains three additional 2024205105   25 Jul 2024 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. 5 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 10 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). 15 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, ap unsaturated carbonyl containing compounds and a-halomethylcarbonyl containing compounds. Examples of maleimides which may be used as molecular scaffolds in the invention include: tris-(2-maleimidoethyl)amine, tris-(2 20 maleimidoethyl)benzene, tris-(maleimido)benzene. An example of an ap unsaturated carbonyl containing compound is 1,T,1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606). An example of an a-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 25 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. Synthesis The peptides of the present invention may be manufactured synthetically by standard 30 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). 35 2024205105   25 Jul 2024 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. 5 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 10 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 15 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 etal. Proc Natl Acad Sci USA. 1994 Dec 20; 91(26):12544-8 or in Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, 20 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, 25 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 peptidepeptide conjugate. 30 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 35 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. 2024205105   25 Jul 2024 Pharmaceutical Compositions According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand as defined herein in combination with one or more 5 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 10 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. 15 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). 20 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 25 "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. 30 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 35 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 2024205105   25 Jul 2024 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 5 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. 10 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 15 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. 20 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 25 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. 30 Therapeutic Uses According to a further aspect of the invention, there is provided a heterotandem bicyclic peptide complex as defined herein for use in preventing, suppressing or treating cancer. Examples of cancers (and their benign counterparts) which may be treated (or inhibited) 35 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 2024205105   25 Jul 2024 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 5 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. 10 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, 15 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 20 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, 25 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 30 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 35 syndromes, congenital or otherwise, which leave the patient susceptible to malignancy (for example Xeroderma Pigmentosum). 2024205105   25 Jul 2024 In a further embodiment, the cancer is selected from a hematopoietic malignancy such as selected from: 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 5 Lymphoma (HL), and chronic myeloid leukemia (CML). 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. 10 "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 15 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. 20 EXAMPLES Example 1: Synthesis of Linkers COM 128 2024205105   25 Jul 2024 Chemical Formula: C30H37N5O8 Exact Mass: 595.26 Chemical Formula: C33H43N9O7 Exact Mass: 677.33 N-ethylethanamine DCM Exact Mass: 455.26 Molecular Weight: 455.51 3 Chemical Formula: C66H108N10O23 Exact Mass: 1408.76 5 COM00000128 A mixture of compound 1 (700.0 mg, 1.18 mmol, 1.0 eq), 3-azidopropan-1-amine (117.66 mg, 1.18 mmol, 1.0 eq), EDCI (270.4 mg, 1.41 mmol, 1.2 eq), HOBt (190.6 mg, 1.41 mmol, 1.2 eq) was dissolved in DCM (20 mL, pre-degassed and purged with N2 for 3 times), and 5 then the mixture was stirred at 20-25 °C for 1 hr under N2 atmosphere. LC-MS showed compound 1 was consumed completely and one main peak with desired m / z (calculated MW: 677.33, observed m / z: 678.2 ([M+H]+)) was detected. The solvent was evaporated to produce compound 2 (600 mg, crude) was obtained as a white solid. 10 A mixture of compound 2 (600.0 mg, 885.3 pmol, 1.0 eq), N-ethylethanamine (1.29 g, 15.19 mmol, 1.50 mL, 17.2 eq) was dissolved in DCM (3 mL, pre-degassed and purged with N2 for 3 times), and then the mixture was stirred at 25-30 °C for 2 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 455.51, observed m / z: 456.3 ([M+H]+)) was detected. The solvent was 15 evaporated to produce compound 3 (400 mg, crude) was obtained as colorless oil. A mixture of compound 3 (150.0 mg, 329.3 pmol, 1.0 eq), compound 4 (320.1 mg, 329.3 pmol, 1.0 eq), HATU (125.2 mg, 329.3 pmol, 1.0 eq), DIEA (42.6 mg, 329.3 pmol, 57.4 pL, 1.0 eq) was dissolved in DMF (2 mL, pre-degassed and purged with N2 for 3 times), and then the mixture was stirred at 25-30 °C for 2 under N2 atmosphere. LC-MS showed 20 compound 3 was consumed completely and one main peak with desired m / z (calculated MW: 1408.76, observed m / z: 705.3 ([M / 2+H]+)) was detected. The solvent was evaporated to produce compound 5 (400 mg, crude) was obtained as yellow oil. 2024205105   25 Jul 2024 Compound 5 (400 mg, 283.77 pmol, 1.0 eq) was dissolved in DMF (4 mL, pre-degassed and purged with N2for 3 times), following by addition of piperidine (862.2 mg, 10.13 mmol, 1 mL, 35.7 eq), and then the mixture was stirred at 25-30 °C for 15 min under N2 atmosphere. LC-MS showed compound 5 was consumed completely and one main peak with desired m / z 5   (calculated MW: 1187.37, observed m / z: 594.4 ([M / 2+H+], 1187.4 ([M+H]+)) was detected. The solvent was evaporated to produce COM128 (250 mg, crude) was obtained as colorless oil. COM129 10 Exact Mass: 950.45 Molecular Weight: 951.03 1 Exact Mass: 1032.51 Molecular Weight: 1033.14 N-ethylethanamine DCM Exact Mass: 810.45 Molecular Weight: 810.90 3 Chemical Formula: C70H111N15O23 Exact Mass: 1529.80 ---- DMF                     O । 9     0         9 COM00000129 A mixture of compound 1 (1.4 g, 1.47 mmol, 1.0 eq), 3-azidopropan-1-amine (162.1 mg, 1.62 mmol, 1.1 eq), EDCI (338.6 mg, 1.77 mmol, 1.2 eq), HOBt (238.7 mg, 1.77 mmol, 1.2 eq) was dissolved in DCM (5 mL, pre-degassed and purged with N2 for 3 times), and then the 15 mixture was stirred at 20-25 °C for 1 hr under N2 atmosphere. LC-MS showed compound 1 was consumed completely and one main peak with desired m / z (calculated MW: 1033.14, observed m / z: 1033.2 ([M+H]+)) was detected. The reaction mixture was treated with a few drops of 1 M HCI, and the organic layer was evaporated under reduced pressure to remove solvent. Compound 2 (1.1 g, crude) was obtained as yellow oil. 20 A mixture of compound 2 (1.1 g, 1.06 mmol, 1 eq), N-ethylethanamine (3.89 g, 53.24 mmol, 5.48 mL, 50 eq) was dissolved in DCM (5 mL, pre-degassed and purged with N2 for 3 times), 2024205105   25 Jul 2024 and then the mixture was stirred at 20-25 °C for 1 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 810.90, observed m / z: 810.9 ([M+H]+)) was detected. The reaction mixture was evaporated under reduced pressure and compound 3 (810 mg, crude) was obtained as a 5 white solid. A mixture of compound 3 (810.0 mg, 998.9 pmol, 1.0 eq), compound 4 (810.7 mg, 1.10 mmol, 1.1 eq), HATU (455.8 mg, 1.20 mmol, 1.2 eq), DIEA (258.2 mg, 2.00 mmol, 348.0 pL, 2.0 eq) was dissolved in DMF (2 mL, pre-degassed and purged with N2 for 3 times), and 10 then the mixture was stirred at 25-30 °C for 2 under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m / z (calculated MW: 1530.72, observed m / z: 765.5 ([M / 2+H]+)) was detected. The reaction mixture was treated with a few drops of 1 M HCI, and the organic layer was collected and evaporated under reduced pressure to remove solvent. Compound 5 (1.1 g, crude) was obtained as a 15 yellow solid. Compound 5 (1 g, 653.29 pmol, 1 eq) was dissolved in DCM (10 mL, pre-degassed and purged with N2 for 3 times), following by addition of piperidine (2.39 g, 32.66 mmol, 3.36 mL, 50 eq), and then the mixture was stirred at 25-30 °C for 2 hr under N2 atmosphere. LC-MS 20 showed Compound 5 was consumed completely and one main peak with desired m / z (calculated MW: 1308.47, observed m / z: 1308.4 ([M+H]+)) was detected. The residue was purified by prep-HPLC (TFA condition: Phase A : 0.075%TFA in H2O, phase B : MeCN, Column: Luna 200*25 mm 10 urn, C18, 110A and Gemin150*30 mm, C18, 5 urn, 110A, connection, 50 °C). COM129 (700 mg, 463.72 pmol, 70.98% yield) was obtained as a 25 yellow solid. COM130 30 2024205105   25 Jul 2024 O Fmoc N3-CH2-CH2CH2NH2 Exact Mass: 1305.64 Molecular Weight: 1306.44 N-ethylethanamine DCM Exact Mass: 1387.70 Molecular Weight: 1388.55 Exact Mass: 1165.63 Molecular Weight: 1166.31 Fmoc Exact Mass: 517.23 Molecular Weight: 517.58 Exact Mass: 1664.85 Molecular Weight: 1665.87 5 COM00000130 A mixture of Compound 1 (291 mg, 222.75 pmol, 1.0 eq), 3-azidopropan-1-amine (24.53 mg, 245.02 pmol, 1.1 eq), EDCI (51.24 mg, 267.30 pmol, 1.2 eq), HOBt (36.12 mg, 267.30 pmol, 1.2 eq) was dissolved in DCM (3 mL, pre-degassed and purged with N2 for 3 times), and 5 then the mixture was stirred at 20-25 °C for 1 hr under N2 atmosphere. LC-MS showed Compound 1 was consumed completely and one main peak with desired m / z (MW: 1388.53, observed m / z: 694.7 ([M / 2+H]+)) was detected. The residue was purified by prep-HPLC (neutral condition). Compound 2 (200 mg, 144.04 pmol, 64.66% yield) was obtained as a white solid. .0 A mixture of Compound 2 (200 mg, 144.04 pmol, 1.0 eq), N-ethylethanamine (210.7 mg, 2.88 mmol, 297 pL, 20.0 eq) was dissolved in DCM (3 mL, pre-degassed and purged with N2 for 3 times), and then the mixture was stirred at 20-25 °C for 1 hr under N2 atmosphere. LC-MS showed Compound 2 was consumed completely and one main peak with desired m / z 2024205105   25 Jul 2024 (MW: 1166.29, observed m / z\ 1166.3 ([M+H]+)) was detected. The reaction mixture was evaporated and Compound 3 (150 mg, crude) was obtained as yellow oil. A mixture of compound 3 (150 mg, 128.61 pmol, 1.0 eq) , compound 4 (75 mg, 144.91 pmol, 5   1.13 eq), HATU (58.7 mg, 154.34 pmol, 1.2 eq) and DIEA (33.24 mg, 257.23 pmol, 44.80 pL, 2.0 eq) was dissolved in DMF (5 mL, pre-degassed and purged with N2 for 3 times), and then the mixture was stirred at 20-25 °C for 2 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m / z (MW: 1665.84, observed m / z: 833.2 ([M / 2+H]+)) was detected. The solvent was removed under reduced 10 pressure and compound 5 (300 mg, crude) was obtained as yellow oil. To crude compound 5 (300 mg, dissolved in 10 mL DMF) was added piperidine (2 mL), and the mixture was stirred at 30 °C for 2 hr. LCMS indicated one main peak with desired m / z (MW: 1443.60 observed m / z: 722.7 ([M / 2+H]+)) was detected. The residue was purified by 15 prep-HPLC (neutral condition). COM130 (140 mg, 58.19 pmol, 32.31% yield, 60% purity) was obtained as a white solid. COM131 O Fmoc Exact Mass: 595.26 Molecular Weight: 595.64 1 N3-CH2-CH2CH2NH2 Exact Mass: 677.33 Molecular Weight: 677.75 Exact Mass: 455.26 Molecular Weight: 455.51 3 HATU, DMF Exact Mass: 954.48 Molecular Weight: 955.06 2Q                               COM00000131 A mixture of compound 1 (700.0 mg, 1.18 mmol, 1.0 eq), 3-azidopropan-1-amine (117.7 mg, 1.18 mmol, 1.0 eq), HOBt (190.6 mg, 1.41 mmol, 1.2 eq), EDCI (270.4 mg, 1.41 mmol, 1.2 eq) was dissolved in DCM (20 mL, pre-degassed and purged with N2 for 3 times), and then the mixture was stirred at 25-30 °C for 2 hr under N2 atmosphere. LC-MS showed compound 2024205105   25 Jul 2024 1 was consumed completely and one main peak with desired m / z (calculated MW: 677.75, observed m / z: 678.2 ([M+H]+)) was detected. The reaction mixture was treatment with a few drops of 1 M HCI, and the organic layer was collected and evaporated under reduced pressure. Compound 2 (600.0 mg, crude) was obtained as a white solid. 5 Compound 2 (600.0 mg, 885.2 pmol, 1.0 eq) was dissolved in DMF (3 mL, pre-degassed and purged with N2 for 3 times), and then piperidine (1.29 g, 15.19 mmol, 1.50 mL, 17.2 eq) was added and the mixture was stirred at 25-30 °C for 2 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z 10 (calculated MW: 455.51 observed m / z: 456.3 ([M+H]+)) was detected. The reaction mixture was purified by prep-HPLC (TFA condition), and compound 3 (400.0 mg, 879.1 pmol) was obtained as colorless oil. A mixture of compound 3 (250.0 mg, 548.83 pmol, 1.0 eq), compound 4 (284.1 mg, 548.83 15 pmol, 1 eq), HATU (229.6 mg, 603.72 pmol, 1.1 eq), DIEA (141.9 mg, 1.10 mmol, 191.19 pL, 2.0 eq) in DCM (20 mL, pre-degassed and purged with N2 for 3 times), and then the mixture was stirred at 25-30 °C for 2 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m / z (calculated MW: 955.06, observed m / z: 955.6 ([M+H]+)) was detected. The residue was purified by prep-HPLC (TFA 20 condition). Compound 5 (400.0 mg, 419.1 pmol) was obtained as a white solid A mixture of Compound 5 (400.0 mg, 418.82 pmol, 1.0 eq) was dissolved in DMF (4 mL, pre-degassed and purged with N2 for 3 times), and then piperidine (862.2 mg, 10.13 mmol, 1 mL, 24.2 eq) was added and the mixture was stirred at 25-30 °C for 2 hr under N2 atmosphere. LC-MS showed Compound 5 was consumed completely and one main peak 25 with desired m / z (MW: 732.83 observed m / z: 733.3 ([M+H]+)) was detected. The residue was purified by prep-HPLC (TFA condition). COM131 (200 mg, 272.9 pmol) was obtained as colorless oil. COM470 0 ।         C?         0 HATU H       ,|| I              / = /                      DIEA +                    OH       DMF 0                          O COM122                             1 H         O I PIPERIDINE       N3^^zN DMF                        O ।        O 3Q                                        COM00000470 0 2 2024205105   25 Jul 2024 To a solution ofCOM122 (228 mg, 149.83 pmol, 1.0 eq), Compound 1 (51.31 mg, 164.82 pmol, 1.1 eq) in DMF (6 mL) was added HATLI (85.40 mg, 224.75 pmol, 1.5 eq) and DIEA (19.37 mg, 149.83 pmol, 26.10 pL, 1.0 eq). The mixture was stirred at 25-30 °C for 2 hr. LC-MS showed Compound 1 was consumed completely and one main peak with desired m / z 5 (MW: 1814.99, observed m / z: 908.2([M / 2+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (neutral condition). Compound 2 (54 mg, 29.75 pmol, 19.86% yield) was obtained as a white solid. 10 To a solution of Compound 2 (54 mg, 29.8 pmol, 1.0 eq) in DMF (2 mL) was added piperidine (61 mg, 715 pmol, 71 pL, 24.0 eq). The mixture was stirred at 25-30 °C for 2 hr. LC-MS showed Compound 2 was consumed completely and one main peak with desired m / z (MW: 1592.75 observed m / z: 796.27([M / 2+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was 15 purified by prep-HPLC (TFA condition). COM470 (40 mg, 25.11 pmol, 84.41% yield) was obtained as a white solid. COM471 Chemical Formula: C30H52N10O11 Exact Mass: 728.38 NH—Fmoc Chemical Formula: C18H17NO4 Exact Mass: 311.12 EDCl.HOBt DCM Chemical Formula: C^HgyN^O^ Exact Mass: 1021.49 1                                     2                                                            3 4 Chemical Formula: C70H111N15O23 Exact Mass: 1529.80 EDCl.HOBt Pip DMF H2N-(B-Ala)-Sar10-Peg10-N3 20                                       COM00000471 A mixture of compound 1 (900 mg, 1.23 mmol, 1.0 eq) and compound 2 (1.0 g, 3.21 mmol, 2.6 eq) was dissolved in DCM (20 mL), following by addition of (284.0 mg, 1.48 mmol, 1.2 2024205105   25 Jul 2024 eq), HOBt (200.2 mg, 1.48 mmol, 1.2 eq).The mixture was stirred at 25 °C for 2 hr. LC-MS showed compound 1 was consumed completely and one peak with desired m / z (calculated MW: 1021.49, observed m / z\ 1022.2 ([M+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by prep- 5 HPLC (TFA condition). Compound 3 (0.900 g, 880.53 pmol, 71.30% yield) was obtained as a white solid. A mixture of compound 3 (500.0 mg, 489.19 pmol, 1.0 eq), compound 4 (257.6 mg, 489.19 pmol, 1.0 eq) was dissolved in DCM (5 mL), following by addition of HOBt (132.2 mg, 978.37 10 pmol, 2.0 eq), EDCI (187.6 mg, 978.37 pmol, 2.0 eq). The mixture was stirred at 25-30 °C for 2 hrs. LC-MS showed compound 3 was consumed completely and one main peak with desired m / z (MW: 1529.80 observed m / z\ 765.9 ([M / 2+H]+) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (neutral condition). Compound 3 (420 mg, 246.94 pmol, 15   50.48% yield) was obtained as colorless oil. Compound 5 (420 mg, 274.38 pmol, 1.0 eq) was dissolved in DMF (4 mL), following by addition of piperidine (865.2 mg, 10.16 mmol, 1 mL, 37 eq). The mixture was stirred at 25-30 °C for 2 hr. LC-MS showed compound 5 was consumed completely and one main peak with 20 desired m / z (calculated MW: 1308.48, observed m / z: 654.8([M / 2+H]+) was detected. The crude product was purified by prep-HPLC (TFA condition). COM471 (386 mg, 265.50 pmol, 96.76% yield) was obtained as colorless oil. COM472 25 L O1___        HATU, DIEA h2nT^ J^N3 -------► DMF DMF Pip Fmoc—N 1    0 Chemical Formula: C30H37N5O8 Exact Mass: 595.26 COM126 Chemical Formula: C32H66N4O15 Exact Mass: 746.45 HATU, DIEA DMF Chemical Formula: C62H101N9O22 Exact Mass: 1323.71 4                                                          5 TFA DCM COM00000472 2024205105   25 Jul 2024 A mixture of compound 1 (0.5 g, 839.43 pmol, 1.0 eq), compound 2 (627.0 mg, 839.43 pmol, 1.0 eq) and DIEA (217.0 mg, 1.68 mmol, 292.4 pL, 2.0 eq) was dissolved in DMF (2 mL), then HATLI (319.2 mg, 839.4 pmol, 1.0 eq) was added to the mixture. The mixture was then 5 stirred at 25 °C for 30 min. TLC (DCM: CHsOH=10:1, Rf=0.24) showed compound 1 was consumed completely and one new spot formed. The solvent was evaporated to produce compound 3 (0.45 g, 339.75 pmol, 40.47% yield, crude) as colorless oil, which was used in next step without further purification. 10 Compound 3 (450.0 mg, 339.75 pmol, 1.0 eq) was dissolved in DMF (8 mL), following by addition of piperidine (2 mL). The mixture was stirred for 15 mins at 25°C. LC-MS showed compound 3 was consumed completely and one main peak with desired (calculated MW: 1102.27, observed m / z\ 552.1 ([M / 2+H]+)) was detected. The residue was purified by prep-HPLC (TFA condition). Compound 3 (370.0 mg, 335.67 pmol, 98.80% yield) was obtained 15 as colorless oil. To a solution of COM126 (60 mg, 54.45 pmol, 1.0 eq), compound 4 (15.5 mg, 81.68 pmol, 1.5 eq) in DMF (5 mL) was added HATU (31 mg, 81.68 pmol, 1.5 eq) and DIEA (10.5 mg, 61.68 pmol, 15 pL, 1.5 eq) .The mixture was stirred at 30 °C for 2 hr. LC-MS showed 20 COM126 was consumed completely and one main peak with desired was detected. The mixture was evaporated to remove solvent, and compound 5 (30 mg, crude) was obtained as colorless oil, which was used in next step without further purification. Compound 5 (30 mg, 23.57 pmol, 1.0 eq) was dissolved DCM (4.5 mL), and then TFA (0.5 25 mL) was added and the mixture was stirred at 25-30 °C for 2 hr. LC-MS showed compound 5 was consumed completely and one main peak with desired was detected. The residue was purified by prep-HPLC (TFA condition). COM472 (10 mg, 8.52 pmol) was obtained as white solid. 30 COM473 2024205105   25 Jul 2024 Exact Mass: 306.19 Exact Mass: 666.30                            Molecular Weight: 306.36 Molecular Weight: 666.72 Pip DMF HOBt, EDCI DCM Exact Mass: 954.48 Molecular Weight: 955.06 Exact Mass: 732.41 Molecular Weight: 732.84 COM00000473 A mixture of compound 1 (300 mg, 449.96 pmol, 1.0 eq), compound 2 (138 mg, 449.96 pmol, 1.0 eq), HOBt (122 mg, 899.93 pmol, 2.0 eq), EDCI (173 mg, 899.93 pmol, 2.0 eq) was dissolved in DCM (10 mL, pre-degassed and purged with N2 for 3 times), and then the 5 mixture was stirred at 20-25 °C for 1 hr under N2 atmosphere. LC-MS showed compound 1 was consumed completely and one main peak with desired (MW: 955.06, observed m / z\ 955.3 ([M+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent. The mixture was evaporated under reduced pressure and compound 3 (300 mg, crude) was obtained as yellow oil. 10 Compound 3 (300 mg, 314.12 pmol, 1.0 eq) was dissolved in DMF (4 mL), and then piperdine (1 mL) was added and the mixture was stirred at 20-25 °C for 1 hr. LC-MS showed compound 3 was consumed completely and one main peak with desired m / z (MW: 732.83 observed m / z: 733.2 ([M+H]+)) was detected. The residue was purified by prep-HPLC 15 (neutral condition). COM473 (160 mg, 218.33 pmol, 69.51% yield) was obtained as a colorless oil. Example 2: Synthesis of EphA2 / CD137 Binding Heterotandem Bicyclic Peptides 2024205105   25 Jul 2024 BCY9173 o o nh2 BCY00009173 Procedure for preparation of BCY9172-PEG12-N3 BCY00009172 + NHS-PEG12-N3 ----------5►  BCY00009172-PEG12-N, MeCN / H2O 5                                    1                                                  2 BCY9172 (520 mg, 248.16 pmol, 1 eq) and compound 1 (370 mg, 499.47 pmol, 2.01 eq), were dissolved in in DMF (5 mL) was added DIEA (48.11 mg, 372.24 pmol, 64.84 pL, 1.5 eq) and then the mixture was strirred at 30°C for 12 hr. LC-MS showed BCY9172 was consumed completely and one main peak with desired m / z (calculated MW: 2721.12 10 observed m / z: 1360.9 ([M / 2+H]+)) was detected. The reaction mixture was purified by prep-HPLC (TFA condition) and compound 2 (284 mg, 101.10 pmol, 40.74% yield, 96.87% purity) was obtained as a white solid. BCY00009172-PEG12-N3 +   BCY00006169 CuSO4 VcNa THPTA BCY00009173 t-BuOH / H2O 2024205105   25 Jul 2024 Procedure for preparation of BCY9173 This reaction was performed in two independent containers in parallel. For one container, 5 Compound 2 (100 mg, 36.75 pmol, 1.0 eq) and BCY6169 (120 mg, 36.78 pmol, 1.0 eq) were first dissolved in 10 mL of t-BuOH / H2O (1:1), and then CuSO4 (0.4 M, 91.9 pL, 1.0 eq), VcNa (0.4 M, 183.8 pL, 2.0 eq) and THPTA (0.4 M, 91.9 pL, 1.0 eq) was added. Finally 1 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 40 °C for 16 hr under N2 atmosphere. LC-MS 10 showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 5983.85 observed m / z: 997.6600 ([M / 6+H]+) and 1197.2300 ([M / 5+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY9173 (218 mg, 34.97 pmol, 47.58% yield, 96% purity) was obtained as a white solid. 15   BCY7985 BCY00007985 2024205105   25 Jul 2024 General procedure for preparation of BCY7859 1) HOSu, EDCI, DMA / DCM N3-PEG12-COOH -----------------------►  BCY00007859 2) BCY00007732, DIEA, DMF To a solution of N3-PEG12-COOH (250 mg, 388 pmol) and HOSu (67.0 mg, 583 pmol) in DMA (4.5 mL) and DCM (1.5 mL) was added EDCI (89.3 mg, 466 pmol) with stirring at 20 °C 5 for 16 hr. To another 50 mL of round flask containing a mixture of BCY7732 (855 mg, 388 pmol) in 5 mL of DMA was added DIEA (186 mg, 1.44 mmol, 250 pL) with stirring for 10 min. Then the initial reaction mixture was added to the flask with further stirring at 20 °C for additional 5 hr. LC-MS (ES8396-307-P1B1) showed BCY7732 was consumed completely and one main peak with desired mass was detected. The resulting reaction mixture was 10 purified directly by prep-HPLC (TFA condition) to give compound BCY7859 (621 mg, 200 pmol, 51.6% yield, TFA salt) as a white solid. General procedure for preparation of BCY6169 PYA-NHS BCY00006099   ------------ BCY00006169 DIEA, DMA 15 To a solution of BCY6099 (300 mg, 94.3 pmol) in DMA (2 mL) was added DIEA (36.6 mg, 283 pmol, 49.3 pL) with stirring for 10 min. After, PYA-NHS (36.8 mg, 189 pmol) was added with further stirring at 20 °C for additional 15 hr. LC-MS showed BCY6099 was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by prep-HPLC (neutral condition) to give compound BCY6169 (299 mg, 86.2 pmol, 20   91.5% yield) as a white solid. General procedure for preparation of BCY7985 Vc, CuSO4 BCY00006169 + BCY00007859 ----1-----BCY00007985 DMF, H2O To a solution of BCY7859 (220 mg, 77.8 pmol) and BCY6169 (251 mg, 77.1 pmol) in DMF 25   (5 mL) purged by nitrogen for 2 hr was added aqueous ascorbic acid solution (0.8 M, 963 pL) follwed by adding aqueous CuSCU (0.8 M, 289 pL) under nitrogen atmosphere. Then the mixture was stirred at 20 °C for 2 hr. LC-MS showed BCY6169 was consumed completely and one main peak with desired mass was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition) to give compound BCY7985 (283 mg, 43.4 pmol, 30   56.3% yield, TFA) as a white solid. BCY8942 2024205105   25 Jul 2024 BCY00008942 General procedure for preparation of BCY8940 EDCI, HOSu BCY00008045 + N3-PEG12-COOH ---------► BCY00008940 DMA. DCM To a solution of N3-PEG12-COOH (120 mg, 186 pmol, 1.0 eq) in DMA (3 mL) and DCM (1 5 mL) was added HOSu (32.2 mg, 280 pmol, 1.5 eq) with stirring. Then EDCI (42.9 mg, 224 pmol, 1.2 eq) was added to the mixture with further stirring for additional 7 hr at 20 °C. LCMS showed the activated ester was formed completely. To another flask with BCY8045 (410 mg, 186 pmol, 1.0 eq) in DMA (3 mL) was added DIEA (120 mg, 932 pmol, 162 pL, 5.0 eq) with stirring, then the activated ester was added and the mixture was stirred for 18 hr at 10   20 °C. LC-MS showed one main peak with desired m / z was detected. The reaction mixture was concentrated in vacuum to remove the DCM. The resulting mixture was purified by prep-HPLC (TFA condition) to give BCY8940 (190 mg, 67.2 pmol, 36.1% yield) as a white solid. 2024205105   25 Jul 2024 General procedure for preparation of BCY8942 BCY00008940 BCY00006169    --------------------►   BCY00008942 Vc, CuSO4, DMF, H2O To a solution of BCY8940 (28.6 mg, 10.1 pmol, 1.1 eq) and BCY6169 (30.0 mg, 9.19 pmol, 1.0 eq) in DMF (2.0 mL) was added (2R)-2-[(1S)-1,2-dihydroxyethyl]-3, 4-dihydroxy-2H-5 furan-5-one (1.0 M, 92.0 pL) and CuSO4 (1.0 M, 27.6 pL) with stirring under nitrogen atmosphere for 2 hr at 20 °C. LC-MS showed BCY6169 was consumed completely and one main peak with desired m / z (calculated MW: 6089.91 observed m / z: 1218.4([M / 5+H]+), 1016.0([M / 6+H]+), 870.7([M / 7+H]+) was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to give compound BCY8942 (15.4 mg, 2.46 pmol, 26.8% yield, 97.3% 10 purity) as a white solid. BCY8943 BCY00008943 General procedure for preparation of BCY8941 2024205105   25 Jul 2024 o. O N—\    DIEA, DMA BCY00006015   + I n / --------►     BCY00008941 o To a solution of BCY6015 (a peptide identical to BCY8941 except for the absence of a PYA moiety; 100 mg, 32.9 pmol) in DMA (2 mL) was added DIEA (12.8 mg, 98.7 pmol, 17.2 pL) with stirring for 10 min. Then (2,5-dioxopyrrolidin-1-yl) pent-4-ynoate (12.8 mg, 65.8 pmol) 5 was added to the mixture, following with further stirring at 20 °C for 16 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired m / z (calculated MW: 3119.60, observed m / z: 1040.5([M / 3+H]+) was detected. The mixture was purified by prep-HPLC (neutral condition) to give compound BCY8941 (90.0 mg, 28.9 pmol, 87.7% yield) as a white solid. 10 General procedure for preparation of BCY8943 BCY00007859 BCY00008941   -------------------*    BCY00008943 Vc, CuSO4, DMF, H2O To a solution of BCY7859 (which may be prepared as described in BCY7985; 40.0 mg, 14.2 pmol) and BCY8941 (42.0 mg, 13.5 pmol) in DMSO (2 mL, pre-purged by nitrogen for 1 hr) 15 was added (2R)-2-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one (1.0 M, 270 pL) and CuSO4 (1.0 M, 80.9 pL). The mixture was purged with nitrogen for 3 times and stirred at 15 °C for 2 hr. LC-MS showed BCY8941 was consumed completely and one main peak with desired m / z (calculated MW: 5946.77, observed m / z: 1190.2 ([M / 5+H]+), 991.5([M / 6+H]+), 849.9([M / 7+H]+) was detected. The reaction mixture was purified by prep-HPLC (A: 0.075% 20 TFA in H2O, B: ACN) to give compound BCY8943 (11.5 mg, 1.90 pmol, 14.1% yield, 98.1% purity) as a white solid. BCY9647 2024205105   25 Jul 2024 Procedure for preparation of compound 2 Exact Mass: 526.32139 Molecular Weight: 526.62144 Exact Mass: 200.98 Molecular Weight: 201.56 1 TEA DCM Exact Mass: 691.32760 Molecular Weight: 691.72446 COM00000134 5 To a solution of COM134 (30.0 mg, 57.0 pmol, 1.0 eq), compound 1 (17.2 mg, 85.3 pmol, 1.5 eq) in DCM (0.5 mL) was added TEA (8.65 mg, 11.9 pL, 1.5 eq). The mixture was stirred at 25 °C for 1 hr. LC-MS showed COM134 was consumed completely and one main peak with desired mass (calculated MW: 691.72, observed m / z: 692.3([M+H]+) and 709.3([M+NH4]+)) was detected. The reaction mixture was concentrated under reduced 10 pressure, and then lyophilized to produce crude compound 2 (30.5 mg, crude) as a white Procedure for preparation of compound 3 Exact Mass: 691.32760 Molecular Weight: 691.72446 BCY00006099     -------- DMF Exact Mass: 3180.55 Molecular Weight: 3182.66 BCY00006099.NAN^p0^N3 H H Exact Mass: 3732.85 Molecular Weight: 3735.28 2024205105   25 Jul 2024 To a solution of compound 2 (10 mg, 1.0 eq) in DMF (1 mL) was added BCY6099 (46 mg, 1.0 eq) and DIEA (5.61 mg, 7.55 pL, 3.0 eq). The mixture was stirred at 30 °C for 2 hr. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 3735.28 observed m / z: 1245.9([M / 3+H]+) and 934.5([M / 4+H]+)) was 5 detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (TFA condition). Compound 3 (34 mg, 62.96% yield, 100% purity) was obtained as a white solid. Procedure for preparation of BCY9647 BCY00006099, A AX n AX n n Exact Mass: 3732.85 Molecular Weight: 3735.28 BCY00007741 CuSO4 VcNa THPTA BCY00009647 t-BuOH:H2O Exact Mass: 2279.93 Molecular Weight: 2281.54 Exact Mass: 6012.79 Molecular Weight: 6016.82 A mixture of Compound 3 (34 mg, 9.10 pmol, 1.0 eq), BCY7741 (23 mg, 10.08 pmol, 1.11 eq), and THPTA (0.4 M, 11.4 pL, 0.5 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 11.4 pL, 0.5 eq) and VcNa (0.4 M, 22.8 pL, 1 eq) were added under N2. The pH of this solution was adjusted to 8 15 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m / z (calculated MW: 6016.82, observed m / z: 1204.1 ([M / 5+H]+), 1003.5([M / 6+H]+), 860.3([M / 7+H]+)). The reaction mixture was directly purified by prep-HPLC (TFA condition). 20   BCY9647 (31.2 mg, 54.67% yield, 95.96% purity) was obtained as a white solid. BCY9648 2024205105   25 Jul 2024 Procedure for preparation of compound 2 h2n n3 Exact Mass: 200.98 Molecular Weight: 201.56 TEA DCM Exact Mass: 1263.66839 Molecular Weight: 1264.40774 2 Exact Mass: 1098.66219 Molecular Weight: 1099.30472 COM00000135 To a solution of COM135 (30 mg, 27.29 pmol, 1.0 eq), compound 1 (8.25 mg, 40.94 pmol, 5   1.5 eq) in DCM (0.5 mL) was added TEA (4.14 mg, 40.94 pmol, 5.70 pL, 1.5 eq). The mixture was stirred at 25~30°C for 1 hr. LC-MS showed COM 135 was consumed completely and one main peak with desired mass [calculated MW: 1264.41, observed m / z: 1281.4([M+NH4]+). 649.8 ([M / 2+H]+)] was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by 10 prep-HPLC (TFA condition) to give compound 2 (18 mg, 14.2 pmol, 52.14% yield). Procedure for preparation of compound 3 Exact Mass: 1263.66839 Molecular Weight: 1264.40774 2 BCY00006099 Exact Mass: 3180.55 Molecular Weight: 3182.66 DIEA                          O DMF *   BCY00006099,nAn^,0^ H H           23 Exact Mass: 4305.19 Molecular V\feight: 4307.96 3 2024205105   25 Jul 2024 To a solution of compound 3 (9 mg, 7.12 pmol, 1 eq) in DMF (1 mL) was added BCY6099 (23 mg, 7.23 pmol, 1.02 eq) and DIEA (2.76 mg, 21.35 pmol, 3.72 pL, 3.0 eq). The mixture was stirred at 30 °C for 2 hr. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 4307.96 observed m / z: 1436.9([M / 3+H]+), 5   1077.9([M / 4+H]+), 862.5([M / 5+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (TFA condition). Compound 3 (14.6 mg, 47.61% yield, 100% purity) was obtained as a white solid. 10 Procedure for preparation of BCY9648 9                                            CuSO4 VcNa THPTA BCY00006099, A                   +        BCY00007741     ------------------►          BCY00009648 t-BuOH:H2O Exact Mass: 4305.19 Molecular Weight: 4307.96 3 Exact Mass: 2279.93 Molecular Weight: 2281.54 Exact Mass: 6585.13 Molecular Weight: 6589.50 A mixture of compound 3 (14.6 mg, 3.39 pmol, 1 eq), BCY7741 (8.5 mg, 3.73 pmol, 1.1 eq) and THPTA (0.4 M, 4.3 pL, 0.5 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, pre-degassed and purged with N2 for 3 times), and then CuSO4 (.4 M, 4.3 pL, 0.5 eq) and VcNa (0.4 M, 8.6 15 pL, 1.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m / z (MW: 6589.50 observed m / z: 1098.8([M / 6+H]+), 942.1 ([M / 7+H]+), 824.6([M / 8+H]+)). The reaction mixture 20 was directly purified by prep-HPLC (TFA condition). BCY9648 (14.7 mg, 63.34% yield, 96.22% purity) was obtained as a white solid. BCY9655 2024205105   25 Jul 2024 Exact Mass: 1186.69 Molecular Weight: 1187.38 COM00000128 Exact Mass: 200.98 Molecular Weight: 201.56 Exact Mass: 1351.70 Molecular Weight: 1352.48 2 5 To a solution of COM128 (120 mg, 101.06 pmol, 1.0 eq), compound 1 (25 mg, 124.03 pmol, 1.25 eq) in DCM (0.5 mL) was added TEA (15.34 mg, 151.59 pmol, 21.10 pL, 1.5 eq). The mixture was stirred at 25 °C for 1 hr. LC-MS showed one new peak with desired m / z (calculated MW: 1352.48, observed m / z: 676.8([M / 2+H]+), 1369.3([M+NH4]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a 10 residue. The residue was purified by prep-HPLC (neutral condition). Compound 2 (14 mg, 8.99 pmol, 8.90% yield, 86.86% purity) was obtained as colorless oil. Procedure for preparation of compound 3 Exact Mass: 1351.70 Molecular Weight: 1352.48 Exact Mass: 3180.55 Molecular Weight: 3182.66 BCY00006099 DMF             3 H Q          h J Exact Mass: 4393.22 Molecular Weight: 4396.03 15 To a solution of compound 2 (7 mg, 5.18 pmol, 1.0 eq) and BCY6099 (16 mg, 5.03 pmol, 1.0 eq) in DMF (2 mL) was added DIEA (2.01 mg, 15.53 pmol, 2.70 pL). The mixture was stirred at 30 °C for 2 hrs. LC-MS showed compound 2 was consumed completely and one main 2024205105   25 Jul 2024 peak with desired m / z (calculated MW: 4396.02, observed m / z\ 879.8([M / 5+H]+) and 1099.8([M / 4+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (0.1% TFA condition). Compound 3 (11.8 mg, 48.29% yield, 93.11% purity) was 5 obtained as a white solid. Procedure for preparation of BCY9655 N3^^nI^^+O^2^Y'bcy000^ +   BCY00007741                 BCY00009655 H O                 0 Exact Mass: 4393.22                               Exact Mass: 2279.93                              Exact Mass: 6663.15 Molecular Weight: 4396.03                          Molecular Weight: 2281.54                         Molecular Weight: 6677.57 3 A mixture of Compound 3 (11.8 mg, 2.69 pmol, 1.0 eq), BCY7741 (7.0 mg, 3.07 pmol, 1.14 10 eq), and THPTA (0.4 M, 6.8 pL, 1 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, pre degassed and purged with N2 for 3 times), and then CuSO4(0.4 M, 6.8 pL, 1.0 eq) and VcNa (0.4 M, 13.6 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS 15 showed compound 3 was consumed completely and one main peak with desired m / z (calculated MW: 6677.57, observed m / z\ 1113.7 ([M / 6+H]+), 954.7 ([M / 7+H]+)). The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY9655 (1.9 mg, 0.26 pmol, 9.65% yield, 91.15% purity) was obtained as a white solid. 20  BCY9656 2024205105   25 Jul 2024 Procedure for preparation of compound 2 Exact Mass: 200.98 Molecular Weight: 201.56 0 Exact Mass: 1307.73 Molecular Weight: 1308.48 Exact Mass: 1472.74 Molecular Weight: 1473.58 COM00000129                            1                                                                          2 To a solution of COM129 (30.0 mg, 22.93 pmol, 1.0 eq), compound 1 (6.9 mg, 34.39 pmol, 5   1.5 eq) in DCM (3 mL) was added TEA (3.5 mg, 34.39 pmol, 4.8 pL, 1.5 eq). The mixture was degassed and purged with N2 for 3 times, and then the mixture was stirred at 25 °C for 1 hr under N2 atmosphere. LC-MS showed COM129 was consumed completely and one main peak with desired m / z (calculated MW: 1473.58, observed m / z: 737.3([M / 2+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent 10 to give a residue. The residue was purified by prep-HPLC (neutral condition) to produce Compound 2 (12.3 mg, 8.35 pmol, 36.41% yield) as a white solid. Procedure for preparation of compound 3 Exact Mass: 1472.74 Molecular Weight: 1473.58 BCY00006099 DIEA DMF Exact Mass: 4514.26 Molecular Weight: 4517.13 Exact Mass: 3732.85 Molecular Weight: 3735.28 15 To a solution of compound 2 (9.26 mg, 6.28 pmol, 1.0 eq) and BCY6099 (10 mg, 3.14 pmol, 0.5 eq) in DMF (3 mL) was added TEA (0.7 mg, 6.93 pmol, 1 pL, 1.1 eq). The mixture was 2024205105   25 Jul 2024 degassed and purged with N2 for 3 times, and then the mixture was stirred at 25-30 °C for 1 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 4517.12, observed m / z\ 1129.8 ([M / 4+H]+),904.1 ([M / 5+H]+), 753.7 ([M / 6+H]+)) was detected. The reaction mixture was 5 filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (TFA condition). Compound 3 (12 mg, 72.36% yield, 85.58% purity) was obtained as a white solid. Procedure for preparation of BCY9656 Exact Mass: 4514.26 Molecular Weight: 4517.13 BCY00007741 CuSO4, VcNa, THPTA t-BuOH:H2O(1:1) BCY00009656 Exact Mass: 2279.93 Molecular Weight: 2281.54 Exact Mass: 6794.19 Molecular Weight: 6798.67 10                       3 A mixture of Compound 3(11 mg, 2.44 pmol, 1.0 eq), BCY7741 (6.0 mg, 2.63 pmol, 1.08 eq), and THPTA (0.4 M, 6.1 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4(0.4 M, 6.1 pL, 1.0 eq) and VcNa (0.4 M, 12.2 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by 15 dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 3 was consumed completely and one main peak with desired m / z (calculated MW: 6798.66, observed m / z: 1133.8 ([M / 6+H]+), 971.9 ([M / 7+H]+), 850.7 ([M / 8+H]+)) was detected. The reaction mixture was directly purified by prep-HPLC (TFA 20 condition). BCY9656 (6.8 mg, 37.36% yield, 90.97% purity) was obtained as a white solid. BCY9657 2024205105   25 Jul 2024 To a solution of COM130 (30.0 mg, 20.78 pmol, 1.0 eq), compound 1 (6.3 mg, 31.17 pmol, 5   1.5 eq) in DCM (3 mL) was added TEA (3.2 mg, 31.17 pmol, 4.4 pL, 1.5 eq). The mixture was stirred at 25-30 °C for 1 hr. LC-MS showed COM 130 was consumed completely and one main peak with desired m / z (calculated MW: 1608.7, observed m / z\ 804.8([M / 2+H]+)) was detected. The reaction mixture was concentrated under reduced pressure and lyophilized to produce compound 2 (7.9 mg, crude) as a white solid. 10 Procedure for preparation of compound 3 T        O                       I O II .           ,               , J, II,                                                                  diea + BCY00006099 ----- »      [BCY00006099]-[COM00000130] H         5              H        3                        DMF 2                                                                                              3 To a solution of compound 2 (7.9 mg, 4.91 pmol, 1.0 eq) and BCY6099 (16 mg, 5.03 pmol, 1.02 eq) in DMF (1 mL) was added DIEA (1.9 mg, 14.73 pmol, 2.6 pL, 3.0 eq). The mixture 15 was stirred at 30 °C for 2 hrs. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 4652.25, observed m / z\ 1551.3([M / 3+H]+), 1163.6([M / 4]+), 931.1([M / 5+H]+), 776.1([M / 6+H]+))was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was 2024205105   25 Jul 2024 purified by reversed-phase HPLC (TFA condition). Compound 3 (13.3 mg, 2.86 pmol, 53.22% yield, 91.42% purity) was obtained as a white solid. Procedure for preparation of BCY9657 [BCY00006099]-[COM00000130]   +   BCY00007741 CuS04 VcNa THPTA»  BCY00009657 t-BuOH / H2O 5                      3 A mixture of Compound 3 (13.3 mg, 2.86 pmol, 1.0 eq), BCY7741 (7.0 mg, 3.07 pmol, 1.03 eq), and THPTA (0.4 M, 7.5 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4(0.4 M, 7.5 pL, 1 eq) and VcNa (0.4 M, 15 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by 10 dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 3 was consumed completely and one main peak with desired m / z [MW: 6933.78, observed m / z: 1156.7([M / 6+H]+), 991.4([M / 7+H]+), 867.4([M / 8+H]+)] was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY9657 (8.4 mg, 15   40.21% yield, 94.9% purity) was obtained as a white solid. BCY9658 2024205105   25 Jul 2024 To a solution of COM131 (167.0 mg, 227.89 pmol, 1.0 eq), compound 1 (55.0 mg, 272.87 5 pmol, 1.2 eq) in DCM (5 mL) was added TEA (36.4 mg, 359.23 pmol, 50.0 pL, 1.6 eq). The mixture was stirred at 25-30 °C for 1 hr. LC-MS showed one main peak with desired m / z (MW: 897.93 observed 920.3([M+Na+]) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (TFA condition). Compound 2 (35 mg, 33.74 pmol, 14.81% yield, 10   86.56% purity) was obtained as colorless oil. Procedure for preparation of compound 3 2 BCY00006099 TEA DMF [BCY00006099HCOM00000131] To a solution of compound 2 (15 mg, 16.71 pmol, 1.0 eq) and BCY6099 (53 mg, 16.65 pmol, 15   1.0 eq) in DMF (2 mL) was added DIEA (6.48 mg, 65.05 pmol, 50.1 pL, 4.0 eq). The mixture 2024205105   25 Jul 2024 was stirred at 30 °C for 2 hrs. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (MW: 3941.47 observed m / z\ 986.0([M / 4+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (TFA condition). 5   [BCY6099]-[COM131] (5 mg, 50.48% yield, 94.96% purity) was obtained as a white solid. Procedure for preparation of BCY9658 [BCY00006099]-[COM00000131]    +   BCY00007741 CuSO4 VcNa THPTA BCY00009658 t-BuOH / H2O 3 A mixture of Compound 3 (35 mg, 8.88 pmol, 1.0 eq), BCY7741 (21 mg, 9.20 pmol, 1.03 10 eq), and THPTA (0.4 M, 22.2 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4(0.4 M, 22.2 pL, 1.0 eq) and VcNa (0.4 M, 44.4 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC- 15 MS showed Compound 3 was consumed completely and one main peak with desired m / z [MW: 6223.01 observed m / z\ 1038.0([M / 6+H]+) and 889.8([M / 8+H]+)] was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY9658 (13.2 mg, 21.54% yield, 90.16% purity) was obtained as a white solid. 20  BCY9659 2024205105   25 Jul 2024 To a solution of COM132 (20.0 mg, 65.28 pmol, 1.0 eq), compound 1 (15.8 mg, 78.34 pmol, 5   1.2 eq) in DCM (5 mL) was added TEA (36.4 mg, 359.23 pmol, 50 pL, 5.5 eq). The mixture was stirred at 25 °C for 1 hr. LC-MS one main peak with desired m / z (MW: 471.46, observed m / z: 489.2([M+NH4]+)) was detected. The reaction mixture was concentrated under reduced pressure to give compound 2 (26 mg, crude) colorless oil. 10 Procedure for preparation of compound 3 TEA pMF *“   [BCY00006099]-[COM00000132] To a solution of compound 2 (15.0 mg, 4.71 pmol, 1.0 eq) and BCY6099 (3.33 mg, 7.07 pmol, 1.5 eq) in DMF (3 mL) was added TEA (0.7 mg, 6.93 pmol, 1 pL, 1.5 eq). The mixture was stirred at 30 °C for 2 hrs. LC-MS showed compound 2 was consumed completely and 15 one main peak with desired m / z (MW: 3515.01, observed m / z: 1172.1 ([M / 3+H]+) 879.5([M / 4+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue, he crude product was purified by reversed-phase HPLC 2024205105   25 Jul 2024 (TFA condition). Compound 3 (12.7 mg, 3.26 pmol, 69.23% yield, 90.3% purity) was obtained as a white solid. Procedure for preparation of BCY9659 [BCY00006099]-[COM00000132]    +   BCY00007741 CuSO4 VcNa THPTA* t-BuOH / H2O BCY00009659 5                    3 A mixture of Compound 3 (12.7 mg, 2.89 pmol, 1.0 eq), BCY7741 (6.80 mg, 2.98 pmol, 1.03 eq), and THPTA (1.3 mg, 2.99 pmol, 1.03 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4(0.4 M, 7.3 pL, 1.0 eq) and VcNa (0.4 M, 14.6 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by 10 dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 3 was consumed completely and one main peak with desired m / z [MW: 5796.54 observed m / z\ 1159.8([M / 5+H]) 966.7([M / 6+H]+)] was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY9659 (6.2 mg, 1.06 pmol, 15   36.58% yield, 98.86% purity) was obtained as a white solid. BCY9758 2024205105   25 Jul 2024 I            j +    BCY00006099    2 0 eq DIPEA- BCY00006099-PEG24-NHS ester o                  o XV                         Dry dmf 0 1                                                                                                                   2 To a solution of compound 1 (5.0 mg, 3.54 pmol, 1.0 eq), BCY6099 (11.3 mg, 3.54 pmol, 1.0 eq) in DMF (3 mL) was added DIEA (0.9 mg, 7.07 pmol, 1.2 pL, 2.0 eq). The mixture was stirred at 25-30 °C for 20 min. LC-MS showed one peak with desired m / z (MW: 4481.11, 5 observed m / z: 1101.3 ([M / 4+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure and lyophilized to give compound 2 (15 mg, crude) as a white solid. Procedure for preparation of BCY9758 2.0 eq DIPEA BCY00006099-PEG24-NHS ester +   BCY00007732   --------------►   BCY00009758 Dry DMF 10                   2 To a solution of compound 2 (15 mg, 3.35 pmol, 1.0 eq) and BCY7732 (14.74 mg, 6.69 pmol, 2.0 eq) in DMF (3 mL) was added DIEA (0.9 mg, 7.07 pmol, 1.2 pL, 2.1 eq). The mixture was stirred at 25-30 °C for 2 hrs. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (MW: 6567.48, observed m / z: 15   1095.1([M / 6+H]), 938.8([M / 7+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (TFA condition). BCY9758 (5.8 mg, 24.26% yield, 91.97% purity) was obtained as a white solid. 20  BCY10568 2024205105   25 Jul 2024 BCY00010568 Procedure for preparation of BCY8919-PEG12-N3 DIEA BCY00008919 + NHS-PEG12-N3 ---------- DMSO 1 BCY00008919-PEG12-N3 2 BCY8919 (80.0 mg, 38.47 pmol, 1.0 eq) and compound 1 (29.6 mg, 40.01 pmol, 1.04 eq) 5 were dissolved in DMSO (1 mL). The solution was then added with DIPEA (7.46 mg, 55.71 pmol, 10.0 pl, 1.5 eq), and then the mixture was stirred at 25-30°C for 2 hr. LC-MS showed majority of BCY8919 was consumed and one main peak with desired m / z (calculated MW: 2705.16, observed m / z: 1353.1([M / 2+H]+)) was detected. The reaction mixture was purified 2024205105   25 Jul 2024 by prep-HPLC (TFA condition) and compound 2 (18.6 mg, 6.86 pmol, 17.83% yield, 99.76% purity) was obtained as a white solid. Procedure for preparation of BCY10568 BCY00008919-PEG12-N3  +    BCY00006169 CuSO4 VcNa THPTA BCY00010568 t-BuOH / H2O 5                      2 Compound 2 (9.0 mg, 3.33 pmol, 1.0 eq) and BCY6169 (11.0 mg, 3.36 pmol, 1.01 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 8.3 pL, 1.0 eq), VcNa (1.4 mg, 7.06 pmol, 2.1 eq) and THPTA (1.4 mg, 3.22 pmol, 1.0 eq) were added. Finally 0.4 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and 10 purged with N2 for 3 times. The reaction mixture was stirred at 30°C for 16 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 5967.90, observed m / z: 995.00([M / 5+H]+) and 1194.70([M / 6+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY10568 (13.4 mg, 2.16 pmol, 69.44% yield, 96.3% purity) was obtained as a white solid. BCY00010570 Procedure for preparation of BCY8920-PEG12-N3 2024205105   25 Jul 2024 DIEA BCY00008920 + NHS-PEG12-N3 ----------------►   BCY00008920-PEG12-N3 DMSO 1                                                           2 To a solution of BCY8920 (37 mg, 17.31 pmol, 1.0 eq) and compound 1 (15 mg, 20.25 pmol, 1.2 eq) in DMSO (2 mL) was added DIEA (3.36 mg, 25.96 pmol, 4.5 pL, 1.5 eq). The mixture was stirred at 30 °C for 12 hr. LC-MS showed BCY8920 was consumed completely and one 5 main peak with desired m / z (calculated MW: 2763.2, observed m / z: 689.07([M / 4-H+])) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent and produced a residue. The residue was then purified by prep-HPLC (neutral condition). Compound 2 (22.8 mg, 8.15 pmol, 47.09% yield, 98.78% purity) was obtained as a white solid. 10 Procedure for preparation of BCY10570 C11SO4 VcNa BCY00008920-PEG12-N3 +    BCY00006169  ---THPTA—-    BCY00010570 t-BuOH / H2O 2 Compound 2 (6 mg, 2.17 pmol, 1.0 eq) and BCY6169 (7.08 mg, 2.17 pmol, 1.0 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 5.4 pL, 1.0 eq), VcNa 15   (0.4 M, 10.8 pL, 2.0 eq) and THPTA (0.4 M, 5.4 pL, 1.0 eq) was added. Finally 0.2 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 30 °C for 4 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (MS: 6025.93, observed m / z: 1004.56([M / 6]+H+) and 861.48([M / 7+H+])) was detected. The 20 reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (TFA condition). BCY10570 (7.2 mg, 1.17 pmol, 53.90% yield, 97.95% purity) was obtained as a white solid. BCY10574 2024205105   25 Jul 2024 DIEA BCY00009594 + NHS-PEG5-N3 1 DMSO BCY00009594-PEG5-N3 2 To a solution of BCY9594 (65 mg, 27.07 pmol, 1 eq), Compound 1 (12.00 mg, 27.75 pmol, 5   1.02 eq) in DMSO (1 mL) was added DIEA (5.25 mg, 40.61 pmol, 7.07 pL, 1.5 eq). The mixture was stirred at 25-30 °C for 2 hr. LC-MS showed BCY9594 was consumed completely and one main peak with desired m / z (calculated MW: 2718.13, observed m / z: 906.04([M / 3+H]+), 1359.07([M / 2+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by 10 prep-HPLC (TFA condition). Compound 2 (42.6 mg, 15.67 pmol, 57.89% yield, 100% purity) was obtained as a white solid. Procedure for preparation of BCY10574 BCY00009594-PEG5-N3   +   BCY00008927 -2^24_VcNajmPTA_^ BCY00010574 t-BuOH / H2O 2024205105   25 Jul 2024 A mixture of Compound 2 (20 mg, 7.36 pmol, 1.0 eq), BCY8927 (17 mg, 7.87 pmol, 1.07 eq), and THPTA (0.4 M, 18.4 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4(0.4 M, 18.4 pL, 1.0 eq) and VcNa (0.4 M, 36.8 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 5   8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 4877.68, observed m / z\ 1219.42 ([M / 4+H]+) and 975.54([M / 5+H]+))was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). 10   BCY10574 (17.6 mg, 3.41 pmol, 46.29% yield, 94.40% purity) was obtained as a white solid. Procedure for preparation of compound 2 15 BCY00009594 + NHS-PEG5-N3 DIEA DMSO BCY00009594-PEG5-N3 2 To a solution of BCY9594 (65 mg, 27.07 pmol, 1 eq), Compound 1 (12.0 mg, 27.75 pmol, 1.02 eq) in DMSO (1 mL) was added DIEA (5.25 mg, 40.61 pmol, 7.07 pL, 1.5 eq). The 2024205105   25 Jul 2024 mixture was stirred at 25-30 °C for 2 hr. LC-MS showed Compound 1 was consumed completely and one main peak with desired m / z [calculated MW:2718.13 observed m / z: 906.04([M / 3+H]+) and 1359.07([M / 2+H]+)] was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was 5 purified by prep-HPLC (TFA condition). Compound 2 (42.6 mg, 15.67 pmol, 57.89% yield, 100% purity) was obtained as a white solid. Procedure for preparation of BCY10575 BCY00009594-PEG5-N3   +   BCY00008928 -2^24_VcNajmPTA_^ BCY00010575 t-BuOH / H2O 2 10 A mixture of Compound 2 (20 mg, 7.36 pmol, 1.0 eq), BCY8928 (17 mg, 7.67 pmol, 1.04 eq), and THPTA (0.4 M, 18.4 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4(0.4 M, 18.4 pL, 1.0 eq) and VcNa (0.4 M, 36.8 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to 15 light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 2 was consumed completely and one main peak with desired m / z [calculated MW: 4935.71, observed m / z: 1234.59 ([M / 4+H]+) and 987.71 ([M / 5+H]+)] was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY10575 (12 mg, 2.37 pmol, 32.27% yield, 97.67% purity) was obtained as a white solid. 20 BCY10576 2024205105   25 Jul 2024 Procedure for preparation of compound 2 BCY00009594 + NHS-PEG5-N3 1 DIEA DMSO BCY00009594-PEG5-N3 2 To a solution of BCY9594 (30.0 mg, 12.50 pmol, 1.0 eq), Compound 1 (5.54 mg, 12.81 5 pmol, 1.02 eq) in DMSO (1 mL) was added DIEA (2.42 mg, 18.74 pmol, 3.3 pL, 1.5 eq). The mixture was stirred at 25-30 °C for 2 hr. LC-MS showed Compound 1 was consumed completely and one main peak with desired m / z [calculated MW: 2718.13, observed m / z: 906.45([M / 3+H]+) and 1359.50([M / 2+H]+)] was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was 10 purified by prep-HPLC (TFA condition). Compound 2 (16 mg, 5.80 pmol, 46.42% yield, 98.54% purity) was obtained as a white solid. Procedure for preparation of BCY10576 BCY00009594-PEG5-N3   +   BCY00011014 -2^24_VcNajmPTA_^ BCY00010576 t-BuOH / H2O 2 15 A mixture of compound 2 (17.0 mg, 6.25 pmol, 1.0 eq), BCY11014 (13.6 mg, 6.25 pmol, 1.0 eq), and THPTA (0.4 M, 1.8 pL, 2.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4(0.4 M, 15.6 pL, 1.0 eq) and 2024205105   25 Jul 2024 VcNa (0.4 M, 1.84 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed majority of compound 2 was consumed and one main peak with desired m / z 5 [calculated MW: 4893.63, observed m / z: 1224.7 ([M / 4+H]+) and 980.0 ([M / 6+H]+)] was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY10576 (20.5 mg, 4.13 pmol, 66.02% yield, 98.57% purity) was obtained as a white solid. BCY10577 10                                                                                          BCY00010577 Procedure for preparation of compound 2 BCY00009172 + N3-CH2-COOH ---------’-------► BCY00009172-CH2-N3 2.DIEA, DMF 1                                                  2 To a solution of compound 1 (5.0 mg, 49.5 pmol, 1.0 eq) in DMF (1 mL) was added EDCI (8.5 mg, 54.8 pmol, 1.1 eq) and HOSu (5.7 mg, 49.5 pmol, 1.0 eq). The mixture was stirred 15 at 25-30 °C for 30 min. TLC indicated compound 1 was consumed completely and one new spot formed. Then BCY9172 (53 mg, 25.29 pmol, 0.47eq) and DIEA (3.27 mg, 25.29 pmol, 4.4 pL, 0.47 eq) were added to the reaction mixture. The mixture was stirred at 25-30 °C for 2 hr. LC-MS showed BCY9172 was consumed completely and one main peak with desired m / z (MW: 2178.46, observed m / z: 1089.5700 ([(M / 2+H+])) was detected. The 20 reaction mixture was concentrated under reduced pressure to remove solvent and produced a residue. The residue was then purified by prep-HPLC (neutral condition). Compound 2 (30 mg, 13.77 pmol, 54.45% yield, 100% purity) was obtained as a white solid. Procedure for preparation of BCY10577 2024205105   25 Jul 2024 CuSO4 VcNa BCY00009172-CH2-N3    +   BCY00006169  ---THPTA—►    BCY00010577 2                                   t-BuOH / H2O Compound 2 (20 mg, 9.18 pmol, 1.0 eq) and BCY6169 (32.95 mg, 10.10 pmol, 1.1 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 23 pL, 1 eq), VcNa (0.4 M, 46 pL, 2.0 eq) and THPTA (0.4 M, 23 pL, 1.0 eq) was added. Finally 1 M NH4HCO3 was 5 added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 30 °C for 4 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 5441.20, observed m / z: 1361.8 ([M / 4+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was 10 purified by prep-HPLC (TFA condition). BCY10577 (16.2 mg, 2.98 pmol, 32.43% yield) was obtained as a white solid. Example 3: Synthesis of Nectin-4 / CD137 Binding Heterotandem Bicyclic Peptides BCY8854 General procedure for preparation of BCY8846 2024205105   25 Jul 2024 BCY00008234    ----------------►    BCY00008846 DEA, DMA To a solution of BCY8234 (a peptide identical to BCY8846 except for the absence of a PYA moiety; 300 mg, 102 pmol, 1.0 eq) in DMA (3 mL) was added DIEA (52.5 mg, 406 pmol, 70.8 pL, 4.0 eq) with stirring for 10 min. Then (2,5-dioxopyrrolidin-1-yl) pent-4-ynoate (25.8 5 mg, 132 pmol, 1.3 eq) was added thereto and the mixture was further stirred at 20 °C for additional 16 hr. LC-MS showed BCY8234 was consumed completely and one main peak with desired m / z (calculated MW: 3034.43, observed m / z: 1011.8 ([M / 3+H]+), 1517.0 ([M / 2+H]+)) was detected. The reaction mixture was purified by prep-HPLC (neutral condition) to give compound BCY8846 (290 mg, 95.6 pmol, 94.1% yield) as a white solid. 10 General procedure for preparation of BCY8854 BCY00007859 BCY00008846    ----------------►    BCY00008854 Vc, CuSO4, DMF, H2O To a solution of BCY8846 (234 mg, 77.1 pmol, 1.0 eq) in DMF (5 mL) was added BCY7859 (which may be prepared as described in BCY7985; 220 mg, 77.8 pmol, 1.0 eq), followed by 15 addition (2R)-2-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one (0.80 M, 963 pL, 1.0 eq) and CuSO4 (0.80 M, 289 pL, 0.3 eq). The mixture was stirred at 20 °C for 2 hr. LC-MS showed BCY8846 was consumed completely and one main peak with desired m / z (Calculated MW: 5861.59, observed m / z: 837.9 ([M / 7+H]+), 977.6 ([M / 6+H]+), 1173.3 ([M / 5+H]+)) was detected. The reaction mixture was purified by prep-HPLC (A: 0.075% TFA 20 in H2O, B: ACN) to give compound BCY8854 (292 mg, 46.8 pmol, 60.8% yield, 95.9% purity, TFA) as a white solid. BCY9350 2024205105   25 Jul 2024 To a solution of BCY8782 (a peptide identical to BCY11942 except for the absence of a PYA 5 moiety; 20.0 mg, 6.77 pmol, 1.0 eq) in DMA (1 mL) was added DIEA (4.37 mg, 33.9 pmol, 5.90 pL, 5.0 eq) and (2,5-dioxopyrrolidin-1-yl) pent-4-ynoate (2.64 mg, 13.5 pmol, 2.0 eq) with stirring for 12 hr at 25 °C. LC-MS showed BCY8782 was consumed completely and one main peak with desired m / z (calculated MW: 3034.43, observed m / z: 1012.1 [M / 3+H]+) was detected. The reaction mixture was purified by prep-HPLC (neutral condition) to give 10   BCY11942 (20.0 mg, 6.00 pmol, 88.6% yield, 91.0% purity) as a white solid. General procedure for preparation of BCY9350 BCY00011942 +  BCY00007859 Vc, CuSO4 DMF BCY00009350 To a solution of BCY11942 (20 mg, 6.59 pmol, 1.0 eq) and BCY7859 (which may be 15 prepared as described in BCY7985; 20.5 mg, 7.25 pmol, 1.1 eq) in DMF (1 mL) was added 2024205105   25 Jul 2024 (2R)-2-[(1S)-1,2-dihydroxyethyl]-3, 4-dihydroxy-2H-furan-5-one (0.4 M, 330 pL, 20.0 eq) and CuS04 (0.4 M, 98.9 pL, 6.0 eq) were added to the mixture. The mixture was stirred at 25 °C for 2 hr. LC-MS showed BCY8782-PYA was consumed completely and one main peak with desired m / z (calculated MW: 5861.59, observed m / z: 1173.3 [M / 5+H]+) was detected. 5 The reaction mixture was purified by prep-HPLC (A: 0.075% TFA in H2O, B: ACN) to give BCY9350 (14.5 mg, 2.40 pmol, 36.5% yield, 97.2% purity) as a white solid. BCY9351 10   General procedure for preparation of BCY9351 Vc, CuSO4 BCY00008940 +  BCY00008846  ----!------ DMF BCY00009351 To a solution of BCY8940 (which may be prepared as described in BCY8942; 9.4 mg, 3.33 pmol, 1.01 eq) and BCY8846 (10.0 mg, 3.30 pmol, 1.0 eq) in DMF (1 mL) was added Vc (0.4 M, 165 pL, 20.0 eq) and CUSO4 (0.4 M, 49.4 pL, 6.0 eq) under nitrogen atmosphere. 15 The mixture was stirred at 25 °C for 1 hr. LC-MS showed BCY8940 was consumed completely and one main peak with desired m / z (calculated MW: 5861.59, observed m / z: 975.4 [M / 6+H]+, 1172.3 [M / 5+H]+) was detected. The reaction mixture was purified by prep-HPLC (A: 0.075% TFA in H2O, B: ACN) to give BCY9351 (5.30 mg, 0.904 pmol, 26.3% yield, 96.0% purity) as a white solid. 2024205105   25 Jul 2024 BCY9399 COM00000134 1 5 To a solution of COM134 (30 mg, 56.97 pmol), Compound 1 (17.22 mg, 85.45 pmol) in DCM (0.5 mL) was added TEA (8.65 mg, 85.45 pmol, 11.9 pL). The mixture was stirred at 25 °C for 1 hr. LC-MS showed COM 134 was consumed completely and one main peak with desired m / z (calculated MW: 691.72, observed m / z\ 692.3([M+H]+) and 709.3 ([M+NH4]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove 10 solvent to give a residue. The residue was purified by prep-HPLC (neutral condition). Compound 2 (30.5 mg) was obtained as a colorless oil. Procedure for preparation of compound 3 2024205105   25 Jul 2024 BCY00008116 —[BCY00008116]-[COM00000134] DMF 3 To a solution of Compound 2 (15 mg, 21.68 pmol) and BCY8116 (47 mg, 21.68 pmol) in DMF (1 mL) was added DIEA (8.41 mg, 65.05 pmol, 11.33 pL). The mixture was stirred at 30 °C for 2 hrs. LC-MS showed Compound 2 was consumed completely and one main peak 5 with desired m / z (MW: 2725.1 observed m / z\ 1362.7([M / 2+H]+), 909.0([M / 3+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (TFA condition). Compound 3 (20 mg, 33.41% yield, 98.71% purity) was obtained as a white solid. 10 Procedure for preparation of BCY9399 [BCY00008116]-[COM00000134]   +   BCY00007741 CuS04 VcNa TH PT A   BCY00009399 t-BuOH / H2O 3 A mixture of Compound 3 (20.0 mg, 5.35 pmol, 1.0 eq), BCY7741 (13.0 mg, 5.70 pmol, 1.01 eq), and THPTA (0.4 M, 13.4 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 13.4 pL, 1.0 eq) and 15 VcNa (0.4 M, 26.8 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 3 was consumed completely and one main peak with desired m / z [MW: 5006.64 observed m / z: 834.9([M / 6+H]+), 1002.3([M / 5+H]+), 1252.4([M / 4+H]+)] was 20 detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY9399 (9.1 mg, 27.20% yield, 96.29% purity) was obtained as a white solid. BCY9400 2024205105   25 Jul 2024 Procedure for preparation of Compound 2 COM00000135 TEA DCM 5 To a solution of COM135 (which may be prepared as described in BCY9648; 30.0 mg, 27.29 pmol), Compound 1 (8.3 mg, 40.94 pmol) in DCM (2 mL) was added TEA (4.14 mg, 40.94 pmol, 5.7 pL). Then the reaction mixture was stirred at 25-30 °C for 1 hr. LC-MS showed COM 135 was consumed completely and one main peak with desired m / z (calculated MW: 1264.40, observed m / z: 1281.4 ([M+NH4D) was detected. The reaction mixture was 10 concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (neutral condition) to give compound 2 (18 mg) as a white solid. Procedure for preparation of Compound 3 BCY00008116 TEA DMF [BCY00008116]-[COM00000135] 3 2024205105   25 Jul 2024 To a solution of Compound 2 (15.5 mg, 7.12 pmol) and BCY8116 (9 mg, 7.12 pmol) in DMF (2 mL) was added DIEA (1.4 mg, 10.68 pmol, 1.9 pL). The mixture was stirred at 30 °C for 2 hrs. LC-MS showed Compound 2 was consumed completely and one main peak with desired m / z (MW: 3297.78, observed m / z: 1099.7([M / 3+H]+)) was detected. The reaction 5 mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (TFA condition). Compound 3 (19.5 mg, 5.91 pmol, 33.41% yield, 83.07% purity) was obtained as a white solid. Procedure for preparation of BCY9400 [BCY00008116]-[COM00000135]  +   BCY00007741 CuS04 VcNa THPTA BCY00009400 t-BuOH / H2O 10                      3 A mixture of Compound 3 (19.5 mg, 5.91 pmol), BCY7741 (14 mg, 6.14 pmol, 1.01eq), and THPTA (0.4 M, 15 pL, 1 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, pre-degassed and purged with N2 for 3 times), and then CuSO4(0.4 M, 15 pL, 1 eq) and VcNa (0.4 M, 30 pL, 2 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 15   0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 3 was consumed completely and one main peak with desired m / z [MW: 5579.31 observed m / z: 930.5([M / 6+H]+), 1116.6([M / 5+H]+)] was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY9400 (13.9 mg, 2.33 pmol, 27.20% yield, 20   93.56% purity) was obtained as a white solid. BCY9401 2024205105   25 Jul 2024 2 Exact Mass: 1591.85429 Molecular Weight: 1592.75832 Exact Mass: 200.98289 Molecular Weight: 201.56396 Exact Mass: 1756.86049 Molecular Weight: 1757.86134 3 COM00000470 To a solution of Compound 1 (50.0 mg, 31.39 pmol, 1 eq), Compound 2 (6.6 mg, 32.96 5 pmol, 1.05 eq) in DCM (2 mL) was added TEA (4.8 mg, 47.09 pmol, 6.6 pL, 1.5 eq). The mixture was stirred at 25-30 °C for 2 hr. LC-MS showed Compound 1 was consumed completely and one main peak with desired m / z (MW: 1757.86 observed m / z: 879.10([M / 2+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (TFA 10 condition). Compound 3 (0.02 g, 6.56 pmol, 20.91% yield, 57.7% purity) was obtained as a white solid. 2024205105   25 Jul 2024 Procedure for preparation of compound 4 3 To a solution of Compound 3 (20 mg, 11.38 pmol, 1 eq), BCY8116 (25 mg, 11.51 pmol, 1.01 5 eq) in DMF (4 mL) was added DIEA (2.2 mg, 17.07 pmol, 2.97 pL, 1.5 eq). The mixture was stirred at 25-30 °C for 12 hr. LC-MS showed Compound 3 was consumed completely and one main peak with desired m / z (MW: 3791.23, observed m / z: 1263.2([M / 3+H]+)) was detected. The reaction was directly purified by prep-HPLC (neutral condition). Compound 4 (10 mg, 2.43 pmol, 21.33% yield, 92% purity) was obtained as colorless oil. 10 Procedure for preparation of BCY9401 CuSO4,VcNa,THPTA [BCY00008116]-[COM00000122]  +  BCY00007741 ------------------► BCY00009401 t-BuOH / H2O 4 A mixture of Compound 4 (10 mg, 2.43 pmol, 0.9 eq), BCY7741 (6.32 mg, 2.77 pmol, 1.0 eq) and THPTA (0.4 M, 6.7 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, pre-15 degassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 6.7 pL, 1.0 eq) and VcNa (0.4 M, 13.4 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 4 was consumed completely and one main peak with desired m / z [MW: 20 MW: 6072.77, observed m / z: 1012.00([M / 6+H]+)] was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY9401 (8.4 mg, 1.56 pmol, 59.31% yield, 95.52% purity) was obtained as a white solid. BCY9403 2024205105   25 Jul 2024 To a solution of COM471 (100.0 mg, 76.42 pmol, 1.0 eq), 4-nitrophenylchloroformate (16.2 5 mg, 80.25 pmol, 1.05 eq) in DCM (10 mL) was added TEA (11.6 mg, 114.64 pmol, 16.0 pL, 1.5 eq). The mixture was stirred at 25-30 °C for 2 hr. LC-MS showed COM471 was consumed completely and one main peak with desired m / z (MW: 1473.58, observed m / z\ 736.83 ([M / 2+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (TFA 10 condition). Compound 2 (62.8 mg, 42.67 pmol, 55.84% yield, 48.37% purity) was obtained as a white oil. Procedure for preparation of Compound 3 2024205105   25 Jul 2024 2                                                                                                            3 To a solution of Compound 2 (44 mg, 29.46 pmol, 1.0 eq), BCY8116 (63 mg, 29.18 pmol, 1.0 eq) in DMF (2 mL) was added DIEA (5.66 mg, 43.77 pmol, 7.62 pL, 1.5 eq). The mixture was stirred at 40 °C for 12 hr. LC-MS showed Compound 2 was consumed completely and 5 one main peak with desired m / z (MW: 3506.95, observed m / z: 1168.58 ([M / 3+H]+))was detected. The residue was purified by prep-HPLC (TFA condition). Compound 3 (20 mg, 5.42 pmol, 18.57% yield, 95.04% purity) was obtained as a white solid. Procedure for preparation of BCY9403 [BCY00008116]-[COM00000471]  +   BCY00007741 CuSO4 VcNa THPTA   BCY00009403 t-BuOH / H2O 10                     3 A mixture of Compound 3 (10.0 mg, 2.71 pmol, 1.0 eq), BCY7741 (6.83 mg, 2.99 pmol, 1.1 eq), and THPTA (0.4 M, 7 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4(0.4 M, 7 pL, 1.0 eq) and VcNa (0.4 M, 14 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by 15 dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 3 was consumed completely and one main peak with desired m / z [MW: 5788.49, observed m / z: 1157.00 ([M / 5+H]+) and 964.60 ([M / 6+H]+)] was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY9403 (2.1 mg, 0.34 20 pmol, 11.93% yield, 93.80% purity) was obtained as a white solid. BCY9405 2024205105   25 Jul 2024 1                                                                2 To a solution of COM472 (44.7 mg, 38.1 pmol), Compound 1 (9.2 mg, 45.72 pmol) in DCM 5   (4 mL) was added TEA (5.8 mg, 57.14 pmol, 8 pL). The mixture was stirred at 25 °C for 2 hr. LC-MS showed COM472 was consumed completely and one main peak with desired m / z (MW: 1338.45, observed m / z: 686.23([M / 2+NH4+])) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (neutral condition). Compound 2 (20 mg, 14.94 pmol, 39.2% yield) 10 was obtained as colorless oil. Procedure for preparation of Compound 3 2024205105   25 Jul 2024 O N N JU      In J     Jk N-»                                   DIEA [f'Y Y if        YY °1         +    BCY00008116     ---------►    [BCY00008116]-[COM00000472] |l J n n LI z-J 4 I-          J15 02N>^  0     0        04                                 dmf 2                                                                                                          3 To a solution of Compound 2 (20 mg, 14.94 pmol) and BCY8116 (38.96 mg, 17.93 pmol) in DMF (4 mL) was added DIEA (1.9 mg, 14.94 pmol, 2.6 pL). The mixture was stirred at 30 °C for 2 hrs. LC-MS showed Compound 2 was consumed completely and one main peak with 5 desired m / z (MW: 3371.82, observed m / z: 1123.94([M / 3+H+])) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (TFA condition). Compound 3 (10 mg, 99.07% yield, 19.66 purity) was obtained as a white solid. 10 Procedure for preparation of BCY9405 [BCY00008116]-[COM00000472]    +   BCY00007741 CuS04 VcNa THPTA „  BCY00009405 t-BuOH / H2O 3 A mixture of Compound 3 (10.0 mg, 2.97 pmol, 1.0 eq), BCY7741 (7.4 mg, 3.26 pmol, 1.1 eq), and THPTA (1.3 mg, 2.97 pmol, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4(0.4 M, 7.5 pL, 1.0 eq) and VcNa 15   (0.4 M, 151 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 3 was consumed completely and one main peak with desired m / z [MW: 5653.36, observed m / z: 1130.47 ([M / 5+H]+)] was detected. The reaction mixture was directly 20 purified by prep-HPLC (TFA condition). BCY9405 (7.8 mg, 46.08% yield, 97.8% purity) was obtained as a white solid. BCY9406 2024205105   25 Jul 2024 To a solution of COM473 (130.0 mg, 177.40 pmol, 1.0 eq), (4-nitrophenyl) carbonochloridate 5   (36.4 mg, 180.59 pmol, 1.02 eq) in DCM (3 mL) was added TEA (27.0 mg, 266.09 pmol, 37 pL, 1.5 eq). The mixture was stirred at 35 °C for 2 hr. LC-MS showed COM473 was consumed completely and one main peak with desired m / z (MW: 897.93, observed m / z\ 897.65([M+H]+), 914.60([M+NH4]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by 2024205105   25 Jul 2024 prep-HPLC (TFA condition). Compound 2 (90 mg, 95.87 pmol, 54.04% yield, 95.65% purity) was obtained as colorless oil. Procedure for preparation of Compound 3 Xi A — JU iNj N                  diea - TN—''Iflt              3 +     BCY00008116      -------------•-     [BCY00008116]-[CC>M00000473] H       I o                                             DMF 5                             2                                                                                 3 To a solution of Compound 2 (10 mg, 11.14 pmol, 1 eq), BCY8116 (25 mg, 11.51 pmol, 1.03 eq) in DMF (2 mL) was added DIEA (2.16 mg, 16.71 pmol, 2.91 pL, 1.5 eq). The mixture was stirred at 25-30 °C for 12 hr. LC-MS showed one main peak with desired m / z (MW:2931.30, observed m / z: 977.00([M / 3+H]+)) was detected. The reaction mixture was 10 filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (FTA condition). Compound 3 (15 mg, 5.12 pmol, 45.79% yield, 99.66% purity) was obtained as a white solid. Procedure for preparation of BCY9406 [BCY00008116]-[COM00000473]  +   BCY00007741 CuSO4 VcNa THPTA   BCY00009406 t-BuOH / H2O 15                   3 A mixture of Compound 3 (15 mg, 5.12 pmol, 1.0 eq), BCY7741 (12 mg, 5.26 pmol, 1.03 eq), and THPTA (0.4 M, 12.8 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 12.8 pL, 1.0 eq) and VcNa (0.4 M, 25.6 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 20   8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 3 was consumed completely and one main peak with desired m / z [MW: 5212.84 observed m / z: 1042.74 ([M / 4+H]+)] was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY9406 (14.4 mg, 2.57 pmol, 50.21% 25 yield, 93.01% purity) was obtained as a white solid. BCY9407 2024205105   25 Jul 2024 BCY00009407 Procedure for preparation of Compound 2 1 2 To a solution of COM128 (60 mg, 50.53 pmol, 1.0 eq), compound 1 (13 mg, 64.50 pmol, 5   1.28 eq), DIEA (9.80 mg, 75.80 pmol, 13.20 pL, 1.5 eq) in DCM (5 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 25-30 °C for 1 hr under N2 atmosphere. LC-MS showed COM128 was consumed completely and one main peak with desired m / z (calculated MW: 1352.48, observed m / z: Q7Q.7 ([M / 2+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a 10 residue. The residue was purified by prep-HPLC (TFA condition). Compound 2 (12 mg, 8.87 pmol, 17.56% yield) was obtained as colorless oil. Procedure for preparation of [BCY8116]-[COM 128] BCY00008116 TEA DMF [BCY00008116]-[COM00000128] 2024205105   25 Jul 2024 To a solution of compound 2 (7 mg, 5.18 pmol, 1.0 eq) and BCY8116 (11 mg, 5.06 pmol, 1.0 eq), DIEA(2.01 mg, 15.53 pmol, 2.70 pL, 3.0 eq) in DMF (3 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 25-30 °C for 1 hr under N2 atmosphere. LC-MS showed one main peak with desired m / z (calculated MW: 3385.85, 5 observed m / z: 1129.3 ([M / 3+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (TFA condition). [BCY8116]-[COM128] (15.6 mg, 4.46 pmol, 86.13% yield, 96.75% purity) was obtained as a white solid 10 Procedure for preparation of BCY9407 [BCY00008116]-[COM00000128]   +   BCY00007741 CuS04 VcNa THPTA» BCY00009407 t-BuOH / H2O A mixture of [BCY8116]-[COM128] (15.6 mg, 4.61 pmol, 1.0 eq), BCY7741 (11 mg, 4.82 pmol, 1.05 eq), and THPTA (0.8 M, 5.8 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, pre-degassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 11.6 pL, 1.0 eq) and 15 VcNa (0.4 M, 23.2 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed one peak with desired m / z (calculated MW: 5667.39, observed m / z: 945.6 ([M / 6+H]+) and 1134.2 ([M / 5+H]+)) was detected. The reaction mixture was directly purified 20 by prep-HPLC (TFA condition). BCY9407 (1.3 mg, 0.23 pmol, 4.33% yield, 86.90% purity) was obtained as a white solid. BCY9408 2024205105   25 Jul 2024 1 To a solution of COM129 (45.0 mg, 34.39 pmol, 1.0 eq), compound 1 (15.0 mg, 74.42 pmol, 5   2.1 eq) in DCM (5 mL) was added TEA (5.5 mg, 53.88 pmol, 7.5 pL, 1.5 eq), and then the mixture was stirred at 25-30 °C for 1 hr under N2 atmosphere. LC-MS showed COM 129 was consumed completely and one main peak with desired m / z (MW: 1473.58, observed m / z\ 737.3 ([M / 2+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (TFA 10 condition). Compound 2 (9 mg, 6.11 pmol, 17.01% yield, 95.76% purity) was obtained as a white solid. 2024205105   25 Jul 2024 Procedure for preparation of Compound 3 ON       O JU N N BCY00008116----—---[BCY00008116]-[COM00000129] JI o                     । O                                              DMF O2N 2                                                                               3 To a solution of compound 2 (9.0 mg, 6.11 pmol, 1.0 eq) and BCY8116 (13.3 mg, 6.11 pmol, 1.0 eq) in DMF (3 mL) was added DIEA (2.4 mg, 18.32 pmol, 3.2 pL, 3.0 eq). All solvents 5 were degassed and purged with N2 for 3 times, and then the mixture was stirred at 25-30 °C for 1 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (MW: 3506.95, observed m / z: 877.4([M / 4+H]+) and m / z: 1169.6([M / 3+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase 10 HPLC (TFA condition). Compound 3 (7.2 mg, 2.05 pmol, 31.93% yield, 95% purity) was obtained as a white solid. Procedure for preparation of BCY9408 [BCY00008116]-[COM00000129] +   BCY00007741                           BCY00009408 t-BuOH / H2O 3 15 A mixture of Compound 3 (7.2 mg, 2.05 pmol, 1.0 eq), BCY7741 (5.0 mg, 2.19 pmol, 1.03 eq), and THPTA (0.4 M, 5.1 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4(0.4 M, 5.1 pL, 1.0 eq) and VcNa (0.4 M, 10.2 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light 20 yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 3 was consumed completely and one main peak with desired m / z [MW: 5788.49 observed m / z: 968.9([M / 6+H]+) and 1158.0([M / 5+H]+)] was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY9408 (3.1 mg, 4.97e-1 pmol, 24.23% yield, 92.87% purity) was obtained as a white solid. 25 BCY9409 2024205105   25 Jul 2024 1 2 To a solution of compound 1 (30 mg, 20.78 pmol), COM130 (6.28 mg, 31.17 pmol) in DCM 5   (3 mL) was added TEA (3.15 mg, 31.17 pmol, 4.34 pL, 1.5 eq). The mixture was stirred at 25-30°C for 1 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired m / z (MW: 1608.70 observed m / z: 804.8 ([M / 2+H]+) was detected. The reaction mixture was concentrated under reduced pressure and then lyophilized to give Compound 2 (10.2 mg, crude) as a white solid. 10 Procedure for preparation of Compound 3 BCY00008116 TEA DMF [BCY00008116]-[COM00000130] 3 To a solution of compound 2 (10.2 mg, 6.34 pmol) and BCY8116 (13.50 mg, 6.22 pmol) in DMF (2 mL) was added DIEA (0.8 mg, 6.22 pmol, 1.1 pL, 1.0 eq).The mixture was stirred at 15   30 °C for 2 hr. LC-MS detected desired m / z (MW: 3642.08, observed m / z: 1214.4([M / 3+H]+). The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (TFA condition). Compound 3 (15.0 mg, 4.12 pmol, 62.94% yield, 95% purity) was obtained as a white solid. 20 Procedure for preparation of BCY9409 [BCY00008116]-[COM00000130]    +   BCY00007741 CuSO4 VcNa THPTA BCY00009409 t-BuOH / H2O A mixture of Compound 3 (15 mg, 4.12 pmol, 1.0 eq), BCY7741 (10 mg, 4.38 pmol, 1.03 eq), and THPTA (0.4 M, 10.3 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 10.3 pL, 1.0 eq) and 2024205105   25 Jul 2024 VcNa (0.4 M, 20.6 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 3 was consumed completely and one main peak with desired m / z 5 [MW: 5923.61, observed m / z: 988.2([M / 6+H]+)] was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY9409 (3.1 mg, 0.52 pmol, 12.62% yield, 90.89% purity) was obtained as a white solid. BCY9410 Procedure for preparation of Compound 2 COM00000131 1 2 To a solution of COM131 (167.0 mg, 227.89 pmol, 1.0 eq), compound 1 (55.0 mg, 272.87 15 pmol, 1.2 eq) in DCM (5 mL) was added TEA (36.4 mg, 359.23 pmol, 50.0 pL, 1.6 eq). The mixture was stirred at 25-30 °C for 1 hr. LC-MS showed one main peak with desired m / z (MW: 897.93 observed 920.3([M+Na+]) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (TFA condition). Compound 2 (35 mg, 33.74 pmol, 14.81% yield, 20   86.56% purity) was obtained as colorless oil. Procedure for preparation of Compound 3 2 BCY00008116 TEA DMF [BCY00008116]-[COM00000131] To a solution of compound 2 (20 mg, 22.27 pmol, 1.0 eq) and BCY8116 (48 mg, 22.09 pmol, 25   1.0 eq) in DMF (2 mL) was added DIEA (8.64 mg, 66.82 pmol, 11.64 pL, 3.0 eq). The mixture was stirred at 30 °C for 2 hr. LC-MS showed compound 3 was consumed completely 2024205105   25 Jul 2024 and one main peak with desired m / z (MW: 2931.32, observed m / z\ 977.7([M+H]+))was detected. The residue was purified by prep-HPLC (TFA condition). Compound 3 (40 mg, 13.08 pmol, 58.7% yield, 95.82% purity) was obtained as a white solid. 5   Procedure for preparation of BCY9410 [BCY00008116]-[COM00000131]   +   BCY00007741 CuS04 VcNa THPTA BCY00009410 t-BuOH / H2O 3 A mixture of Compound 3 (40 mg, 13.08 pmol, 1.0 eq), BCY7741 (35 mg, 15.34 pmol, 1.17 eq), and THPTA (0.4 M, 34 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuSO4(0.4 M, 34 pL, 1.0 eq) and VcNa 10   (0.4 M, 68 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O) and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 3 was consumed completely and one main peak with desired m / z [MW: 5212.85, observed m / z: 1043.2 ([M / 5+H]+)] was detected. The reaction mixture was directly 15 purified by prep-HPLC (TFA condition). BCY9410 (38.6 mg, 6.78 pmol, 49.71% yield, 91.6% purity) was obtained as a white solid. 2024205105   25 Jul 2024 Procedure for preparation of Compound 2 1 TEA DCM 2 COM00000132 To a solution of COM132 (5 mg, 16.32 pmol, 1 eq), Compound 1 (4 mg, 19.85 pmol, 1.22 eq) in DCM (5 mL) was added TEA (2.8 mg, 24.48 pmol, 3.4 pL, 1.5 eq). The mixture was 5 stirred at 25 °C for 1 hr. LC-MS showed one peak with desired m / z (calculated MW: 471.46, observed m / z: 489.2([M+NH4]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent, and then lyophilized to give compound 2 (8 mg, crude) as a white solid. 10   Procedure for preparation of Compound 3 °2Ny^ ° k X Jk                    +     BCY00008116    _____DIEA____[BCY00008116]-[COM00000132] O N1         N3                             DMF rl 2                                                                                       3 To a solution of Compound 2 (3.3 mg, 6.9 pmol, 1.5 eq) and BCY8116 (10.0 mg, 4.6 pmol, 1.0 eq) in DMF (5 mL) was added DIEA (0.7 mg, 6.90 pmol, 1 pL, 1.5 eq). The mixture was stirred at 30 °C for 2 hrs. LC-MS showed Compound 2 was consumed completely and one 15 main peak with desired m / z (calculated MW: 2504.83, observed m / z: 1252.3([M / 2+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (TFA condition). Compound 3 (4.2 mg, 1.51 pmol, 32.78% yield, 90% purity) was obtained as a white solid. 20   Procedure for preparation of BCY9411 [BCY00008116]-[COM00000132]   +   BCY00007741 CuSO4 VcNa THPTA » BCYoooo9411 t-BuOH / H2O 3 A mixture of Compound 3 (4.2 mg, 1.68 pmol, 1.0 eq), BCY7741 (4.0 mg, 1.75 pmol, 1.05 eq), and THPTA (0.04 M, 84 pL, 2.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, predegassed and purged with N2 for 3 times), and then CuS04(0.04 M, 84 pL, 2.0 eq) and 25 VcNa (0.04 M, 168 pL, 4.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 3 was consumed completely and one main peak with desired m / z [MW: 4786.37 observed m / z: 1596.2([M / 3+H]+), 1196.9([M / 4+H]+)] was detected. The 2024205105   25 Jul 2024 reaction mixture was directly purified by prep-HPLC (TFA condition). BCY9411 (4.1 mg, 0.86 pmol, 50.20% yield, 98.26% purity) was obtained as a white solid. Procedure for preparation of Compound 2 BCY00008116 2.0 eq DI PEA Dry DMF * BCY00008116-PEG24-NHS ester To a solution of compound 1 (5.0 mg, 3.54 pmol, 1.0 eq), BCY8116 (7.7 mg, 3.54 pmol, 1.0 10 eq) in DMF (3 mL) was added DIEA (0.9 mg, 7.07 pmol, 1.2 pL, 2.0 eq). The mixture was stirred at 0 °C for 20 min. LC-MS detected mass corresponding to compound 2 with NHS group falling off (calculated MW: 3470.95, hydrolyzed MW: 3373.81, observed m / z: 1125.0([M / 3+H]+). The reaction mixture was filtered and concentrated under reduced pressure and lyophilized to give compound 2 (15 mg, crude) was obtained as a white solid. Procedure for preparation of BCY9759 2024205105   25 Jul 2024 2 0 ea DIPEA BCY00008116-PEG24-NHS ester +   BCY00007732    —:►    BCY00009759 Dry DMF 2 To a solution of compound 2 (20 mg, 5.76 pmol, 1.0 eq) and BCY7732 (12.7 mg, 5.76 pmol, 1.0 eq) in DMF (3 mL) was added DIEA (1.5 mg, 11.52 pmol, 2.0 pL, 2.0 eq). The mixture was stirred at 25-30 °C for 2 hrs. LC-MS showed compound 2 was consumed completely 5 and one main peak with desired m / z (MW: 5557.3, observed m / z\ 927.0 ([M / 6+H]+) and 1112.2([M / 5+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (TFA condition). BCY9759 (2.3 mg, 6.92% yield, 96.29% purity) was obtained as a white solid. .0 BCY10000 2024205105   25 Jul 2024 BCY00010000 Procedure for preparation of BCY9172-PEG12-N3 DEA BCY00009172 + NHS-PEG12-N3 ----------------► BCY00009172-PEG12-N, DMF 1                                                            2 BCY9172 (520 mg, 248.16 pmol, 1.0 eq) and compound 1 (370 mg, 499.47 pmol, 2.01 eq) 5 were dissolved in DMF (5 mL), then the mixture was added with DIEA (48.11 mg, 372.24 pmol, 64.84 pL, 1.5 eq) and stirred at 30°C for 12 hr. LC-MS showed BCY9172 was consumed completely and one main peak with desired m / z (calculated MW: 2721.12, observed m / z: 1360.9 ([M / 2+H]+)) was detected. The reaction mixture was purified by prep-HPLC (TFA condition) and compound 2 (284 mg, 101.10 pmol, 40.74% yield, 96.87% purity) 10 was obtained as a white solid. 2024205105   25 Jul 2024 Procedure for preparation of BCY10000 CuSO4 VcNa BCY00009172-PEG12-N3 +   BCY00008846  ---THPTA—►    BCY00010000 t-BuOH / H2O 2 This reaction was performed in two independent containers in parallel. For one container, 5 Compound 2 (142 mg, 52.18 pmol, 1.0 eq) and BCY8846 (157 mg, 51.74 pmol, 1.0 eq) were first dissolved in 10 mL of t-BuOH / H2O (1:1), and then CuSO4 (0.4 M, 130.5 pL, 1.0 eq), VcNa (0.4 M, 261.0 pL, 2.0 eq) and THPTA (0.4 M, 130.5 pL, 1.0 eq) were added. Finally 1 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 30 °C for 12 hr under N2 atmosphere. LC-10 MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 5755.54, observed m / z: 959.60 ([M / 6+H]+) and 1151.55 ([M / 5+H]-)) was detected. The reaction mixture was purified by prep-HPLC (TFA condition) and BCY10000 (314.9 mg, 51.99 pmol, 49.82% yield, 95.03% purity) was obtained as a white solid. 15   BCY10567 2024205105   25 Jul 2024 BCY00010567 Procedure for preparation of BCY8919-PEG12-N3 DIEA BCY00008919 + NHS-PEG12-N3 ----------------►  BCY00008919-PEG12-N3 DMSO 1                                                           2 BCY8919 (60.0 mg, 28.85 pmol, 1.0 eq) and compound 1 (22.2 mg, 30.01 pmol, 1.04 eq) 5 were dissolved in DMSO (1 mL). The solution was added with DIPEA (5.6 mg, 43.28 pmol, 7.6 pl, 1.5 eq), and then the mixture was stirred at 25-30°C for 2 hr. LC-MS showed BCY8919 was consumed completely and one main peak with desired m / z (calculated MW: 2705.16, observed m / z: 1353.15([M / 2+H]+)) was detected. The reaction mixture was purified by prep-HPLC (TFA condition) and compound 2 (BCY8919-PEG12-N3, 18.5 mg, 6.77 pmol, 10   23.47% yield, 99.04% purity) was obtained as a white solid. 2024205105   25 Jul 2024 Procedure for preparation of BCY10567 CuSO4 VcNa BCY00008919-PEG12-N3  +    BCY00008846  ---THPTA—►    BCY00010567 t-BuOH / H2O 2 Note: This reaction has been performed twice, and the first one is described below. Compound 2 (9.0 mg, 3.33 pmol, 1.0 eq) and BCY8846 (10.1 mg, 3.33 pmol, 1.0 eq) were 5 first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 8.3 pL, 1.0 eq), VcN (1.3 mg, 6.56 pmol, 2.0 eq) and THPTA (1.4 mg, 3.22 pmol, 1.0 eq) were added. Finally 0.4 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 30°C for 16 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z 10 (calculated MW: 5739.58, observed m / z: 956.75([M / 6+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY10567 (6.85 mg, 1.18 pmol, 35.48% yield, 98.91% purity) was obtained as a white solid. BCY10569 2024205105   25 Jul 2024 BCY00010569 Procedure for preparation of Compound 3 DIEA BCY00008920    + NHS-PEG12-N3 -----—---►   BCY00008920-PEG12-N3 2                                                3 A mixture of compound BCY8920 (40.0 mg, 18.71 pmol, 1.0 eq.), compound 2 (16.0 mg, 5   21.6 pmol, 1.15 eq.) and DIEA (5.0 pL, 28.0 pmol, 1.5 eq.) was dissolved in DMF. The reaction mixture was stirred at 40 °C for 1 hr, till LC-MS showed one main peak with desired m / z (calculated MW:2763.2, observed m / z: 912.17([(M-28) / 2+H]+) was detected. The reaction mixture was then concentrated under reduced pressure to remove solvent and 2024205105   25 Jul 2024 produced a residue, following by purification by prep-HPLC (TFA condition). Compound 3 (23.4 mg, 8.47 pmol, 45.25% yield, 99.0% purity) was obtained as a white solid. Procedure for preparation of BCY10569 5 C11SO4 VcNa THPTA BCY00008920-NHS-PEG12-N3  +   BCY00008846   ---------------------►   BCY00010569 ,                                       DMF A mixture of compound 3 (5.0 mg, 1.81 pmol, 1.0 eq.), BCY8846 (5.8 mg, 1.9 pmol, 1.05 eq.), and THPTA (1.0 mg, 2.3 pmol, 1.3 eq.) was dissolved in t-BuOH / H2O (1:1, 1 mL, predegassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 5.0 pL, 1.0 eq.) and 10 VcNa (0.4 M, 5.0 pL, 1.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 40 °C for 2 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m / z (calculated MW: 5797.62, observed m / z: 1160.7 ([M / 5+H]+) was detected. The reaction 15 mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (TFA condition), and BCY10569 (5.7 mg, 1.18 pmol, 52.25% yield, 96.16% purity) was obtained as a white solid. BCY10571 0    0 BCY00010571 Procedure for preparation of BCY8116-PEG5-N3 2024205105   25 Jul 2024 DIEA BCY00008116 + NHS-PEG5-N3 ----------------►   BCY00008116-PEG5-N3 DMSO 1                                                            2 BCY8116 (60 mg, 27.62 pmol, 1.0 eq) and compound 1 (12.0 mg, 27.75 pmol, 1.0 eq) were first dissolved in DMSO (1 mL), then the mixture was added with DIEA (5.4 mg, 41.43 pmol, 7.22 pL, 1.5 eq). The mixture was stirred at 30 °C for 12 hr. LC-MS showed one main peak 5 with desired m / z (MW: 2489.82. observed m / z: 1245.1700 ([M / 2+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (TFA condition). Compound 2 (48 mg, 19.28 pmol, 69.80% yield, 100% purity) was obtained as a white solid. 10 Procedure for preparation of BCY10571 CuSO4 VcNa BCY00008116-PEG5-N3   +    BCY00008927 ---THPTA—►    BCY00010571 t-BuOH / H2O 2 This reaction was performed in two independent containers in parallel. For one container, Compound 2 (10 mg, 4.02 pmol, 1.0 eq) and BCY8927 (9 mg, 4.17 pmol, 1.04 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 10.0 pL, 1.0 eq), VcNa (0.4 15 M, 20.1 pL, 2.0 eq) and THPTA (0.4 M, 10.0 pL, 1 eq) were added. Finally 0.4 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 30 °C for 4 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m / z (MW: 4649.36, observed m / z: 1162.57 ([M / 4+H]+), 1549.69 ([M / 3+H]+)) was detected. The residue 20 was purified by prep-HPLC (TFA condition). BCY10571 (13 mg, 2.79 pmol, 34.88% yield, 96.48% purity) was obtained as a white solid. BCY10572 2024205105   25 Jul 2024 '2 BCY00010572 Procedure for preparation of BCY8116-PEG5-N3 DIEA BCY00008116 + NHS-PEG5-N3 DMSO BCY00008116-PEG5-N3 BCY8116 (60 mg, 27.62 pmol, 1.0 eq) and compound 1 (12.0 mg, 27.75 pmol, 1.0 eq) were 5 first dissolved in DMSO (1 mL), then the mixture was added with DIEA (5.4 mg, 41.43 pmol, 7.22 pL, 1.5 eq). The mixture was stirred at 30 °C for 12 hr. LC-MS showed one main peak with desired m / z (MW: 2489.82. observed m / z: 1245.1700 ([M / 2+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (TFA condition). Compound 2 (48 mg, 10   19.28 pmol, 69.80% yield, 100% purity) was obtained as a white solid. Procedure for preparation of BCY10572 C11SO4 VcNa BCY00008116-PEG5-N3   +    BCY00008928 ---THPTA—►    BCY00010572 t-BuOH / H2O 2 This reaction was performed in two independent containers in parallel. For one container, 15 Compound 2 (10 mg, 4.02 pmol, 1.0 eq) and BCY8928 (9 mg, 4.06 pmol, 1.01 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 10.1 pL, 1 eq), VcNa (0.4 M, 20.2 pL, 2.0 eq) and THPTA (0.4 M, 10.1 pL, 1.0 eq) was added. Finally 0.4 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 2024205105   25 Jul 2024 times. The reaction mixture was stirred at 30 °C for 4 hr under N2 atmosphere. LC-MS showed compound 1 was consumed completely and one main peak with desired m / z (MW: 4707.40. observed m / z: 1568.29 ([M / 3+H]+) and 1176.83 ([M / 4+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a 5 residue. The residue was purified by prep-HPLC (TFA condition). BCY10572 (21 mg, 4.46 pmol, 55.7% yield, 97.51% purity) was obtained as a white solid. BCY10573 BCY00010573 10   Procedure for preparation of Compound 2 BCY00008116 + NHS-PEG5-N3 1 DIEA DMSO BCY00008116-PEG5-N3 2 To a solution of BCY8116 (35 mg, 16.11 pmol, 1 eq), Compound 1 (7.00 mg, 16.19 pmol, 1 eq) in DMSO (1 mL) was added DIEA (3.12 mg, 24.17 pmol, 4.21 pL, 1.5 eq). The mixture was stirred at 25-30 °C for 2 hr. LC-MS showed majority of BCY8116 was consumed and 15 one main peak with desired m / z (calculated MW: 2489.82, observed m / z: 1245.37 ([M / 2+H]+) and 830.25([M / 3+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (TFA condition). Compound 2 (26.8 mg, 10.76 pmol, 66.81% yield, 100% purity) was obtained as a white solid. 2024205105   25 Jul 2024 Procedure for preparation of BCY10573 BCYOOOO8H6-PEG5-N3 +   BCY00011014 CuS°4 VcNa THPTA „    BCY00010573 t-BuOH / H2O 2 A mixture of Compound 2 (15 mg, 6.02 pmol, 1.0 eq), BCY11014 (13.50 mg, 6.21 pmol, 1.03 eq), and THPTA (0.4 M, 15.1 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, pre-5 degassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 15.1 pL, 1.0 eq) and VcNa (0.4 M, 30.2 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 2 was consumed completely and one main peak with desired 10 m / z [MW: 4665.32, observed m / z: 1167.50 ([M / 4+H+])] was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY10573 (11.5 mg, 2.42 pmol, 40.14% yield, 98.11% purity) was obtained as a white solid. BCY10578 2024205105   25 Jul 2024 BCY00010578 Procedure for preparation of Compound 2 ,n.n KI                   1« EDOI, HOSlJ BCY00009172 + N3-CH2-COOH ---------’-------► BCY00009172-CH2-N3 2.DIEA, DMF 1                                                  2 Compound 1 (5.0 mg, 49.5 pmol, 1.0 eq) was first activated by mixing with EDCI (8.5 mg, 5   54.8 pmol, 1.1 eq) and HOSu (5.7 mg, 49.5 pmol, 1.0 eq). The mixture was stirred at 25-30 °C for 30 min. TLC indicated compound 1 was consumed completely and one new spot formed. Then compound BCY9172 (80.0 mg, 38.18 pmol, 0.8 eq.) and DIEA (6.3 mg, 8.5 pL, 49.5 pmol, 1.0 eq.) were added to this mixture, and stirred at 40 °C for 1 hr, till LC-MS showed one main peak with desired m / z (calculated MW:2178.46, observed m / z: 1089.44 10   ([M / 2+H]+) was detected. The reaction mixture was then concentrated under reduced pressure to remove solvent and produced a residue, following by purification by prep-HPLC 2024205105   25 Jul 2024 (TFA condition). Compound 2 (15 mg, 6.88 pmol, 18.66% yield, 73.3% purity) was obtained as a white solid. Procedure for preparation of BCY10578 C11SO4 VcNa THPTA BCY00009172-CH2-N3   +   BCY00008846   ---------------------►   BCY00010578 ,                                      DMF 5                   2 A mixture of compound 2 (9.8 mg, 4.5 pmol, 1.0 eq.), BCY8846 (14.0 mg, 4.6 pmol, 1.0 eq.), and THPTA (2.0 mg, 4.6 pmol 1.0 eq.) was dissolved in t-BuOH / H2O (1:1, 1 mL, predegassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 12 pL, 1.0 eq.) and VcNa (0.4 M, 24 pL, 2.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by 10 dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 40 °C for 2 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 5212.88, observed m / z: 1304.2 ([M / 4+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude 15 product was purified by prep-HPLC (TFA condition), and BCY10578 (13.78 mg, 2.64 pmol, 58.66% yield, 96.23% purity) was obtained as a white solid. BCY10917 2024205105   25 Jul 2024 Procedure for preparation of BCY8831-PEG12-N3 DIEA BCY00008831 + NHS-PEG12-N3 DMF BCY00008831-PEG12-N3 2 BCY8831 (40.0 mg, 13.29 pmol, 1.0 eq) and compound 1 (10.5 mg, 14.17 pmol, 1.07 eq) 5 were dissolved in DMF (1 mL). The solution was added with DIPEA (2.6 mg, 20.09 pmol, 3.5 pl, 1.5 eq), and then the mixture was stirred at 30°C for 16 hr. LC-MS showed BCY8831 was consumed completely and one main peak with desired m / z (calculated MW: 3635.16 observed m / z: 1212.0([M / 3+H]+)) was detected. The reaction mixture was purified by prep-HPLC (TFA condition) and compound 2 (22.0 mg, 5.83 pmol, 43.85% yield, 96.39% purity) 10 was obtained as a white solid. 2024205105   25 Jul 2024 Procedure for preparation of BCY10917 CuSO4 VcNa BCY00008831-PEG12-N3 +    BCY00011014 ---THPTA—►    BCY00010917 t-BuOH / H2O 2 Note: Two batches were made, and the first one was written for final report. Compound 2 (10.0 mg, 2.75 pmol, 1.0 eq) and BCY11014 (5.98 mg, 2.75 pmol, 1.0 eq) , 5 were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 13.7 pL, 2.0 eq), VcNa (1.1 mg, 5.55 pmol, 2.0 eq) and THPTA (1.2 mg, 2.76 pmol, 1.0 eq) was added. Finally 1 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 30°C for 16 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak 10 with desired m / z (calculated MW: 5810.66 observed m / z: 1163.0([M / 5+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY10917 (6.4 mg, 1.07 pmol, 39.03% yield, 97.49% purity) was obtained as a white solid. BCY11020 2024205105   25 Jul 2024 Procedure for preparation of BCY8831-PEG5-N3 DIEA DMF BCY00008831 + NHS-PEG5-N3 1 BCY00008831 -PEG5-N3 2 BCY8831 (25.0 mg, 8.31 pmol, 1.0 eq) and compound 1 (3.9 mg, 9.02 pmol, 1.09 eq), were 5 dissolved in DMF (1 mL). The solution was added with DIPEA (1.6 mg, 12.46 pmol, 2.2 pl, 1.5 eq), and then the mixture was stirred at 35°C for 2 hr. LC-MS showed BCY8831 was consumed completely and one main peak with desired m / z (calculated MW: 3326.79 observed m / z: 1109.66([M / 3+H]+)) was detected. The reaction mixture was purified by prep-HPLC (TFA condition) and compound 2 (7.3 mg, 2.09 pmol, 25.20% yield, 95.41% purity) 10 was obtained as a white solid. Procedure for preparation of BCY11020 2024205105   25 Jul 2024 CuSO4 VcNa BCY00008831-PEG5-N3   +    BCY00011014 ---THPTA—►    BCY00011020 t-BuOH / H2O 2 Compound 2 (7.3 mg, 2.19 pmol, 1.0 eq) and BCY11014 (4.8 mg, 2.19 pmol, 1.0 eq) , were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 5.5 pL, 1.0 eq), VcNa (1.0 mg, 5.05 pmol, 2.3 eq) and THPTA (1.0 mg, 2.30 pmol, 1.0 eq) was added. 5 Finally 1 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 30°C for 12 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 5502.29, observed m / z: 1101.74([M / 5+H]+)) was detected. The reaction mixture was purified by prep-HPLC (TFA condition) and BCY11020 (3.3 mg, 10   0.577 pmol, 26.30% yield, 96.24% purity) was obtained as a white solid. BCY00011373 Procedure for preparation of Compound 2 2024205105   25 Jul 2024 m zxU rnnu 1.EDCI, HOSu BCY00008116 + N3-CH2-COOH ---------’-------► BCYOOOO8H6-CH2-N3 2.DIEA, DMF 1                                                    2 To a solution of compound 1 (5.0 mg, 49.5 pmol, 1.0 eq) in DMF (1 mL) was added EDCI (8.5 mg, 54.8 pmol, 1.1 eq) and HOSu (5.7 mg, 49.5 pmol, 1.0 eq). The mixture was stirred at 25-30 °C for 30 min. TLC indicated compound 1 was consumed completely and one 5 new spot formed. Then 0.3 mL of this mixture was added with BCY8116 (30.0 mg, 13.81 pmol, 0.28 eq.) and DIEA (2.4 pL, 13.81 pmol, 0.28 eq.), and stirred at 25-30 °C for 2 hr. LC-MS showed BCY8116 was consumed completely and one main peak with desired m / z (calculated MW:2255.53, observed m / z: 1128.34([M / 2+H]+) was detected. The reaction mixture was then concentrated under reduced pressure to remove solvent and produced a 10 residue, following by purification by prep-HPLC (TFA condition). Compound 2 (21 mg, 8.9 pmol, 64.43% yield, 95.56% purity) was obtained as a white solid. Procedure for preparation of BCY11373 CuSO4 VcNa THPTA BCYOOOO8H6-CH2-N3 +    BCY00008927   ----—► BCY00011373 2 3                              t-BuOH / H2O 2 15 A mixture of compound 2 (5 mg, 2.22 pmol, 1.0 eq.), BCY8928 (4.79 mg, 2.22 pmol, 1.0 eq.), and THPTA (1.0 mg, 2.30 pmol, 1.0 eq.) was dissolved in t-BuOH / H2O (1:1, 1 mL, predegassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 5.6 pL, 1.0 eq.) and VcNa (0.4 M, 5.6 pL, 1.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to 20 light yellow. The reaction mixture was stirred at 40 °C for 2 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 4415.07, observed m / z: 1471.5([M / 3+H]+ and 1103.8([M / 4+H]+) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (TFA condition), and BCY11373 25   (4.9 mg, 1.03 pmol, 46.26% yield, 92.4% purity) was obtained as a white solid. BCY11374 2024205105   25 Jul 2024 Procedure for preparation of BCY11374 CuSO4 VcNa THPTA t-BuOH / H2O BCY00008116-CH2-N3    +    BCY00008928 BCY00011374 A mixture of compound 2 (which may be prepared as described in the procedure for 5 preparing BCY11373; 5 mg, 2.22 pmol, 1.0 eq.), BCY8928 (4.9 mg, 2.22 pmol, 1.0 eq.), and THPTA (1.0 mg, 2.30 pmol, 1.0 eq.) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 5.6 pL, 1.0 eq.) and VcNa (0.4 M, 5.6 pL, 1.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The 10 reaction mixture was stirred at 40 °C for 2 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 4473.11, observed m / z: 1491.5([M / 3+H]+ and 1118.5([M / 4+H]+) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (TFA condition), and BCY11374 (4.1 mg, 1.27 15 pmol, 38.04% yield, 92.0% purity) was obtained as a white solid. 2024205105   25 Jul 2024 BCY00011375 Procedure for preparation of BCY11375 CuSO4 VcNa THPTA _ BCYOOOO8H6-CH2-N3 +    BCY00011014   --------==-----►    BCY00011375 2 J                                    DMF 2 5 A mixture of compound 2 (which may be prepared as described in BCY11373; 5 mg, 2.22 pmol, 1.0 eq.), BCY11014 (4.8 mg, 2.22 pmol, 1.0 eq.), and THPTA (0.5 mg, 2.30pmol, 1.0 eq.) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 5.6 pL, 1.0 eq.) and VcNa (0.4 M, 5.6 pL, 1.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 10 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 40 °C for 2 hr under N2 atmosphere. LC-MS detected some desired m / z (calculated MW: 4431.03, observed m / z: 1107.59([M / 4+H]+ and 1477.90([M / 3+H]+). The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was 2024205105   25 Jul 2024 purified by prep-HPLC (TFA condition), and BCY11375 (6 mg, 1.31 pmol, 59.13% yield, 96.8% purity) was obtained as a white solid. BCY11616 Procedure for preparation of Compound 3 BCY00008116 1 NHS-Peg5-N3 2 DEA DMF BCY00008116-Peg5-N3 3 A mixture of compound BCY8116 (30.0 mg, 13.81 pmol, 1.0 eq.), compound 2 (6.0 mg, 13.88 pmol, 1.0 eq.) and DIEA (2.4 pL, 13.82 pmol, 1.0 eq.) was dissolved in DMF. The 10 reaction mixture was stirred at 40 °C for 1 hr, till LC-MS showed compound 1 was consumed completely and one main peak with desired m / z (calculated MW:2489.82, observed m / z: 1245.4 ([M / 2+H]+) was detected. The reaction mixture was then concentrated under reduced pressure to remove solvent and produced a residue, following by purification by prep-HPLC (TFA condition). Compound 3 (27 mg, 10.29 pmol, 74.52% yield, 94.9% purity) was obtained 15 as a white solid. 2024205105   25 Jul 2024 Procedure for preparation of BCY11616 CuSO4 VcNa THPTA BCY00008116-Peg5-N3   +   BCY00007744   ---------- ------->►   BCY00011616 ,                                       DMF 5 A mixture of compound 3 (5 mg, 2.01 pmol, 1.0 eq.), BCY7744 (5.2 mg, 2.21 pmol, 1.1 eq.), and THPTA (1.0 mg, 2.30 pmol, 1.0 eq.) was dissolved in t-BuOH / H2O (1:1, 1 mL, predegassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 5.0 pL, 1.0 eq.) and VcNa (0.4 M, 5.0 pL, 1.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCOs (in 1:1 t-BuOH / H2O), and the solution turned to 10 light yellow. The reaction mixture was stirred at 40 °C for 2 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m / z (calculated MW: 4827.46, observed m / z: 1207.12 ([M / 4+H]+) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (TFA condition), and BCY11616 (4.7 mg, 1.0 pmol, 15   48.48% yield, 94.7% purity) was obtained as a white solid. BCY11617 BCY00011617 Procedure for preparation of BCY11617 2024205105   25 Jul 2024 C11SO4 VcNa thpta BCY00008116-Peg5-N3   +   BCY00011506   --------- -------►   BCY00011617 ,                                      DMF w A mixture of compound 3 (which may be prepared as described in the procedure for preparing BCY11616; 5 mg, 2.01 pmol, 1.0 eq.), BCY11506 (5.2 mg, 2.21 pmol, 1.1 eq.), and THPTA (1.0 mg, 2.30 pmol, 1.1 eq.) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre- 5 degassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 5.0 pL, 1.0 eq.) and VcNa (0.4 M, 5.0 pL, 1.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 40 °C for 2 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m / z 10 (calculated MW: 4828.45, observed m / z: 1206.97 ([M / 4+H]+) and 965.91 ([M / 5+H]+)) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (TFA condition), and BCY11617 (3.2 mg, 0.63 pmol, 31.37% yield, 95.05% purity) was obtained as a white solid. 15   BCY11857 o BCY00011857 Procedure for preparation of BCY11414-PEG5-N3 2024205105   25 Jul 2024 NaHCO3 BCY00011414 + NHS-PEG5-N3 ----------------►   BCY00011414-PEG5-N3 MeCN / H2O 1                                                            2 BCY11414 (60.0 mg, 29.06 pmol, 1.0 eq) and compound 1 (13.0 mg, 30.06 pmol, 1.03 eq) were dissolved in 2 mL of MeCN / H2O (1:1). Adjust pH to 8 with NaHCOs(0.4 M), and then the mixture was stirred at 25-30°C for 2 hr. LC-MS showed one main peak with desired m / z 5 (calculated MW: 2381.72, observed m / z: 1191.07([M / 2+H]+)) was detected. The reaction mixture was purified by prep-HPLC (TFA condition) and compound 2 (38.0 mg, 15.9 pmol, 54.71% yield, 97.35% purity) was obtained as a white solid. Procedure for preparation of BCY11857 CuSO4 VcNa BCY00011414-PEG5-N3   +    BCY00007744 ---THPTA—►    BCY00011857 t-BuOH / H2O 10                    2 Compound 2 (10.0 mg, 4.20 pmol, 1.0 eq) and BCY7744 (11.5 mg, 4.92 pmol, 1.2 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 11.0 pL, 1.0 eq), VcNa (2.0 mg, 10 pmol, 2.4 eq) and THPTA (2.0 mg, 4.6 pmol, 1.1 eq) were added. Finally 0.2 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged 15 with N2 for 3 times. The reaction mixture was stirred at 30°C for 16 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 4719.37, observed m / z: 1180.24([M / 4+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY11857 (10.3 mg, 2.18 pmol, 51.90% yield, 96.02% purity) was obtained as a white solid. 20 BCY11858 2024205105   25 Jul 2024 BCY00011858 Procedure for preparation of BCY11414-PEG5-N3 NaHCO3 BCY00011414 + NHS-PEG5-N3 ----------------►   BCY00011414-PEG5-N3 MeCN / H2O 1                                                            2 BCY11414 (60.0 mg, 29.06 pmol, 1.0 eq) and compound 1 (13.0 mg, 30.06 pmol, 1.03 eq), 5 were dissolved in 2 mL of MeCN / H2O (1:1). Adjust pH to 8 with NaHCOs(0.4 M), and then the mixture was stirred at 25-30°C for 2 hr. LC-MS showed one main peak with desired m / z (calculated MW: 2381.72, observed m / z: 1191.07([M / 2+H]+)) was detected. The reaction mixture was purified by prep-HPLC (TFA condition) and compound 2 (38.0 mg, 15.9 pmol, 54.71% yield, 97.35% purity) was obtained as a white solid. 10 Procedure for preparation of BCY11858 CuSO4 VcNa BCY00011414-PEG5-N3   +    BCY00008928  ---THPTA—► t-BuOH / H2O BCY00011858 Compound 2 (20.0 mg, 8.40 pmol, 1.0 eq) and BCY8928 (22.0 mg, 9.92 pmol, 1.1 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 21.0 pL, 1.0 eq), 2024205105   25 Jul 2024 VcNa (4.0 mg, 20.19 pmol, 2.4 eq) and THPTA (4.0 mg, 9.20 pmol, 1.1 eq) were added. Finally 0.4 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 30°C for 16 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak 5 with desired m / z (calculated MW: 4599.30, observed m / z: 920.38([M / 5+H]+), 1150.79 ([M / 4+H]+),1533.35([M / 3+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY11858 (16.9 mg, 3.67 pmol, 43.43% yield, 99.25% purity) was obtained as a white solid. 10  BCY11859 BCY00011859 Procedure for preparation of BCY11415-PEG5-N3 BCY00011415 + NHS-PEG5-N3 1 NaHCO3 MeCN / H2O BCY00011415-PEG5-N3 2 2024205105   25 Jul 2024 BCY11415 (30.0 mg, 13.81 pmol, 1.0 eq) and compound 1 (6.0 mg, 30.06 pmol, 1.0 eq), were dissolved in 2 mL of MeCN / H2O (1:1). Adjust pH to 8 with NaHCOs(0.4 M), and then the mixture was stirred at 25-30°C for 2 hr. LC-MS showed one main peak with desired m / z (calculated MW: 2489.82, observed m / z: 1245.18([M / 2+H]+)) was detected. The reaction 5 mixture was purified by prep-HPLC (TFA condition) and compound 2 (24.0 mg, 9.63 pmol, 69.7% yield, 99.28% purity) was obtained as a white solid. Procedure for preparation of BCY11859 CuSO4 VcNa BCY00011415-PEG5-N3   +    BCY00008928  ---THPTA—►    BCY00011859 t-BuOH / H2O 2 10 Compound 2 (20.0 mg, 8.03 pmol, 1.0 eq) and BCY8928 (21.0 mg, 9.47 pmol, 1.1 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 21.0 pL, 1.0 eq), VcNa (4.0 mg, 2.5 eq) and THPTA (4.0 mg, 1.1 eq) was added. Finally 1 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 30°C for 16 hr under N2 atmosphere. LC-MS showed 15 compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 4707.40, observed m / z: 941,7([M / 5+H]+), 1176.9([M / 4+H]+), 1569.6([M / 3+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY11859 (19.2 mg, 4.01 pmol, 49.87% yield, 98.22% purity) was obtained as a white solid. 20 Example 4: Synthesis of PD-L1 / CD137 Binding Heterotandem Bicyclic Peptides BCY8939 2024205105   25 Jul 2024 o General procedure for preparation of BCY8939 LIA m-SAXA AAAU     1) HOSU, EDCI N3-PEG12-COOH ----------!------►  BCY00007859 2) BCY00007732, DEA To a solution of N3-PEG12-COOH (250 mg, 388 pmol) and HOSu (67.0 mg, 583 pmol) in 5 DMA (4.5 mL) and DCM (1.5 mL) was added EDCI (89.3 mg, 466 pmol) with stirring at 20 °C for 16 hr. LCMS showed the desired intermediate was formed completely. BCY7732 (854.97 mg, 388.37 pmol, 1 eq) and DIEA (186 mg, 1.44 mmol, 250 pL) were added to the mixture with further stirring at 20 °C for additional 5 hr. LC-MS showed BCY7732 was consumed completely and one main peak with desired mass was detected. The reaction mixture was 10 purified by prep-HPLC (TFA condition) to give compound BCY7859 (621 mg, 200.58 pmol, 51.65% yield, 95% purity, TFA) as a white solid. Calculated MW: 2817.16, observed m / z: 942.7 [M / 3+H]+ General procedure for preparation of BCY8939 BCY00007859 15 BCY00008938 Vc,CuSO4,DMF BCY00008939 To a solution of BCY7859 (31.1 mg, 11.0 pmol) and BCY8938 (30.0 mg, 10.0 pmol) in DMF (2 mL) was added (2R)-2-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxyl -2H-furan-5-one (1 M, 100 pL) and CuSO4 (1 M, 30.0 pL) with stirring under nitrogen atmosphere for 2 hr at 20 °C. LC MS showed BCY7859 was consumed completely and one main peak with desired mass was 20 detected. The reaction mixture was purified by prep-HPLC (TFA condition) to give compound BCY8939 (16.1 mg, 2.72 pmol, 27.1% yield, 98.3% purity) as a white solid. Calculated MW: 5823.49, observed m / z: 1165.4 [M / 5+H]+, 971.0 [M / 6+H]+, 832.9[M / 7+H]+ 2024205105   25 Jul 2024 BCY10580 BCY00010580 '"OH° Procedure for preparation of BCY9172-PEG12-N3 DIEA BCY00009172 + NHS-PEG12-N3 DMSO BCY00009172-PEG12-N3 5   BCY9172 (100.0 mg, 47.72 pmol, 1 eq) and compound 1 (40.0 mg, 54.00 pmol, 1.13 eq) in DMSO (2 mL) was added DIEA (9.25 mg, 71.58 pmol, 12.47 pL, 1.5 eq). The mixture was stirred at 30 °C for 12 hr. LC-MS showed BCY9172 was consumed completely and one main peak with desired m / z (MW: 2721.12, observed m / z: 1361.07([(M / 2+H+])) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent 10 and produced a residue. The residue was then purified by prep-HPLC (neutral condition). Compound 2 (48 mg, 17.44 pmol, 45.68% yield, 98.87% purity) was obtained as a white solid. Procedure for preparation of BCY10580 CuSO4 VcNa BCY00009172-PEG12-N3 +   BCY00010043 ---THPTA—► t-BuOH / H2O BCY00010580 2024205105   25 Jul 2024 Compound 2 (20 mg, 7.35 pmol, 1.0 eq) and BCY10043 (23.1 mg, 7.35 pmol, 1.0 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 18.4 pL, 1.0 eq), VcNa(0.4 M, 36.8 pL, 2.0 eq) and THPTA (0.4 M, 18.4 pL, 1.0 eq) were added. Finally 1 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 5 for 3 times. The reaction mixture was stirred at 30 °C for 4 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m / z (MW: 5855.74 observed m / z: 976.40 ([M / 6+H]+) and 1171.67 ([M / 5+H]+)) was detected. The residue was purified by prep-HPLC (TFA condition). BCY10580 (29 mg, 4.85 pmol, 65.95% yield, 97.879% purity) was obtained as a white solid. 10 BCY10581 o 0 BCY00010581 Procedure for preparation of BCY9172-PEG12-N3 DIEA BCY00009172 + NHS-PEG12-N3 1 DMSO BCY00009172-PEG12-N3 2 15   BCY9172 (100 mg, 47.72 pmol, 1 eq) and compound 1 (40.00 mg, 54.00 pmol, 1.13 eq) in DMSO (2 mL) was added DIEA (9.25 mg, 71.58 pmol, 12.47 pL, 1.5 eq). The mixture was stirred at 30 °C for 12 hr. LC-MS showed BCY9172 was consumed completely and one main peak with desired m / z (MW: 2721.12, observed m / z: 1361.07([(M / 2+H+])) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent 20 and produced a residue. The residue was then purified by prep-HPLC (neutral condition). 2024205105   25 Jul 2024 Compound 2 (48 mg, 17.44 pmol, 45.68% yield, 98.87% purity) was obtained as a white solid. Procedure for preparation of BCY10581 BCY00009172-PEG12-N3 +    BCY00010044 CuSO4 VcNa THPTA BCY00010581 t-BuOH / H2O 5                          2 Compound 2 (12 mg, 4.41 pmol, 1 eq) and BCY10044 (14.08 mg, 4.41 pmol, 1eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 11.02 pL, 1 eq), VcNa (0.4 M, 22.05 pL, 2 eq) and THPTA (0.4 M, 10.04 pL, 1 eq) was added. Finally 1 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 10 times. The reaction mixture was stirred at 30 °C for 4 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m / z (MW: 5912.84, observed m / z: 985.90 ([M / 6+H]+) and 1183.28 ([M / 5+H]+)) was detected. The residue was purified by prep-HPLC (TFA condition). BCY10581 (9.3 mg, 1.47 pmol, 33.36% yield, 93.541% purity) was obtained as a white solid. BCY10582 2024205105   25 Jul 2024 Procedure for preparation of Compound 2 BCY00009172 + NHS-PEG12-N3 ------------►  BCY00009172-PEG12-N3 DMSO 1                                                       2 To a solution of BCY9172 (100.0 mg, 47.7 pmol, 1.0 eq), Compound 1 (40.0 mg, 54.0 pmol, 1.13 eq) in DMSO (2 mL) was added DIEA (9.2 mg, 71.6 pmol, 12.5 pL, 1.5 eq). The mixture 5 was stirred at 30°C for 12 hr. LC-MS showed BCY9172 was consumed completely and one main peak with desired m / z (calculated MW: 2721.12, observed m / z: 1361.07([M / 2+H]+)) was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (TFA condition). Compound 2 (37 mg, 13.60 pmol, 28.49% yield) was obtained as a white solid. 10 Procedure for preparation of BCY10582 BCY00009172-PEG12-N3 +   BCY00010045 -2y§O4_\ / cNajmPTA_^ BCY00010582 t-BuOH / H2O 2 A mixture of Compound 2 (16.0 mg, 5.9 pmol, 1.0 eq), BCY10045 (14.0 mg, 6.0 pmol, 1.01 eq), and THPTA (0.4 M, 14.7 pL, 1.0 eq) was dissolved in t-BuOH / H2O (1:1,2 mL, pre-15 degassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 14.7 pL, 1.0 eq) and VcNa (0.4 M, 29.4 pL, 2.0 eq) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 25-30 °C for 12 hr under N2 atmosphere. LC-MS showed Compound 2 was consumed completely and one main peak with desired 20 m / z [calculated MW: 5073.89, observed m / z: 1015.24 ([M / 5+H]+) and 1268.97([M / 4+H]+) was detected. The reaction mixture was directly purified by prep-HPLC (TFA condition). BCY10582 (10 mg, 1.92 pmol, 32.58% yield, 97.21% purity) was obtained as a white solid. BCY11017 2024205105   25 Jul 2024 BCY00011017 Procedure for preparation of BCY11017 CuSO4 VcNa BCY00008919-PEG12-N3 +    BCY00010861   ---THPTA—►    BCY00011017 t-BuOH / H2O 2 Compound 2 (which may be prepared as described in the procedure for preparing 5   BCY10567; 7.0 mg, 2.59 pmol, 1.0 eq) and BCY10861 (7.03 mg, 2.59 pmol, 1.0 eq) , were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 13.0 pL, 2.0 eq), VcNa (1.0 mg, 5.03 pmol, 2.0 eq) and THPTA (1.1 mg, 2.53 pmol, 1.0 eq) were added. Finally 1 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 35°C for 16 hr under N2 atmosphere. LC-MS 10 showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 5421.30, observed m / z\ 1084.7([M / 5+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY11017 (6.6 mg, 1.17 pmol, 45.24% yield, 96.16% purity) was obtained as a white solid. 2024205105   25 Jul 2024 BCY00011018 Procedure for preparation of BCY11018 BCY00008920-PEG12-N3 BCY00010861 C11SO4 VcNa THPTA BCY00011018 t-BuOH / H2O Compound 2 (which may be prepared as described in the procedure for preparing 5   BCY10570; 6.0 mg, 2.17 pmol, 1.0 eq) and BCY10861 (5.9 mg, 2.17 pmol, 1.0 eq) , were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 11.0 pL, 2.0 eq), VcNa (1.0 mg, 2.3 eq) and THPTA (1.1 mg, 1.0 eq) was added. Finally 1 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 35°C for 16 hr under N2 atmosphere. LC-MS showed 10 compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 5479.34, observed m / z: 1096.40([M / 5+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY11018 (2.3 mg, 0.40 pmol, 18.31% yield, 94.73% purity) was obtained as a white solid. 2024205105   25 Jul 2024 BCY00011019 Procedure for preparation of BCY11019 BCY00009172-PEG12-N3  +    BCY00010861 CUSO4 VcNa THPTA BCY00011019 t-BuOH / H2O 5 Compound 2 (which may be prepared as described in the procedure for preparing BCY10581; 8.0 mg, 2.94 pmol, 1.0 eq) and BCY10861 (8.0 mg, 2.95 pmol, 1.0 eq) , were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 14.7 pL, 2.0 eq), VcNa (1.2 mg, 6.05 pmol, 2.0 eq) and THPTA (1.3 mg, 2.99 pmol, 1.0 eq) was added. Finally 1 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 10 for 3 times. The reaction mixture was stirred at 35°C for 16 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 5437.26, observed m / z: 1088.09([M / 5+H]+) and 1360.19([M / 4+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY11019 (7.6 mg, 1.36 pmol, 46.09% yield, 96.95% purity) was obtained as a white solid. 2024205105   25 Jul 2024 BCY11376 o O 0 BCY00011376 Procedure for preparation of Compound 2 BCY00008919 + N3-CH2-COOH ---1EDCI,HOSu—BCY00008919-CH2-N3 2.DIEA, DMF 5                                          1                                                  2 To a solution of compound 1 (5.0 mg, 49.5 pmol, 1.0 eq) in DMF (1 mL) was added EDCI (8.5 mg, 54.8 pmol, 1.1 eq) and HOSu (5.7 mg, 49.5 pmol, 1.0 eq). The mixture was stirred at 25-30 °C for 30 min. TLC indicated compound 1 was consumed completely and one new spot formed. Then 0.2 mL of this mixture was added with BCY8919 (20.0 mg, 9.62 10 pmol) and DIEA (1.7 pL, 9.62 pmol). The mixture was stirred at 25-30 °C for 2 hr. LC-MS showed BCY8919 was consumed completely and one main peak with desired m / z (calculated MW:2162.51, observed m / z: 1081.8 ([M / 2+H]+)) was detected. The reaction mixture was then concentrated under reduced pressure to remove solvent and produced a residue, following by purification by prep-HPLC (TFA condition). Compound 2 (12 mg, 5.55 15 pmol, 56.28% yield, 97.54% purity) was obtained as a white solid. 2024205105   25 Jul 2024 Procedure for preparation of BCY11376 CuSO4 VcNa THPTA BCY00008919-CH2-N3 +   BCY00010861   ---------------------►   BCY00011376 2                                       t-BuOH / H2O A mixture of compound 2 (3 mg, 1.39 pmol, 1.0 eq.), BCY10861 (3.8 mg, 1.40 pmol, 1.0 5   eq.), and THPTA (1.2 mg, 2.76 pmol, 2.0 eq.) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre degassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 3.5 pL, 1.0 eq.) and VcNa (0.4 M, 3.5 pL, 1.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 40 °C for 2 hr under N2 atmosphere. LC-MS 10 showed BCY10861 was consumed completely and one main peak with desired m / z (calculated MW: 4878.64, observed m / z: 1220.8([M / 4+H]+) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (TFA condition), and BCY11376 (1.9 mg, 1.0 pmol, 27.01% yield, 96.2% purity) was obtained as a white solid. 15 BCY11377 2024205105   25 Jul 2024 BCY00011377 Procedure for preparation of Compound 2 ki u            1.EDCI, HOSu BCY00008920 + N3-CH2-COOH ---------’-------». BCY00008920-CH2-N3 2.DIEA, DMF 1                                                     2 To a solution of compound 1 (5.0 mg, 49.5 pmol, 1.0 eq) in DMF (1 mL) was added EDCI 5   (8.5 mg, 54.8 pmol, 1.1 eq) and HOSu (5.7 mg, 49.5 pmol, 1.0 eq). The mixture was stirred at 25-30 °C for 30 min. TLC indicated compound 1 was consumed completely and one new spot formed. Then 0.2 mL of this mixture was added with BCY8920 (20.0 mg, 9.36 pmol) and DIEA (1.2 mg, 9.36 pmol). The mixture was stirred at 25-30 °C for 2 hr. LC-MS showed BCY8920 was consumed completely and one main peak with desired m / z 10 (calculated MW:2220.54, observed m / z: 1110.90 ([M / 2+H]+) was detected. The reaction mixture was then concentrated under reduced pressure to remove solvent and produced a residue, following by purification by prep-HPLC (TFA condition). Compound 2 (12 mg, 5.15 pmol, 56.28% yield, 95.3% purity) was obtained as a white solid. 2024205105   25 Jul 2024 Procedure for preparation of BCY11377 CuSO4 VcNa THPTA BCY00008920-CH,-N, +  BCY00010861   ---------------------►   BCY00011377 2                                     t-BuOH / H2O A mixture of compound 2 (3 mg, 1.35 pmol, 1.0 eq.), BCY10861 (3.8 mg, 1.35 pmol, 1.0 5   eq.), and THPTA (0.6 mg, 1.0 eq.) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 3.4 pL, 1 eq.) and VcNa (0.4 M, 3.4 pL, 1 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 40 °C for 2 hr under N2 atmosphere. LC-MS showed one 10 main peak with desired m / z (calculated MW: 4936.68, observed m / z: 1234.9([M / 4+H]+) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (TFA condition), and BCY11377 (3.5 mg, 0.66 pmol, 48.86% yield, 93.1% purity) was obtained as a white solid. 15   BCY11378 2024205105   25 Jul 2024 BCY00011378 Procedure for preparation of Compound 2 KI r'u r'oou 1.EDCI, HOSu BCY00009172 + N3-CH2-COOH ----------------» BCY00009172-CH2-N3 2.DIEA, DMF 1                                                     2 To a solution of compound 1 (5.0 mg, 49.5 pmol, 1.0 eq) in DMF (1 mL) was added EDCI 5   (8.5 mg, 54.8 pmol, 1.1 eq) and HOSu (5.7 mg, 49.5 pmol, 1.0 eq). The mixture was stirred at 25-30 °C for 30 min. TLC indicated compound 1 was consumed completely and one new spot formed. Then 0.2 mL of this mixture was added to BCY9172 (20.0 mg, 9.54 pmol) and DIEA (1.7 pL, 9.62 pmol). The mixture was stirred at 25-30 °C for 2 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired m / z (calculated 10   MW:2176.49, observed m / z: 1090.0 ([M / 2+H]+) was detected. The reaction mixture was then concentrated under reduced pressure to remove solvent and produced a residue, following by purification by prep-HPLC (TFA condition). Compound 2 (20.2 mg, 7.48 pmol, 78.34% yield, 80.57% purity) was obtained as a white solid. 15 Procedure for preparation of BCY11378 2024205105   25 Jul 2024 CuSO4 VcNa THPTA BCY00009172-CH2-N3     +   BCY00010861 ----. D _UfU ►   BCY00011378 l-DUUn / n2 U 2 A mixture of compound 2 (5 mg, 2.30 pmol, 1.0 eq.), BCY10861 (6.24 mg, 2.30 pmol, 1.0 eq.), and THPTA (1.0 mg, 1.0 eq.) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged with N2 for 3 times), and then CuSO4 (0.4 M, 5.8 pL, 1.0 eq.) and VcNa (0.4 M, 5   5.8 pL, 1.0 eq.) were added under N2. The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned to light yellow. The reaction mixture was stirred at 40 °C for 2 hr under N2 atmosphere. LC-MS showed compound 3 was consumed completely and one main peak with desired m / z (calculated MW: 4894.61, observed m / z: 1224.3([M / 4+H]+) was detected. The reaction mixture was 10 filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (TFA condition), and BCY11378 (1.2 mg, 0.34 pmol, 10.07% yield, 94.3% purity) was obtained as a white solid. BCY11379 BCY00011379 Procedure for preparation of BCY8919-PEG5-N3 2024205105   25 Jul 2024 NaHCO3 BCY00008919 + NHS-PEG5-N3 ----------------►  BCY00008919-PEG5-N3 MeCN / H2O 1                                                        2 BCY8919 (30.0 mg, 14.43 pmol, 1.0 eq) and compound 1 (6.3 mg, 14.57 pmol, 1.01 eq), were dissolved in a mixture of MeCN (1 mL) and H2O (1 mL). The solution was added with 1 M NaHCOs to adjust pH to 8, and then the mixture was stirred at 35°C for 2 hr. LC-MS 5 showed BCY8919 was consumed completely and one main peak with desired m / z (calculated MW: 2396.79, observed m / z: 1198.74([M / 2+H]+) and 799.50([M / 4+H]+)) was detected. The reaction mixture was purified by prep-HPLC (TFA condition) and compound 2 (20 mg, 8.07 pmol, 55.92% yield, 96.68% purity) was obtained as a white solid. 10 Procedure for preparation of BCY11379 C11SO4 VcNa BCY00008919-PEG5-N3   +   BCY00010861  ---THPTA—►    BCY00011379 t-BuOH / H2O 2 Compound 2 (3.0 mg, 1.25 pmol, 1.0 eq) and BCY10861 (3.4 mg, 1.25 pmol, 1.0 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 7 pL, 2.24 eq), VcNa (1 mg, 5.04 pmol, 4.03 eq) and THPTA (1 mg, 2.30 pmol, 1.84 eq) was added. Finally 1 M 15 NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 25-30°C for 16 hr under N2 atmosphere. LC-MS showed compound 2 was consumed completely and one main peak with desired m / z (calculated MW: 5112.93 observed m / z: 1022.96([M / 5+H]+) and 1278.74([M / 4+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY11379 (3.4 mg, 0.615 20 pmol, 52.00% yield, 97.88% purity) was obtained as a white solid. BCY11380 2024205105   25 Jul 2024 BCY00011380 Molecular Weight: 4994.77 Procedure for preparation of BCY8920-PEG5-N3 NaHCO3 BCY00008920 + NHS-PEG5-N3 ----------------►   BCY00008920-PEG5-N3 MeCN / H2O 1                                                         2 BCY8920 (30.0 mg, 14.04 pmol, 1.0 eq) and compound 1 (6.1 mg, 14.11 pmol, 1.01 eq), 5 were dissolved in a mixture of MeCN (1 mL) and H2O (1 mL). The solution was added with 1 M NaHCOs to adjust pH to 8, and then the mixture was stirred at 35°C for 2 hr. LC-MS showed BCY8920 was consumed completely and one main peak with desired m / z (calculated MW: 2454.83, observed m / z: 1227.63([M / 2+H]+) and 818.66([M / 3+H]+)) was detected. The reaction mixture was purified by prep-HPLC (TFA condition) and compound 2 10   (20 mg, 8.03 pmol, 57.21% yield, 98.56% purity) was obtained as a white solid. Procedure for preparation of BCY11380 2024205105   25 Jul 2024 C11SO4 VcNa BCY00008920-PEG5-N 3   +    BCY00010861   ---THPTA—►    BCY00011380 t-BuOH / H2O 2 Compound 2 (3.5 mg, 1.43 pmol, 1.0 eq) and BCY10861 (3.9 mg, 1.44 pmol, 1.0 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 8 pL, 2.24 eq), VcNa (1 mg, 5.04 pmol, 3.52 eq) and THPTA (1 mg, 2.30 pmol, 1.61 eq) were added. Finally 1 M 5 NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 for 3 times. The reaction mixture was stirred at 25-30°C for 16 hr under N2 atmosphere. LC-MS showed majority of compound 2 was consumed and one main peak with desired m / z (calculated MW: 5170.97, observed m / z: 1034.28([M / 5+H]+) and 1293.10([M / 4+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY11380 (1.6 mg, 0.296 10 pmol, 20.77% yield, 96.77% purity) was obtained as a white solid. BCY11381 Procedure for preparation of BCY8920-PEG5-N3 BCY00009172 + NHS-PEG5-N3 NaHCO3 MeCN / H2O BCY00009172-PEG5-N3 2 2024205105   25 Jul 2024 BCY9172 (30.0 mg, 14.32 pmol, 1.0 eq) and compound 1 (6.2 mg, 14.34 pmol, 1.0 eq), were dissolved in a mixture of MeCN (1 mL) and H2O (1 mL). The solution was added with 1 M NaHCOs to adjust pH to 8, and then the mixture was stirred at 35°C for 2 hr. LC-MS showed BCY9172 was consumed completely and one main peak with desired m / z 5 (calculated MW: 2412.75, observed m / z: 1206.72([M / 2+H]+)) was detected. The reaction mixture was purified by prep-HPLC (TFA condition) and compound 2 (15 mg, 6.14 pmol, 42.87% yield, 98.75% purity) was obtained as a white solid. Procedure for preparation of BCY11381 C11SO4 VcNa BCY00009172-PEG5-N3   +    BCY00010861   ---THPTA—►    BCY00011381 t-BuOH / H2O 10                    2 Compound 2 (3.0 mg, 1.24 pmol, 1.0 eq) and BCY10861 (3.4 mg, 1.25 pmol, 1.01 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4(0.4 M, 7 pL, 2.25 eq), VcNa (1 mg, 5.04 pmol, 4.06 eq) and THPTA (1 mg, 2.30 pmol, 1.85 eq) were added. Finally 1 M NH4HCO3 was added to adjust pH to 8. All solvents here were degassed and purged with N2 15 for 3 times. The reaction mixture was stirred at 25-30°C for 16 hr under N2 atmosphere. LC- MS showed one peak with desired m / z (calculated MW: 5128.89, observed m / z: 1026.05([M / 5+H]+) and 1282.50([M / 4+H]+)). The reaction mixture was purified by prep-HPLC (TFA condition) and BCY11381 (1.6 mg, 0.295 pmol, 23.73% yield, 94.59% purity) was obtained as a white solid. 20 Example 5: Production of CD137 monoclonal antibody agonist The sequence of the CD137 monoclonal antibody agonist that was used for comparison to CD137 multimers in the experiments presented herein was disclosed in US Patent Number US 7,288,638. The lgG4 isotype antibody was expressed using the ExpiCHO Expression 25 System (Thermo Fisher Scientific) following transient transfection of the DNA expression construct. The antibody was purified by Protein A affinity chromatography and formulated in phosphate-buffered solution (PBS) pH 7.2. Purity analysis using HPLC-SEC (column GF-250, Agilent) indicated that the monomer rate of CD137 monoclonal antibody is approximately 95%. Binding activity analysis indicated that the CD137 monoclonal antibody 30 with a concentration higher than 1pg / ml can bind to CHO cells expressing CD137. Endotoxin analysis using the ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (Genscript) indicated that the CD 137 monoclonal antibody preparation contained <7 EU / mg of endotoxin. 2024205105   25 Jul 2024 BIOLOGICAL DATA 1.     CD137 Biacore Experimental Description Biacore experiments were performed to determine ka (M'1s-1), kd (s-1), Kd (nM) values of 5 heterotandem peptides binding to human CD137 protein. Recombinant human CD137 (R&D systems) was resuspended in PBS and biotinylated using EZ-Link™ Sulfo-NHS-LC-LC-Biotin reagent (Thermo Fisher) as per the manufacturer’s suggested protocol. The protein was desalted to remove uncoupled biotin using spin columns into PBS. 10 For analysis of peptide binding, a Biacore T200 or a Biacore 3000 instrument was used with a XanTec CMD500D chip. Streptavidin was immobilized on the chip using standard amine-coupling chemistry at 25°C with HBS-N (10 mM HEPES, 0.15 M NaCI, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated with a 7 min injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1 M / V- 15 hydroxy succinimide (NHS) at a flow rate of 10 pl / 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 120pl of onto the activated chip surface. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5) and biotinylated CD137 captured to a level of 270-1500 RU. Buffer was changed to PBS / 0.05% Tween 20 and a dilution series of the peptides was 20 prepared in this buffer with a final DMSO concentration of 0.5%. The top peptide concentration was 500nM with 6 further 2-fold or 3-fold dilutions. The SPR analysis was run at 25°C at a flow rate of 90|jl / min with 60 seconds association and 900 seconds dissociation. After each cycle a regeneration step (1 Opl of 10mM glycine pH 2) was employed. Data were corrected for DMSO excluded volume effects as needed. All data were double-referenced for blank 25 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. 30 Certain heterotandem peptides were tested in this assay and the results are shown in Table 1 below Table 1: CD137 Biacore Assay Data with Heterotandem Peptides Complex ID SPR (KD)(nM) 2024205105   25 Jul 2024 BCY9173 7.98 BCY7985 143 BCY8942 853 BCY8943 156 BCY9647 206 BCY9648 202 BCY9655 199 BCY9656 159 BCY9657 256 BCY9658 152 BCY9659 88.1 BCY9758 189 BCY8854 108 BCY9350 69.4 BCY9351 3640 BCY9399 73 BCY9400 53 BCY9408 105 BCY9409 97.7 BCY9410 65.8 BCY9411 71.1 BCY9759 44.3 BCY10000 6.19 BCY10571 12.03 BCY10572 5.00 BCY10573 3.39 2. Nectin-4 Biacore Experimental Description Biacore experiments were performed to determine ka (M'1s-1), kd (s-1), Kd (nM) values of heterotandem peptides binding to human Nectin-4 protein (obtained from Charles River). 5 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 10 Anti-FLAG M2 affinity agarose resin (Sigma) washed in PBS and the resin subsequently 2024205105   25 Jul 2024 transferred to a column and washed extensively with PBS. The protein was eluted with 100pg / 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. 5 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 with a CM5 chip (GE Healthcare). Streptavidin was immobilized on the chip using standard amine-coupling 10 chemistry at25°C with HBS-N (10 mM HEPES, 0.15 M NaCI, 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 / V-hydroxy succinimide (NHS) at a flow rate of 10 pl / 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 120pl of 15 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 Rll. 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 20 at a flow rate of 50pl / 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 25 binding model allowing for mass transport effects where appropriate. Certain heterotandem peptides of the invention were tested in the above mentioned Nectin-4 binding assays and the results are shown in Table 2 below: 30 Table 2: Nectin-4 Biacore Assay Data with Heterotandem Peptides Complex ID SPR KD(nM) BCY8854 2.76 BCY9350 > 200 nM BCY9351 2.47 BCY9399 1.67 2024205105   25 Jul 2024 BCY9400 1.8 BCY9408 1.57 BCY9409 1.66 BCY9410 1.49 BCY9411 1.48 BCY9759 2.14 BCY10000 2.26 3. EphA2 Biacore Experimental Description Biacore experiments were performed to determine ka (M'1s-1), kd (s-1), Kd (nM) values of heterotandem peptides binding to human EphA2 protein. 5 EphA2 were biotinylated with EZ-Link™ Sulfo-NHS-LC-Biotin for 1 hour in 4mM sodium acetate, 100mM NaCI, pH 5.4 with a 3x molar excess of biotin over protein. The degree of labelling was determined using a Fluorescence Biotin Quantification Kit (Thermo) after dialysis of the reaction mixture into PBS. For analysis of peptide binding, a Biacore T200 instrument was used with a XanTec CMD500D chip. Streptavidin was immobilized on the chip using 10 standard amine-coupling chemistry at 25°C with HBS-N (10 mM HEPES, 0.15 M NaCI, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated with a 7 min 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 pl / min. For capture of streptavidin, the protein was diluted to 0.2 mg / ml in 10 mM sodium acetate (pH 4.5) 15 and captured by injecting 120pl onto the activated chip surface. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5):HBS-N (1:1). Buffer was changed to PBS / 0.05% Tween 20 and biotinylated EphA2 was captured to a level of 500-1500 Rll using a dilution of protein to 0.2|jM in buffer. A dilution series of the peptides was prepared in this buffer with a final DMSO concentration of 0.5% with a top peptide concentration was 20   50 or 100nM and 6 further 2-fold dilutions. The SPR analysis was run at 25°C at a flow rate of 90|jl / min with 60 seconds association and 900-1200 seconds dissociation. 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). 25 Data were fitted using simple 1:1 binding model allowing for mass transport effects where appropriate. Certain heterotandem peptides of the invention were tested in the EphA2 binding assays and the results are shown in Table 3 below: 2024205105   25 Jul 2024 Table 3: EphA2 Biacore Assay Data with Heterotandem Peptides Complex ID SPR KD(nM) BCY9173 2.1 BCY7985 2 BCY8942 1.7 BCY8943 > 200 nM BCY9647 1.69 BCY9648 1.75 BCY9655 1.33 BCY9656 0.75 BCY9657 1.1 BCY9658 1.9 BCY9659 1.03 BCY9758 1.5 5   4. CD137 reporter assay co-culture with tumour cells Culture medium referred to as R1 media is prepared by adding 1 % FBS to RPMI-1640 (component of Promega kit CS196005). Serial dilutions of test articles in R1 are prepared in a sterile 96 well-plate. Use 25 pl per well of test articles or R1 (as a background control) to designated wells in white cell culture plate. Tumour cells* are harvested and resuspended at 10 a concentration of 400,000 cells / mL in R1 media. Twenty five (25) pL / well tumour cells are used in white cell culture plate. Jurkat cells (Promega kit CS196005, 0.5 mL) are thawed in the water bath and then added to 5 ml pre-warmed R1 medium. Twenty five (25) pL / well Jurkat cells are used in white cell culture plate. Incubate the cells and test articles for 6h at 37°C, 5 % CO2. At the end of 6h, add 75 pl / well Bio-Gio™ (Promega) and incubate for 10 min before 15 reading luminescence in a plate reader (Clariostar, BMG). The fold change relative to cells (Jurkat cells + Cell line used in co-culture) is calculated and plotted in GraphPad Prism as log(agonist) vs response to determine EC50(nM) and Fold Induction over background (Max) The tumour cell type used in co-culture is dependent on the tumour target specific for 20 heterotandem as is shown in Table 4 below: Table 4: Cell Lines Used for each Tumour Target 2024205105   25 Jul 2024 Tumour target Cell line used in co-culture EphA2 Nectin-4 PD-L1 A549, SC-OV-3, PC3, LNCaP HT1376, NCI-H292 RKO Data is presented in Figure 3 which shows that the EphA2-CD137 heterotandem BCY7985 showed strong induction of CD137 cell activity in the Promega CD137 luciferase reporter assay in the presence of EphA2-expressing HT1080 cells. There is no CD137 induction by 5 the heterotandem in the absence of HT1080 cells. Data is presented in Figure 4 which shows that EphA2 / CD137 heterotandems induce strong CD137 activation in CD137 reporter assay and the fold induction of activation is dependent on tumour target expression level on the cell line (A549 and SC-OV-3:EphA2 High and 10 LNCaP:EphA2 Low) used in co-culture. Data is presented in Figure 6 which shows that Nectin-4 / CD137 heterotandems induce strong CD137 activation in CD137 reporter assay and the fold induction of activation is dependent on tumour target expression level on the cell line (HT1376:Nectin-4 high and NCI-H292: 15 Nectin-4 Medium) used in co-culture. Data is presented in Figure 9 which shows that PD-L1 / CD137 heterotandems induce strong activation of CD137 in the CD137 reporter assay in presence of PD-L1 expressing cell line. A summary of the EC50(nM) and Fold Induction induced by heterotandem peptides in CD137 20 reporter assay in co-culture with different cells lines are reported in Table 5 below: Table 5: Fold Induction induced by Heterotandem Peptides in CD137 Reporter Assay Complex ID Tumour Target Cell Line used in Coculture EC50 (nM) Fold Induction over Background BCY9173 EphA2 SC-OV-3 0.94 21 BCY7985 EphA2 SC-OV-3 4.0 15 BCY8942 EphA2 PC3 - < 2 fold induction at 100 nM BCY8943 EphA2 PC3 - < 2 fold induction at 100 nM BCY9647 EphA2 SC-OV-3 7.2 24 2024205105   25 Jul 2024 BCY9648 EphA2 SC-OV-3 9.3 20 BCY9655 EphA2 SC-OV-3 4.1 6 BCY9656 EphA2 SC-OV-3 1.1 3 BCY9657 EphA2 SC-OV-3 9.0 26 BCY9658 EphA2 SC-OV-3 6.2 11 BCY9659 EphA2 SC-OV-3 9.9 7 BCY9758 EphA2 SC-OV-3 1.2 7 BCY10568 EphA2 PC3 0.25 32 BCY10570 EphA2 PC3 0.41 38 BCY10574 EphA2 PC3 1.0 32 BCY10575 EphA2 PC3 0.62 38 BCY10576 EphA2 PC3 0.51 38 BCY10577 EphA2 PC3 0.28 37 BCY8854 Nectin4 H1376 1.2 30 BCY9350 Nectin4 H1376 - < 2 fold induction at 100 nM BCY9351 Nectin4 H1376 - < 2 fold induction at 100 nM BCY9399 Nectin4 H1376 11 13 BCY9400 Nectin4 H1376 2.9 13 BCY9401 Nectin4 H1376 18 70 BCY9407 Nectin4 H1376 3.4 29 BCY9408 Nectin4 H1376 1.1 20 BCY9409 Nectin4 H1376 1.2 24 BCY9410 Nectin4 H1376 1.3 24 BCY9411 Nectin4 H1376 14 41 BCY9759 Nectin4 H1376 2.7 15 BCY10000 Nectin4 H1376 0.58 61 BCY10567 Nectin4 H1376 1.7 45 BCY10569 Nectin4 H1376 1.2 52 BCY10571 Nectin4 H1376 3.5 60 BCY10572 Nectin4 H1376 0.44 55 BCY10573 Nectin4 H1376 0.90 55 BCY10578 Nectin4 H1376 0.42 58 BCY10917 Nectin4 H1376 0.27 54 2024205105   25 Jul 2024 BCY11020 Nectin4 H1376 0.26 47 BCY11373 Nectin4 H1376 0.16 74 BCY11374 Nectin4 H1376 0.091 72 BCY11375 Nectin4 H1376 0.23 72 BCY8939 mouse PD- L1 MC38 - < 2 fold induction at 100 nM BCY10580 PD-L1 RKO 28 3 BCY10581 PD-L1 RKO 18 6 BCY10582 PD-L1 RKO 28 4 BCY11017 PD-L1 RKO 66 4 BCY11018 PD-L1 RKO 27 7 BCY11019 PD-L1 RKO 18 6 BCY11376 PD-L1 RKO 127 9 BCY11377 PD-L1 RKO 40 6 BCY11378 PD-L1 RKO 80 3 BCY11379 PD-L1 RKO 68 6 BCY11380 PD-L1 RKO 34 7 BCY11381 PD-L1 RKO 105 7 5. Primary human T cells-A549 co-culture (Tumour cell killing) PBMC were isolated from three healthy donors and added to Nuclight Red labelled tumour target cells (human lung carcinoma cells A549®, ATCC CLL-185™) at two defined ratios in the 5 presence of anti-CD3 stimulation at two concentrations. Tumour cell: PBMC co-cultures were incubated with the lead bicycles at three concentrations. All test conditions were also plated onto tumour cells in the absence of stimulated PBMC in order to detect direct tumour cell cytotoxicity. Tumour killing was evaluated by counting viable Nuclight red positive tumour cells over time. In addition, a Caspase 3 / 7 dye was used to identify apoptotic tumour cells. Cultures 10 were analysed using an IncuCyte S3 machine which allows real-time live cell fluorescence imaging. Co-cultures were imaged for 72 hours. Each condition was established in triplicate. Data is presented in Figure 5 which demonstrates that EphA2 / CD137 heterotandems induce tumour cell killing in primary human T-cell and cancer cell co-culture assay. Anti-CD137 mAb 15 agonist is used as a control. 6. Human PBMC-4T1 co-culture (cytokine release) assay 2024205105   25 Jul 2024 Mouse mammary gland tumor cell line 4T1-1 (4T1-Parental) and murine Nectin-4 overexpressing 4T1 (4T1-D02) were cultured in RPMI1640 supplemented with 10% heat-inactivated Fetal Bovine Serum, 100 l.ll / ml Penicillin and 100 I.U / Streptomycin, 20 mM HEPES, 1X Non-Essential Amino Acids, and 2 mM L-Glutamine (RPMI working medium). 5 Frozen PBMCs from healthy human donors were thawed and washed one time in room temperature PBS, and then resuspended in RPMI working medium. For tumor cell and PBMC co-culture, 10000 PBMCs and 2000 tumor cells (5:1) were mixed and plated in each well of a 384 well plate. For stimulating human PBMCs, 125 ng / ml of soluble anti-CD3 mAb (clone OKT3) was added to the culture on day 0. Test, control compounds or vehicle controls were 10 added to respective wells and brought the final volume per well to 100ul. Plates were incubated in a 37°C cell culture incubator with 5% CO2 for up to three days. Supernatants were collected 48 hours after stimulation, and human IL-2 and IFNy were detected using HTRF assays. Raw data were analyzed using Excel or Prism software to generate standard curves to interpolate protein concentrations. Data represents one study with three different donor 15 PBMC tested in experimental duplicates. Data presented in Figure 7 demonstrates that Nectin-4 / CD137 heterotandems induce robust IL-2 and IFN-y cytokine secretion in a PBMC-4T1 co-culture assay. BCY9350 and BCY9351 are non-binding controls for Nectin-4 and CD137 respectively. 20 A summary of the EC50(nM) and maximum IFN-y cytokine secretion (pg / ml) induced by selected Nectin-4 / CD137 heterotandem peptides in Human PBMC-4T1 co-culture (cytokine release) assay is reported in Table 6 below: 25 Table 6: EC50 and maximum IFN-y cytokine secretion induced by selected Nectin-4 / CD137 heterotandem peptides in Human PBMC-4T1 co-culture (cytokine release) assay Complex ID Cell line EC50 (nM) max IFN-y (pg / ml) BCY8854 4T1- D02(Nectin4+) 0.89 15962 BCY9350 4T1- D02(Nectin4+) - No Activity up to 1 pM BCY9351 4T1- D02(Nectin4+) - No Activity up to 1 pM 2024205105   25 Jul 2024 BCY10000 4T1- D02(Nectin4+) 0.21 19642 BCY10571 4T1- D02(Nectin4+) 0.44 18349 BCY10572 4T1- D02(Nectin4+) 0.25 17915 7. Ex vivo culture protocol Primary patient derived tumour cells from Discovery Life Sciences (DLS) are thawed gently in 10mL pre-warmed wash medium spiked fresh with Benzonase. The 3D spheroid kit from 5 Greiner (cat# 655840) is used to maintain cells in culture for 2 days. Briefly, tumour cells are counted with trypan blue using a haemocytometer. The cells are centrifuged at 1500rpm for 5min to wash, and the pellet is resuspended in lOOpL per 1X106 cells N3D nanoshuttle. To make them magnetic, cells are spun down at 1500rpm for 5 min and resuspended; this process is repeated for a total of 4 times. After the final spin, cells are resuspended in the 10 appropriate amount of fresh Lung DTC medium (DLS) to give 50,000-100,000 cells per well in 10OpL / well. Greiner cell-repellent, 96-well plates (cat #655976) are used for this experiment. If there are cell clumps or debris visible, sample is applied to a 70-100|jm filter before plating. At least 50,000 cells per sample are reserved for a Day 0 flow cytometry panel, these cells are stained, fixed, and stored at 4°C for later flow analysis. Control / test compound dilutions 15 are prepared in a separate plate at 2x in Lung DTC medium, and lOOpL / well of these 2X drug solutions are added to the wells as described by the plate map. The assay plate is then placed onto the 96-well magnetic spheroid drive in a humidified chamber at 37C, 5% CO2. At 24h, the magnetic spheroid drive is removed. At 48h, medium is collected for cytokine analysis and cells are collected for a Day 2 flow cytometry panel. Cytokines are quantified using a 20 custom-built cytokine / chemokine panel (IP-10, Granzyme B, IFNy, IL-2, IL-6, TNFa, IL-8, MIP- 1a, MIP-1b, MCP-1, IL-10, MIG) from R&D systems on a Luminex reader. Flow panels: Day 0 = Live / Dead, CD45, EpCAM, Nectin4, CD3, CD4, CD8, CD137; Day 2 = Live / Dead, CD45, EpCAM, Nectin4, CD3, CD8, Ki67, and counting beads. Flow data is analysed with Flowjo software. 25 Data shown in Figure 8 demonstrate that Nectin-4 / CD137 heterotandems induce target dependent cytokine release in ex-vivo cultures of primary patient-derived lung tumours. Treatment of BCY10572 induced Nectin-4 dependent change in several immune markers (normalized to vehicle) and in %CD8 +ki67+ T cells in patient-derived samples. 30 8. Pharmacokinetics of CD 137 Bispecifics in SD Rats 2024205105   25 Jul 2024 Male SD Rats were dosed with 2 mg / kg of each Bicycle multimer formulated in 25 mM Histidine HCI, 10% sucrose pH 7. Serial bleeding (about 80 pL blood / time point) was performed via submadibular or saphenous vein at each time point. All blood samples were immediately transferred into prechilled microcentrifuge tubes containing 2 pL K2-EDTA 5   (0.5M) as anti-coagulant and placed on wet ice. Blood samples were immediately processed for plasma by centrifugation at approximately 4°C, 3000g. The precipitant including internal standard was immediately added into the plasma, mixed well and centrifuged at 12,000 rpm, 4°C for 10 minutes. The supernatant was transferred into pre-labeled polypropylene microcentrifuge tubes, and then quick-frozen over dry ice. The samples were stored at 70 °C 10 or below as needed until analysis. 7.5 pL of the supernatant samples were directly injected for LC-MS / MS analysis using an Orbitrap Q Exactive in positive ion mode to determine the concentrations of Bicycle multimer. Plasma concentration versus time data were analyzed by non-compartmental approaches using the Phoenix WinNonlin 6.3 software program. CO, Cl, Vdss, 7½. AUC(O-last), AUC(O-inf), MRT(O-last) , MRT(O-inf) and graphs of plasma 15 concentration versus time profile were reported. Figure 10 shows the plasma concentration vs time curve of BCY10572 and BCY10000 from a 2 mg / kg IV dose in SD Rat (n =3). The pharmacokinetic parameters from the experiment are as shown in Table 7: 20 Table 7: Pharmacokinetic Parameters of plasma concentration vs time curve of BCY10572 and BCY10000 Compound T1 / 2(h) Clp (ml / min / kg) Vdss (L / kg) BCY10000 0.357 16.1 0.395 BCY10572 0.926 15.6 0.882

Claims

1. A heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof comprising:(a) a first peptide ligand which binds to a component present on an immune cell, wherein said component present on an immune cell is CD137, and wherein the CD137 binding bicyclic peptide ligand comprises an amino acid sequence selected from:CiIEEGQYCiiFADPY[Nle]Ciii (SEQ ID NO: 1);Ci[tBuAla]PE[D-Ala]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 3);CiIEEGQYCiiF[D-Ala]DPY[Nle]Ciii (SEQ ID NO: 4);Ci[tBuAla]PK[D-Ala]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 5);Ci[tBuAla]PE[D-Lys]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 6); Ci[tBuAla]P[K(PYA)][D-Ala]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 7);Ci[tBuAla]PE[D-Lys(PYA)]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 8);CiIEE[D-Lys(PYA)]QYCiiFADPY(Nle)Ciii (SEQ ID NO: 9);[dCi][dI][dE][dE][K(PYA)][dQ][dY][dCii][dF][dA][dD][dP][dY][dNle][dCiii] (SEQ ID NO: 10);conjugated via a linker to(b) a second peptide ligand which binds to a component present on a cancer cell, wherein said component present on a cancer cell is EphA2, PD-L1, or Nectin-4, wherein(b)(i) the EphA2 binding bicyclic peptide ligand comprises an amino acid sequence selected from:Ci[HyP]LVNPLCiiLHP[dD]W[HArg]Ciii (SEQ ID NO: 2); and CiLWDPTPCiiANLHL[HArg]Ciii (SEQ ID NO: 11);or a modified derivative thereof,(b)(ii) the PD-L1 binding bicyclic peptide ligand comprises an amino acid sequence selected from:Ci[HArg]DWCiiHWTFSHGHPCiii (SEQ ID NO: 12);CiSAGWLTMCiiQKLHLCiii (SEQ ID NO: 13); and CiSAGWLTMCiiQ[K(PYA)]LHLCiii (SEQ ID NO: 14); or a modified derivative thereof, or(b)(iii) the Nectin-4 binding bicyclic peptide ligand comprises an amino acid sequence selected from:CiP[1Nal][dD]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 15; hereinafter referred to as BCY8116);2024205105   08 Sep 2026CiP[1Nal][dD]CiiM[HArg]D[dW]STP[HyP][dW]Ciii (SEQ ID NO: 16; hereinafter referred to as BCY11415); andCiP[1Nal][dK](Sar10-(B-Ala))CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 17);CiPFGCiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 18; hereinafter referred to as BCY11414);or a modified derivative thereof;wherein Ci, Cii and Ciii represent first, second and third cysteine residues, respectively, Nle represents norleucine, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid, HyP represents hydroxyproline, dD represents aspartic acid in D-configuration and HArg represents homoarginine, 1Nal represents 1-naphthylalanine, Sar10 represents 10 sarcosine units, B-Ala represents beta-alanine, or a pharmaceutically acceptable salt thereof;wherein each of said peptide ligands comprises a polypeptide comprising at least three cysteine residues, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the cysteine residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

2. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in claim 1, wherein the immune cell is selected from: white blood cells; lymphocytes (e.g. T lymphocytes or T cells, B cells or natural killer cells); CD8 or CD4; CD8; dendritic cells, follicular dendritic cells and granulocytes.

3. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in claim 1, wherein the CD137 binding bicyclic peptide ligand comprises N-and C-terminal modifications and comprises:Ac-A-(SEQ ID NO: 1)-Dap (hereinafter referred to as BCY7732);Ac-A-(SEQ ID NO: 1)-Dap(PYA) (hereinafter referred to as BCY7741);Ac-(SEQ ID NO: 3)-Dap (hereinafter referred to as BCY9172);Ac-(SEQ ID NO: 3)-Dap(PYA) (hereinafter referred to as BCY11014);Ac-A-(SEQ ID NO: 4)-Dap (hereinafter referred to as BCY8045);Ac-(SEQ ID NO: 5)-A (hereinafter referred to as BCY8919);Ac-(SEQ ID NO: 6)-A (hereinafter referred to as BCY8920);Ac-(SEQ ID NO: 7)-A (hereinafter referred to as BCY8927);Ac-(SEQ ID NO: 8)-A (hereinafter referred to as BCY8928);Ac-A-(SEQ ID NO: 9)-A (hereinafter referred to as BCY7744); andAc-[dA]-(SEQ ID NO: 10)-[dA]-NH2 (hereinafter referred to as BCY11506);wherein Ac represents an acetyl group, Dap represents diaminopropionic acid and PYA represents 4-pentynoic acid, or a pharmaceutically acceptable salt thereof.2024205105   08 Sep 20264. The heterotandem bicyclic peptide complex or pharmaceutically acceptable salt thereof as defined in claim 3, wherein the CD137 binding bicyclic peptide ligand comprises:Ac-(SEQ ID NO: 8)-A (hereinafter referred to as BCY8928);wherein Ac represents an acetyl group.

5. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 4, wherein the cancer cell is selected from an HT1080, SC-OV-3, PC3, H1376, NCI-H292, LnCap, MC38 and RKO tumor cell.

6. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 5, wherein the component present on a cancer cell is EphA2 and the second peptide ligand comprises an EphA2 binding bicyclic peptide ligand.

7. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in claim 1, wherein the EphA2 binding bicyclic peptide ligand comprises N-terminal modifications and comprises:A-HArg-D-(SEQ ID NO: 2) (hereinafter referred to as BCY9594);[B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (hereinafter referred to as BCY6099);[PYA]-[B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (hereinafter referred to as BCY6169); and[PYA]-[B-Ala]-[Sar10]-VGP-(SEQ ID NO: 11) (hereinafter referred to as BCY8941);or a modified derivative thereof;wherein HArg represents homoarginine, PYA represents 4-pentynoic acid, Sar10 represents 10 sarcosine units, B-Ala represents beta-alanine, or a pharmaceutically acceptable salt thereof.

8. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 5, wherein the component present on a cancer cell is PD-L1 and the second peptide ligand comprises PD-L1 binding bicyclic peptide ligand.

9. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in claim 1, wherein the PD-L1 binding bicyclic peptide ligand comprises N-terminal and / or C-terminal modifications and comprises:[PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 12) (hereinafter referred to as BCY8938);[PYA]-[B-Ala]-[Sar10]-SDK-(SEQ ID NO: 13) (hereinafter referred to as BCY10043);2024205105   08 Sep 2026NH2-SDK-(SEQ ID NO: 13)-[Sar10]-[K(PYA)] (hereinafter referred to as BCY10044); NH2-SDK-(SEQ ID NO: 14) (hereinafter referred to as BCY10045); and Ac-SDK-(SEQ ID NO: 14)-PSH (hereinafter referred to as BCY10861);or a modified derivative thereof;wherein PYA represents 4-pentynoic acid, B-Ala represents beta-alanine, Sar10 represents 10 sarcosine units, or a pharmaceutically acceptable salt thereof.

10. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 5, wherein the component present on a cancer cell is Nectin-4 and the second peptide ligand comprises Nectin-4 binding bicyclic peptide ligand.

11. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in claim 1, wherein the Nectin-4 binding bicyclic peptide ligand optionally comprises N-terminal modifications and comprises:SEQ ID NO: 15 (hereinafter referred to as BCY8116);[PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 15) (hereinafter referred to as BCY8846);SEQ ID NO: 16 (hereinafter referred to as BCY11415);[PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 16) (hereinafter referred to as BCY11942);Ac-(SEQ ID NO: 17) (hereinafter referred to as BCY8831); andSEQ ID NO: 18 (hereinafter referred to as BCY11414);or a modified derivative thereof;wherein PYA represents 4-pentynoic acid, B-Ala represents beta-alanine, Sar10 represents10 sarcosine units, or a pharmaceutically acceptable salt thereof.

12. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in claim 10 or 11, wherein the Nectin-4 binding bicyclic peptide ligand comprises:SEQ ID NO: 15 (hereinafter referred to as BCY8116).

13. The heterotandem bicyclic peptide complex or pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 12 which is:(i) a CD137 / Nectin-4 complex comprising a first peptide ligand comprising a CD137 binding bicyclic peptide ligand attached to a TATA scaffold, and a second peptide ligand comprising an Nectin-4 binding bicyclic peptide ligand attached to a TATA scaffold, wherein said complex comprises:2024205105   08 Sep 2026• [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 15) (BCY8846) and Ac-A-(SEQ ID NO: 1)-Dap (BCY7732), wherein the N-terminal PYA of BCY8846 is linked to the C-terminal Dap of BCY7732 via a -PEG12- linker;• [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 16) (BCY11942) and Ac-A-(SEQ ID NO: 1)-Dap (BCY7732), wherein the N-terminal PYA of BCY11942 is linked to the C-terminal Dap of BCY7732 via a -PEG12- linker;• [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 15) (BCY8846) and Ac-A-(SEQ ID NO: 4)-Dap (BCY8045), wherein the N-terminal PYA of BCY8846 is linked to the C-terminal Dap of BCY8045 via a -PEG12- linker;• SEQ ID NO: 15 (BCY8116) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY8116 is linked to the C-terminal Dap(PYA) of BCY7741 via a -PEG10- linker;• SEQ ID NO: 15 (BCY8116) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY8116 is linked to the C-terminal Dap(PYA) of BCY7741 via a -PEG23- linker;• SEQ ID NO: 15 (BCY8116) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY8116 is linked to the C-terminal Dap(PYA) of BCY7741 via a -B-Ala-Sar20- linker;• SEQ ID NO: 15 (BCY8116) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY8116 is linked to the C-terminal Dap(PYA) of BCY7741 via a -B-Ala-Sar10-PEG10- linker;• SEQ ID NO: 15 (BCY8116) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY8116 is linked to the C-terminal Dap(PYA) of BCY7741 via a -B-Ala-Sar5-PEG15- linker;• SEQ ID NO: 15 (BCY8116) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY8116 is linked to the C-terminal Dap(PYA) of BCY7741 via a -B-Ala-Sar5-PEG5- linker;• SEQ ID NO: 15 (BCY8116) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY8116 is linked to the C-terminal Dap(PYA) of BCY7741 via a -PEG15-Sar5- linker;• SEQ ID NO: 15 (BCY8116) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY8116 is linked to the C-terminal Dap(PYA) of BCY7741 via a -PEG10-Sar10- linker;• SEQ ID NO: 15 (BCY8116) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY8116 is linked to the C-terminal Dap(PYA) of BCY7741 via a -PEG5-Sar15 linker;2024205105   08 Sep 2026• SEQ ID NO: 15 (BCY8116) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY8116 is linked to the C-terminal Dap(PYA) of BCY7741 via a -PEG5-Sar5- linker;• SEQ ID NO: 15 (BCY8116) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY8116 is linked to the C-terminal Dap(PYA) of BCY7741 via a -PEG5- linker;• SEQ ID NO: 15 (BCY8116) and Ac-A-(SEQ ID NO: 1)-Dap (BCY7732), wherein the N-terminus of BCY8116 is linked to the C-terminal Dap of BCY7732 via -PEG24- linker;• [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 15) (BCY8846) and Ac-(SEQ ID NO: 3)-Dap (BCY9172), wherein the N-terminal PYA of BCY8846 and the C-terminal Dap of BCY9172 are linked via a -PEG12- linker;• [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 15) (BCY8846) and Ac-(SEQ ID NO: 5)-A (BCY8919), wherein the N-terminal PYA of BCY8846 and the Lys3 of BCY8919 are linked via a -PEG12- linker;• [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 15) (BCY8846) and Ac-(SEQ ID NO: 6)-A (BCY8920), wherein the N-terminal PYA of BCY8846 and the dLys4 of BCY8920 are linked via a -PEG12- linker;• SEQ ID NO: 15 (BCY8116) and Ac-(SEQ ID NO: 7)-A (BCY8927), wherein the N-terminus of BCY8116 and the Lys(PYA)3 of BCY8927 are linked via a -PEG5 linker;• SEQ ID NO: 15 (BCY8116) and Ac-(SEQ ID NO: 8)-A (BCY8928), wherein the N-terminus of BCY8116 and the dLys(PYA)4 of BCY8928 are linked via a -PEG5- linker;• SEQ ID NO: 15 (BCY8116) and Ac-(SEQ ID NO: 3)-Dap(PYA) (BCY11014), wherein the N-terminus of BCY8116 and the C-terminal Dap(PYA) of BCY11014 are linked via a -PEG5- linker;• [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 15) (BCY8846) and Ac-(SEQ ID NO: 3)-Dap (BCY9172), wherein the N-terminal PYA of BCY8846 and the C-terminal Dap of BCY9172 are linked via a -CH2- linker;• Ac-(SEQ ID NO: 17) (BCY8831) and Ac-(SEQ ID NO: 3)-Dap(PYA) (BCY11014), wherein the dLys(Sar10)-(B-Ala))4 of BCY8831 and the C-terminal Dap(PYA) of BCY11014 are linked via a -PEG12- linker;• Ac-(SEQ ID NO: 17) (BCY8831) and Ac-(SEQ ID NO: 3)-Dap(PYA) (BCY11014), wherein the dLys(Sar10)-(B-Ala))4 of BCY8831 and the C-terminal Dap(PYA) of BCY11014 are linked via a -PEG5- linker;• SEQ ID NO: 15 (BCY8116) and Ac-(SEQ ID NO: 7)-A (BCY8927), wherein the N-terminus of BCY8116 and the Lys(PYA)3 of BCY8927 are linked via a -CH2- linker;2024205105   08 Sep 2026• SEQ ID NO: 15 (BCY8116) and Ac-(SEQ ID NO: 8)-A (BCY8928), wherein the N-terminus of BCY8116 and the dLys(PYA)4 of BCY8928 are linked via a -CH2- linker;• SEQ ID NO: 15 (BCY8116) and Ac-(SEQ ID NO: 3)-Dap(PYA) (BCY11014), wherein the N-terminus of BCY8116 and the C-terminal Dap(PYA) of BCY11014 are linked via a -CH2- linker;• SEQ ID NO: 15 (BCY8116) and Ac-A-(SEQ ID NO: 9)-A (BCY7744), wherein the N-terminus of BCY8116 and the dLys (PYA)4 of BCY7744 are linked via a -PEG5- linker;• SEQ ID NO: 15 (BCY8116) and Ac-[dA]-(SEQ ID NO: 10)-[dA]-NH2 (BCY11506), wherein the N-terminus of BCY8116 and the Lys(PYA)4 of BCY11506 are linked via a -PEG5- linker;• SEQ ID NO: 18 (BCY11414) and Ac-A-(SEQ ID NO: 9)-A (BCY7744), wherein the N-terminus of BCY11414 and the dLys(PYA)4 of BCY7744 are linked via a -PEG5-linker;• SEQ ID NO: 18 (BCY11414) and Ac-(SEQ ID NO: 8)-A (BCY8928), wherein the N-terminus of BCY11414 and the dLys(PYA)4 of BCY8928 are linked via a -PEG5-linker; or• SEQ ID NO: 16 (BCY11415) and Ac-(SEQ ID NO: 8)-A (BCY8928), and wherein the N-terminus of BCY11415 and the dLys(PYA)4 of BCY8928 are linked via a -PEG5-linker;or a pharmaceutically acceptable salt thereof; or(ii) a CD137 / EphA2 complex comprising a first peptide ligand comprising a CD137 binding bicyclic peptide ligand attached to a TATA scaffold, and a second peptide ligand comprising an EphA2 binding bicyclic peptide ligand attached to a TATA scaffold, wherein said complex comprises:• [PYA]-[B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6169) and Ac-(SEQ ID NO: 3)-Dap (BCY9172), wherein the N-terminal PYA of BCY6169 is linked to the C-terminal Dap of (BCY9172) via a -PEG12- linker;• [PYA]-[B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6169) and Ac-A-(SEQ ID NO: 1)-Dap (BCY7732), wherein the N-terminal PYA of BCY6169 is linked to the C-terminal Dap of BCY7732 via a -PEG12- linker;• [PYA]-[B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6169) and Ac-A-(SEQ ID NO: 4)-Dap, (BCY8045) wherein the N-terminal PYA of BCY6169 is linked to the C-terminal Dap of BCY8045 via a -PEG12- linker;• [PYA]-[B-Ala]-[Sar10]-VGP-(SEQ ID NO: 11) (BCY8941) and Ac-A-(SEQ ID NO: 1)-Dap (BCY7732), wherein the N-terminal PYA of BCY8941 is linked to the C-terminal Dap of BCY7732 via a -PEG12- linker;2024205105   08 Sep 2026• [B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY6099 is linked to the C-terminal Dap(PYA) of BCY7741 via a -PEG10- linker;• [B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY6099 is linked to the C-terminal Dap(PYA) of BCY7741 via a -PEG23- linker;• [B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY6099 is linked to the C-terminal Dap(PYA) of BCY7741 via a -PEG15-Sar5- linker;• [B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY6099 is linked to the C-terminal Dap(PYA) of BCY7741 via a -PEG10-Sar10- linker;• [B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY6099 is linked to the C-terminal Dap(PYA) of BCY7741 via a -PEG5-Sar15- linker;• [B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY6099 sequence is linked to the C-terminal Dap(PYA) of BCY7741 via a -PEG5-Sar5- linker;• [B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099) and Ac-A-(SEQ ID NO: 1)-Dap(PYA) (BCY7741), wherein the N-terminus of BCY6099 is linked to the C-terminal Dap(PYA) of BCY7741 via a -PEG5- linker;• [B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6099) and Ac-A-(SEQ ID NO: 1)-Dap (BCY7732), wherein the N-terminus of BCY6099 is linked to the C-terminal Dap of BCY7732 via a -PEG24- linker;• [PYA]-[B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6169) and Ac-(SEQ ID NO: 5)-A (BCY8919), wherein the N-terminal PYA of BCY6169 is linked to the Lys3 of BCY8919 via a -PEG12- linker;• [PYA]-[B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6169) and Ac-(SEQ ID NO: 6)-A (BCY8920), wherein the N-terminal PYA of BCY6169 is linked to the dLys4 of BCY8920 via a -PEG12- linker;• A-HArg-D-(SEQ ID NO: 2) (BCY9594) and Ac-(SEQ ID NO: 7)-A (BCY8927), wherein the N-terminus of BCY9594 is linked to the Lys(PYA)3 of BCY8927 via a -PEG5- linker;• A-HArg-D-(SEQ ID NO: 2) (BCY9594) and Ac-(SEQ ID NO: 8)-A (BCY8928), wherein the N-terminus of BCY9594 is linked to the dLys(PYA)4 of BCY8928 via a -PEG5-linker;2024205105   08 Sep 2026• A-HArg-D-(SEQ ID NO: 2) (BCY9594) and Ac-(SEQ ID NO: 3)-Dap(PYA) (BCY11014), wherein the N-terminus of BCY9594 is linked to the C-terminal Dap(PYA) of BCY11014 via a -PEG5- linker; or• [PYA]-[B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 2) (BCY6169) and Ac-(SEQ ID NO: 3)-Dap (BCY9172) wherein the N-terminus of BCY6169 is linked to the C-terminal Dap of BCY9172 via a -CH2- linker;or a pharmaceutically acceptable salt thereof; or(iii) a CD137 / PD-L1 complex comprising a first peptide ligand comprising a PD-L1 binding bicyclic peptide ligand attached to a TATA scaffold, and a second peptide ligand comprising a PD-L1 binding bicyclic peptide ligand attached to a TATA scaffold, wherein said complex comprises:• [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 12) (BCY8938) and Ac-A-(SEQ ID NO: 1)-Dap (BCY7732), wherein the N-terminal PYA of BCY8938 and the C-terminal Dap of BCY7732 are linked via a -PEG12- linker;• [PYA]-[B-Ala]-[Sar10]-SDK-(SEQ ID NO: 13) (BCY10043) and Ac-(SEQ ID NO: 3)-Dap (BCY9172), wherein the N-terminal PYA of BCY10043 and the C-terminal Dap of BCY9172 are linked via a -PEG12- linker;• NH2-SDK-(SEQ ID NO: 13)-[Sar10]-[K(PYA)] (BCY10044) and Ac-(SEQ ID NO: 3)-Dap (BCY9172), wherein the C-terminal Lys(PYA) of BCY10044 and the C-terminal Dap of BCY9172 are linked via a -PEG12- linker;• NH2-SDK-(SEQ ID NO: 14) (BCY10045) and and Ac-(SEQ ID NO: 3)-Dap (BCY9172), wherein the Lys(PYA)9 of BCY10044 and the C-terminal Dap of BCY9172 are linked via a -PEG12- linker;• Ac-SDK-(SEQ ID NO: 14)-PSH (BCY10861) and Ac-(SEQ ID NO: 5)-A (BCY8919), wherein the Lys(PYA)9 of BCY10861 and the Lys3 of BCY8919 are linked via a -PEG12- linker;• Ac-SDK-(SEQ ID NO: 14)-PSH (BCY10861) and Ac-(SEQ ID NO: 6)-A (BCY8920), wherein the Lys(PYA)9 of BCY10861 and the dLys4 of BCY8920 are linked via a -PEG12- linker;• Ac-SDK-(SEQ ID NO: 14)-PSH (BCY10861) and Ac-(SEQ ID NO: 3)-Dap (BCY9172), wherein the Lys(PYA)9 of BCY10861 and the C-terminal Dap of BCY9172 are linked via a -PEG12- linker;• Ac-SDK-(SEQ ID NO: 14)-PSH (BCY10861) and Ac-(SEQ ID NO: 5)-A (BCY8919), wherein the Lys(PYA)9 of BCY10861 and the Lys3 of BCY8919 are linked via a -CH2-linker;2024205105   08 Sep 2026• Ac-SDK-(SEQ ID NO: 14)-PSH (BCY10861) and Ac-(SEQ ID NO: 6)-A (BCY8920), wherein the Lys(PYA)9 of BCY10861 and the dLys4 of BCY8920 are linked via a -CH2-linker;• Ac-SDK-(SEQ ID NO: 14)-PSH (BCY10861) and Ac-(SEQ ID NO: 3)-Dap (BCY9172), wherein the Lys(PYA)9 of BCY10861 and the C-terminal Dap of BCY9172 are linked via a -CH2- linker;• Ac-SDK-(SEQ ID NO: 14)-PSH (BCY10861) and Ac-(SEQ ID NO: 5)-A (BCY8919), wherein the Lys(PYA)9 of BCY10861 and the Lys3 of BCY8919 are linked via a -PEG5-linker;• Ac-SDK-(SEQ ID NO: 14)-PSH (BCY10861) and Ac-(SEQ ID NO: 6)-A (BCY8920), wherein the Lys(PYA)9 of BCY10861 and the dLys4 of BCY8920 are linked via a -PEG5- linker; or• Ac-SDK-(SEQ ID NO: 14)-PSH (BCY10861) and Ac-(SEQ ID NO: 3)-Dap (BCY9172), wherein the Lys(PYA)9 of BCY10861 and the C-terminal Dap of BCY9172 are linked via a -PEG5- linker;or a pharmaceutically acceptable salt thereof.

14. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 13 which is:(i) a CD137 / Nectin-4 complex selected from:164O) UIBCY9399NH HNOH HN.BCY9401BCY94002024205105   08 Sep 2026BCY9405NHONHOHOHNOH2NOO'' N H2024205105   08 Sep 20260BCY9407NH2024205105   08 Sep 2026oBCY9409BCY9410170BCY9759BCY94112024205105   08 Sep 2026BCY10567172BCY10571BCY105692024205105   08 Sep 2026BCY105732024205105   08 Sep 2026BCY109172024205105   08 Sep 2026' N-OHXN OI x OHN^OH ON JLNH2NHBCY11373'OH HONHN'N-NH22024205105   08 Sep 2026BCY11374BCY11375NHOHN ■ OSN.O^N"'^SN^ / NN_N17B6CY000113752024205105   08 Sep 2026BCY11616BCY11617OHOOOHNOHOBCY00011617178BCY11858BCY118572024205105   08 Sep 2026and BCY11859or a pharmaceutically acceptable salt thereof; or(ii) a CD137 / EphA2 complex selected from:BCY9173180BCY9647BCY8943coBCY9655BCY9648182BCY9657BCY96562024205105   08 Sep 2026BCY9658OSx oJXH N nHz,.,h,,.,,1 hn^nhNH2n^n hO. XHNH2N^NHO. ZH NO-< .''oXS 0  \ O S 0 VJ                         OHVnhHN'X10NZO^\NHoA^\HN' N'"’\    0 / ^H / r-NH^OJ c’           NH 'V-OH O y-"^ o T^y--'O      HO-\_J'iNOO■ N^Nr     N"\X  VH^-nh / 0    nho( NAyNH2..... S 0,BCY00009658BCY9659NH?NH HOBCY10568BCY9758185BCY10574BCY10570186BCY10576BCY105752024205105   08 Sep 2026and BCY10577or a pharmaceutically acceptable salt thereof; or (iii) a CD137 / PD-L1 complex selected from:BCY8939o2024205105   08 Sep 2026BCY105812024205105   08 Sep 20262024205105   08 Sep 2026BCY11019BCY11377BCY11376192BCY11379BCY113782024205105   08 Sep 2026BCY11380oand BCY113812024205105   08 Sep 2026or a pharmaceutically acceptable salt thereof.

15. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 14, wherein the linker is selected from: -CH2-, -PEG5-, -PEG10-, -PEG12-, -PEG23-, -PEG24-, -PEG15-Sar5-, -PEG10-Sar10-, -PEG5-Sar15-, -PEG5-Sar5-, -B-Ala-Sar20-, -B-Ala-Sar10-PEG10-, -B-Ala-Sar5-PEG15- and -B-Ala-Sar5-PEG5-.

16. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 15, wherein the molecular scaffold is selected from 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).

17. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 16, wherein the pharmaceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium, ammonium salt.

18. The heterotandem bicyclic peptide complex or pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 17, further comprising a cytotoxic agent, a radiochelator and / or a chromophore.

19. The heterotandem bicyclic peptide complex or pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 18, wherein one or more of the polypeptides comprised in said peptide ligands comprises a C-terminal amide group.

20. A pharmaceutical composition which comprises the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof of any one of claims 1 to 19 in combination with one or more pharmaceutically acceptable excipients.

21. The heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 20 for use in preventing, suppressing or treating cancer, wherein:(i) the second peptide ligand binds to EphA2 and the cancer is an EphA2 associated cancer; (ii) the second peptide ligand binds to PD-L1 and the cancer is a PD-L1 associated cancer; or (iii) the second peptide ligand binds to Nectin-4 and the cancer is a Nectin-4 associated cancer.

22. A method of preventing, suppressing or treating cancer comprising administering to a subject in need thereof an effective amount of the heterotandem bicyclic peptide complex or2024205105   08 Sep 2026a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 19, or the pharmaceutical composition as defined in claim 20, wherein:(i) the second peptide ligand binds to EphA2 and the cancer is an EphA2 associated cancer; (ii) the second peptide ligand binds to PD-L1 and the cancer is a PD-L1 associated cancer; or (iii) the second peptide ligand binds to Nectin-4 and the cancer is a Nectin-4 associated cancer.

23. Use of the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 19 in the manufacture of a medicament for preventing, suppressing or treating cancer, wherein:(i) the second peptide ligand binds to EphA2 and the cancer is an EphA2 associated cancer; (ii) the second peptide ligand binds to PD-L1 and the cancer is a PD-L1 associated cancer; or (iii) the second peptide ligand binds to Nectin-4 and the cancer is a Nectin-4 associated cancer.

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