EA044626B1 - Bicyclic peptide ligands with specificity for EphA2 - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to polypeptides that are covalently linked to non-aromatic molecular scaffolds such that two or more peptide loops are interposed between the points of attachment to the scaffold. In particular, the invention describes peptides that bind with high affinity the Eph receptor tyrosine kinase A2 (EphA2). The invention also relates to drug conjugates comprising said peptides conjugated to one or more effector and/or functional groups, pharmaceutical compositions comprising said peptide ligands and drug conjugates, and the use of said peptide ligands and drug conjugates for the prevention, inhibiting the development or treatment of a disease or disorder characterized by overexpression of EphA2 in the affected tissue (such as a tumor).

State of the art

Cyclic peptides are able to bind with high affinity and specificity to target proteins and, therefore, are a promising class of molecules for the development of therapeutic agents. Indeed, several cyclic peptides are already successfully used in the clinic, such as, for example, the antibacterial peptide vancomycin, the immunosuppressant cyclosporine or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24) . The high binding characteristics are due to the relatively large interaction surface formed between the peptide and the target, as well as the reduced conformational flexibility of the cyclic structures. Typically, macrocycles bind to surfaces of several hundred square angstroms, for example, in the case of the CVX15 antagonist cyclic peptide CXCR4, the binding area is 400 A ² (Wu et al. (2007), Science 330, 1066-71), in the case of the cyclic peptide with Arg-Gly-Asp binding motif to integrin aVb3-355 A ² (Xiong et al. (2002), Science 296 (5565), 151-5) or in the case of the cyclic peptide inhibitor of upain-1 binding to urokinase-type plasminogen activator - 603 A ² (Zhao et al. (2007), J Struct Biol 160 (1), 1-10).

Due to the cyclic configuration, peptide macrocycles are less flexible than linear peptides, resulting in less entropy loss after binding to targets and resulting in higher binding affinities. Reduced flexibility also leads to fixation of target-specific conformations, which increases binding specificity compared to linear peptides. This effect was illustrated by the highly active and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which lost its selectivity for other matrix metalloproteinases (MMPs) when ring opened (Cherney et al. (1998), J Med Chem 41 ( 11), 1749-51). The favorable binding characteristics that result from macrocyclization are even more pronounced for polycyclic peptides having more than one peptide ring, such as vancomycin, nisin and actinomycin.

Previously, various groups of researchers have linked polypeptides containing cysteine residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and colleagues used tris(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide loops on synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for the synthesis of drug compounds of interest, in which the compounds are synthesized by coupling cysteine-containing polypeptides to a molecular scaffold, such as, for example, TATA (1,1',1''-(1,3,5-triazinan-1,3, 5-triyl)triprop-2-en-1-one, described in Heinis et al. Angew Chem, Int Ed. 2014;53:1602-1606).

Phage display techniques based on combinatorial approaches have been developed to generate and image large libraries of bicyclic peptides for targets of interest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO 2009/098450). Briefly, combinatorial libraries of linear peptides containing three cysteine residues and two regions of six randomized amino acids (Cys-(Xaa) ₆ -Cys-(Xaa) ₆ -Cys) were displayed on phage and cyclized by covalently linking cysteine side chains to the small molecule backbone.

The essence of the invention

According to a first aspect of the invention, there is provided a peptide ligand having specificity for EphA2, comprising a polypeptide containing at least three cysteine residues separated by at least two loop sequences, and a non-aromatic molecular scaffold that forms covalent bonds with the residues cysteine of a polypeptide, resulting in the formation of at least two polypeptide loops on a molecular framework, where the peptide ligand includes the amino acid sequence:

Ci(HyP)LVNPLCiiLHP(D-Asp)W(HArg)Ciii (SEQ Ш NO: 1);

wherein HyP represents hydroxyproline, HArg represents homoarginine and Ci, Cii and Ciii represent the first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

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In accordance with yet another aspect of the invention, there is provided a drug conjugate comprising a peptide ligand as defined above conjugated to one or more effector and/or functional groups.

According to yet another aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand or drug conjugate as defined above in combination with one or more pharmaceutically acceptable excipients.

In accordance with yet another aspect of the invention, there is provided a peptide ligand or drug conjugate as defined above for use in preventing, inhibiting, or treating a disease or disorder characterized by overexpression of EphA2 in a diseased tissue (such as a tumor).

Brief description of drawings

Fig. 1. General diagram demonstrating the concept of preparing bicyclic drug conjugates (BDCs).

Fig. Fig. 2. Graphical dependence of the average tumor volume on time after the start of BCY6136 dosing in mice with a xenograft of HT1080 cells. Doses (2, 3, and 5 mg/kg) were administered on days 0 and 7.

Fig. 3. Graphical dependence of the average tumor volume on time after the start of BCY6136 dosing in mice with a xenograft of NCI-H1975 cells. Doses (1, 2, and 3 mg/kg) were administered on days 0, 7, 14, 21, 28, and 35.

Fig. 4. Graphical dependence of the average tumor volume on time after the start of BCY6136 dosing in mice with a xenograft of MDA-MB-231 cells. Doses (1, 2, and 3 mg/kg) were administered on days 0, 7, 14, 21, 28, 35, and 45.

Fig. 5 and 6. Volume of residual tumor after administration of BCY6136 (Fig. 5) and ADC (Fig. 6) to female BALB/c nude mice bearing a PC-3 xenograft.

Fig. 7. Residual tumor volume after administration of BCY6136, EphA2-ADC, or docetaxel to male Balb/c nude mice BALB/c bearing a PC-3 xenograft.

Fig. 8. Volume of residual tumor after administration of BCY6136 to female BALB/c nude mice bearing an NCI-H1975 xenograft. Experimental points represent the average tumor volume in the group.

Fig. 9 and 10. Residual tumor volume after administration of BCY6136 and ADC to female BALB/c nude mice bearing the LU-01-0251 xenograft. Experimental points represent the average tumor volume in the group.

Fig. 11. Volume of residual tumor after administration of BCY6136 to female BALB/c nude mice carrying xenograft LU-01-0046. Experimental points represent the average tumor volume in the group.

Fig. 12. Residual tumor volume after administration of BCY6136 or ADC to female BALB/c nude mice bearing the LU-01-0046 xenograft in the NSCLC PDX model. Experimental points represent the average tumor volume in the group.

Fig. 13-15. Volume of residual tumor after administration of BCY6136 (Fig. 13), BCY6173 (Fig. 14) and BCY6175 (Fig. 15) to female BALB/c nude mice carrying LU-01-0046.

Fig. 16. Volume of residual tumor after administration of BCY6136 (designated BT5528 in FIG. 16), BCY8245, or BCY8781 to female BALB/c nude mice bearing the LU01-0412 xenograft. Experimental points represent the average tumor volume in the group.

Fig. 17. Volume of residual tumor after administration of BCY6136 to female BALB/c nude mice carrying xenograft LU-01-0486.

Fig. 18. Volume of residual tumor after administration of BCY6136 to female BALB/c nude mice carrying an MDA-MB-231-luc xenograft. Experimental points represent the average tumor volume in the group.

Fig. 19. Volume of residual tumor after administration of BCY6136 to female BALB/c nude mice carrying syngeneic EMT-6 cells. The dose in group 3 and group 4 was changed to 5 mg/kg and 3 mg/kg from day 14.

Fig. 20. Volume of residual tumor after administration of BCY6136 to female BALB/c nude mice bearing an NCI-N87 xenograft.

Fig. 21. Volume of residual tumor after administration of BCY6136 to female BALB/c nude mice bearing the SK-OV-3 xenograft.

Fig. 22. Volume of residual tumor after administration of BCY6136 to female BALB/c nude mice carrying an OE21 xenograft.

Fig. 23. Volume of residual tumor after administration of BCY6136 to female CB17-SCID mice bearing a MOLP-8 xenograft.

Fig. 24-29. Volume of residual tumor after administration of BCY6173 (Fig. 24), BCY6135 (Fig. 25), BCY6136 (Fig. 26), BCY6174 (Fig. 27), BCY6175 (Fig. 28) and ADC (Fig. 29) to female BALB nude mice /c, bearing the HT1080 xenograft.

Where error bars are shown in the figures described above, they represent the standard error of the mean (SEM).

Detailed Description of the Invention

In one embodiment, the peptide ligand includes the amino acid sequence:

(3-Ala)-Sario-A(HArg)D-Ci(HyP)LWPLCiiLHP(D-Asp)W(HArg)Ciii (SEQ ID NO: 2) (BCY6099);

where Sar is sarcosine, HArg is homoarginine and HuP is hydroxyproline.

In one embodiment, the molecular scaffold is 1,1',G'-(1,3,5triazinan-1,3,5-triyl)-triprop-2-en-1-one (TATA).

In yet another embodiment, the molecular scaffold is 1,1',1''-(1,3,5triazinan-1,3,5-triyl)-triprop-2-en-1-one (TATA), and the peptide the ligand includes the amino acid sequence:

(3-Ala)-Sario-A(HArg)D-Ci(HyP)LWPLCiiLHP(D-Asp)W(HArg)Ciii (SEQ Ш NO: 2) (BCY6099); and where Sar is sarcosine, HArg is homoarginine and HuP is hydroxyproline.

Unless otherwise specified, all technical and scientific terms used in the invention have the meaning that is commonly accepted by those of ordinary skill in the fields of peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. Standard techniques used in molecular biology, genetic and biochemical methods are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999) ^4th ed., John Wiley & Sons, Inc., the entire contents of which are incorporated herein by reference.

Nomenclature.

Numbering.

When specifying amino acid residue positions in the peptides of the invention, cysteine residues (Ci, Cii and Ciii) are omitted in the numbering since they are invariant, and therefore, the numbering of amino acid residues in the peptides of the invention is indicated as shown below:

-Ci-HyPi-L2-V3-N4-P5-L6-Cii-L ₇ -H ₈ -P9-(D-Asp)io-Wii-(HArg)i2-Ciii- (SEQ ID NO: 1).

For purposes of this invention, it is contemplated that all bicyclic peptides are cyclized with 1,1',1''-(1,3,5-triazinan-1,3,5-triyl)triprop-2-en-1-one ( TATA) to obtain the structure of trisubstituted 1,1',1''-(1,3,5-triazinan-1,3,5-triyl)tripropan-1-one. Cyclization with TATA occurs at

Ci, Cii, and Ciii.

Molecular format.

N- or C-terminal extensions to the bicyclic core sequence are added to the left and right sides of the sequence via a hyphen. For example, the N-terminal (e-Ala)-Sar ₁₀ -Ala tail may be designated as:

(3-Ala)-Sano-A-(SEQ Ш NO: X).

Inversion peptide sequences.

In light of Nair et al (2003) J Immunol 170(3), 1362-1373, it is believed that the peptide sequences disclosed herein may also find use in their retro-inverse form. For example, the sequence is subject to inversion (ie, the N-terminus becomes the C-terminus and vice versa), and their stereochemistry is similarly also subject to inversion (ie, D-amino acids become L-amino acids and vice versa).

Peptide ligands.

A peptide ligand as used herein refers to a peptide, peptide fragment or peptidomimetic covalently linked to a molecular framework. Typically, such peptides, peptide fragments or peptidomimetics include a peptide having natural or artificial amino acids in which two or more reactive groups (i.e. cysteine residues) are capable of forming covalent bonds with the framework, and a sequence located between these reactive groups, which is called a loop sequence because it forms a loop when a peptide, peptide fragment, or peptidomimetic binds to the scaffold. Here, the peptides, peptide fragments or peptidomimetics comprise at least three cysteine residues (referred to herein as C _i , C _ii and C _iii ) and form at least two loops on the framework.

Advantages of peptide ligands.

The particular bicyclic peptides of the present invention have a number of advantageous properties that make them suitable drug-like molecules for injection, inhalation, nasal, ophthalmic, oral or topical administration. Such beneficial properties include:

cross-reactivity with other species. It is a common requirement for preclinical evaluation of pharmacodynamics and pharmacokinetics;

resistance to protease. Bicyclic peptide ligands should generally demonstrate resistance to plasma proteases, epithelial (membrane-anchored) proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases and other similar proteases. Protease resistance must be maintained between different species so that a promising prototype bicyclic peptide can be developed in animal models and can be safely administered to humans;

required solubility profile. It represents the ratio of charged and hydrophilic residues to hydrophobic residues and internal/intermolecular H-bonds, which is important for formulation and absorption;

optimal half-life from blood plasma. Depending on the clinical indication and treatment regimen, the development of a bicyclic peptide with a short or prolonged in vivo exposure time may be required to treat either chronic or acute disease states. The optimal exposure time will be determined, on the one hand, by taking into account the requirement to use prolonged exposure times (for maximum therapeutic efficacy), and on the other hand, by taking into account the requirement of using short exposure times to minimize the toxicological effects arising from the use of prolonged exposure of the drug;

selectivity. Specific peptide ligands of the invention are characterized by high selectivity for other Eph receptor tyrosine kinases, such as EphA1, EphA3, EphA4, EphA5, EphA6, EphA7, EphB1, factor XIIA, carbonic anhydrase 9 and CD38 (selectivity data for selected peptide ligands of the invention are presented in tables 11 and 12). It should also be noted that the selected peptide ligands of the invention exhibit cross-reactivity with other species (eg, mouse and rat), allowing testing in animal models (Tables 3, 78, 10 and 12); and safety. Bleeding events have been reported during preclinical in vivo model studies and clinical trials using EphA2 antibody conjugates. For example, a phase 1 open-label study of MEDI-547 was stopped due to bleeding and coagulation events that occurred in 5 of 6 patients (Annunziata et al, Invest New Drugs (2013) 31:77-84). The observed bleeding events in patients were consistent with the effects on the coagulation system observed in preclinical studies in rats and monkeys: increased activated partial thromboplastin time and increased fibrinogen/fibrin degradation products (Annunziata et al IBID). Cases of apparent bleeding have been reported in toxicology studies in monkeys (Annunziata et al, IBID). Taken together, these results suggest that MEDI-547 induces disseminated intravascular coagulation (DIC) in both preclinical animal species and patients. The bicyclic drug conjugates (BDCs) described herein have short half-lives in vivo (<30 min) and are therefore unlikely to cause disseminated intravascular coagulation (DIC) in patients. The results presented in the invention (see data from biological activity studies, sections 5 and 6 and table 15) show that the selected bicyclic drug conjugates according to the invention do not affect blood coagulation parameters and do not cause cases of bleeding during preclinical studies.

Pharmaceutically acceptable salts.

It should be understood that salt forms are included within the scope of this invention, and references to peptide ligands include salt forms of said ligands.

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

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

One particular group of salts consists of salts formed with acetic, hydrochloric, hydroiodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalene sulfonic , valeric, propane, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

If the compound is anionic or has a functional group that can be anionic (eg, -COOH can be ^-COO- ), then a salt can 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 cations such as Li ⁺ , Na ⁺ and K ⁺ , alkaline earth metal cations such as Ca ²⁺ and Mg ²⁺ , and other cations such as Al ³⁺ or Zn2 ⁺ . Examples of suitable organic cations include, but are not limited to, ammonium ion (ie, NH4 ⁺ ) and substituted ammonium ions (eg, NH3R ⁺ , NH2R2+, NHR3 ⁺ , NR4+). Examples of some suitable substituted ammonium ions are those formed from methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine and tromethamine, as well as amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3) ₄ ⁺ .

When the peptides of the invention contain amine functionality, they can form quaternary ammonium salts, for example, by reaction with an alkylating reagent in accordance with methods that are well known in the art. Such quaternary ammonium compounds are included within the scope of the peptides of the invention.

Isotopic options.

The present invention includes all pharmaceutically acceptable isotope-labeled (radioactive isotope) peptide ligands of the invention in which 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 commonly found in nature, and the peptide ligands of the invention to which are attached metal chelating groups (called effectors) that are capable of retaining the corresponding isotopes (radioactive isotopes), and the peptide ligands of the invention in which specific functional groups are covalently replaced by corresponding isotopes (radioactive isotopes) or isotope-labeled functional groups.

Examples of isotopes used for introduction into the peptide ligands of the invention include isotopes of hydrogen such as ^2H (D) and ^3H (T), carbon such as 11C, ^13C and ^14C , chlorine such as ^36Cl , fluorine , such as ¹⁸ F, iodine, such as ¹²³ I, ¹²⁵ I and ¹³¹ I, nitrogen, such as ¹³ N and ¹⁵ N, oxygen, such as ¹⁵ O, ¹⁷ O and ¹⁸ O, phosphorus, such as ³² P, sulfur , such as ³⁵ S, copper, such as ⁶⁴ Cu, gallium, such as ⁶⁷ Ga or ⁶⁸ Ga, yttrium, such as ⁹⁰ Y, and lutetium, such as ¹⁷⁷ Lu, and bismuth, such as ²¹³ Bi.

The specific isotope-labeled peptide ligands of the invention, for example, peptide ligands into which a radioactive isotope is incorporated, are used in studies of drug and/or substrate tissue distribution and for clinical assessment of the presence and/or absence of an EphA2 target in diseased tissues. In addition, the peptide ligands of the invention may have diagnostically important properties such that they can be used to detect or identify complex formation between a labeled compound and other molecules, peptides, proteins, enzymes or receptors. Detection or identification methods may employ compounds that are labeled with labeling reagents such as radioactive isotopes, enzymes, fluorescent substances, luminescent substances (eg, luminol, luminol derivatives, luciferin, aequorin and luciferase), and the like. The radioactive isotopes tritium, i.e. ³ H (T), and carbon-14, i.e. ¹⁴ C, are especially suitable for this purpose from the point of view of the ease of their administration and the availability of means for their detection.

Substitution with heavier isotopes such as deuterium, i.e. ^2H (D), may provide certain beneficial therapeutic effects due to greater resistance to inactivation by metabolism, such as increased in vivo half-life or decreased levels of

- 5044626 required doses, and therefore may be preferred in some circumstances.

Substitution with positron-active isotopes such as ^nC , ^18F , ^15O and ^13N can be used in positron emission tomography (PET) studies to determine the extent of target occupancy.

The isotope-labeled peptide ligand compounds of the invention can be prepared generally by conventional methods known to those skilled in the art, or by methods similar to those described in the examples of the invention by using an appropriate isotope-labeled reagent in place of a previously used non-isotope-labeled reagent.

Non-aromatic molecular framework.

As used herein, the term non-aromatic molecular framework refers to any molecular framework as defined above which does not contain an aromatic (ie unsaturated) carbocyclic or heterocyclic ring system.

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

As noted in the documents mentioned above, the molecular framework may be a small molecule, such as a small organic molecule.

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

In one embodiment, the molecular framework includes reactive groups that are capable of reacting with the functional group(s) of the polypeptide to form covalent bonds.

The molecular framework may include chemical groups that form a bond with the peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

An example of an α unsaturated carbonyl-containing compound is 1,1',1”-(1,3,5-triazinan1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014) , 53(6), 16021606).

Effector and functional groups.

In accordance with yet another aspect of the invention, there is provided a drug conjugate comprising a peptide ligand as defined above conjugated to one or more effector and/or functional groups.

Effector and/or functional groups can be attached, for example, to the N- and/or C-termini of the polypeptide, to an amino acid in the polypeptide, or to a molecular framework.

Suitable effector groups include antibodies and parts or fragments thereof. For example, an effector group may include an antibody light chain constant region (CL), an antibody heavy chain CH1 domain, an antibody heavy chain CH2 domain, an antibody heavy chain CH3 domain, or any combination thereof, in addition to one or more constant region domains. The effector group may also include the hinge region of the antibody (such a region is usually found between the CH1 and CH2 domains in the IgG molecule).

In yet another embodiment of this aspect of the invention, the effector group of the present invention is the Fc region of an IgG molecule. Preferably, the peptide ligand-effector group of the present invention includes or consists of a peptide ligand-Fc fusion construct having a half-life of one day or more, two days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. More preferably, the peptide ligand of the present invention includes or consists of a peptide ligand-Fc fusion construct having a half-life of one day or more.

Functional groups typically include linking groups, drugs, reactive groups for attaching other structural moieties, functional groups that promote the uptake of macrocyclic peptides into cells, and other similar groups.

The ability of peptides to penetrate cells allows peptides to be effective against intracellular targets. Targets accessed by peptides through their ability to enter cells include transcription factors, intracellular signaling molecules such as tyrosine kinases, and molecules involved in the apoptotic signaling pathway. Functional groups that are able to enter cells include peptides or chemical groups that have been added to either the peptide or the molecular scaffold. Peptides, such as those derived from VP22, HIV-Tat, the Drosophila homeobox protein (Antennapedia locus), are described, for example, in Chen and Harrison, Biochemical Society Transactions (2007) Volume 35, part 4, p821; Gupta et al. in Advanced Drug Discovery Reviews (2004) Volume 57 9637. Examples of short peptides that have been shown to be effective in translocation across cellular cytoplasmic

- 6 044626 membranes, include the penetratin peptide from the Drosophila protein locus Antennapedia with a length of 16 amino acids (Derossi et al (1994) J Biol. Chem. Volume 269 p10444), a model amphipathic peptide with a length of 18 amino acids (Oehlke et al (1998) Biochim Biophys Acts Volume 1414 p127) and arginine-enriched regions of the human immunodeficiency virus (HIV) TAT protein. Non-peptide approaches include the use of small molecule mimics or second messenger-operated calcium channels (SMOCs), which can be easily attached to biomolecules (Okuyama et al (2007) Nature Methods Volume 4 p153). Other chemical strategies of adding guanidinium groups to molecules also enhance cell penetration (Elson-Schwab et al (2007) J Biol Chem Volume 282 p13585). Low molecular weight molecules, such as steroids, can be added to the molecular scaffold to enhance cellular uptake.

One class of functional groups that can be attached to peptide ligands are antibodies and their binding fragments, such as Fab, Fv, or single domain fragments. In particular, antibodies that bind to proteins capable of increasing the half-life of the peptide ligand in vivo can be used.

In one embodiment, the peptide effector group ligand of the invention has a half-life selected from the group consisting of 12 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more or 20 days or more. Preferably, the peptide ligand effector group or composition of the invention has a te in the range of 12 to 60 hours. In yet another embodiment, the tp half-life is one day or more. In yet another embodiment, the tp half-life is from 12 to 26 hours.

In one particular embodiment of the invention, the functional group is selected from a metal chelating reagent that is used to complex radioactive isotopes of medical grade metals.

Possible effector groups also include enzymes, such as carboxypeptidase G2, for use in antibody-mediated enzyme and prodrug therapy (ADEPT), in which antibodies are replaced by a peptide ligand.

In one particular embodiment of the invention, the functional group is selected from a drug, such as a cytotoxic agent for anticancer therapy. Suitable examples include alkylating agents such as cisplatin and carboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites, including purine analogs, azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids, including vinca alkaloids such as vincristine, vinblastine, vinorelbine and vindesine; podophyllotoxin and its derivatives etoposide and teniposide; taxanes, including paclitaxel, formerly known as Taxol; topoisomerase inhibitors, including camptothecins; irinotecan and topotecan, and type II inhibitors, including amsacrine, etoposide, etoposide phosphate and teniposide. Other agents may include antitumor antibiotics, which include the immunosuppressant dactinomycin (which is used in kidney transplants), doxorubicin, epirubicin, bleomycin, calicheamicins, and others.

In yet another specific embodiment of the invention, the cytotoxic agent is selected from maytansinoids (such as DM1) or monomethyl auristatins (such as MMAE).

DM1 is a cytotoxic agent that is a thiol-containing derivative of maytansine and has the following structure:

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

- 7 044626

In one embodiment, the cytotoxic agent is linked to the bicyclic peptide by a readily cleavable bond, such as a disulfide bond or a protease-sensitive bond. In yet another embodiment, the groups adjacent to the disulfide bond are modified to control the steric hindrance of access to the disulfide bond and thereby the rate of cleavage and concomitant release of the cytotoxic agent.

Research has been published on the possibility of modifying the susceptibility of a disulfide bond to breakage by introducing steric hindrance on both sides of the disulfide bond (Kellogg et al (2011) Bioconjugate Chemistry, 22, 717). A higher degree of steric hindrance leads to a decrease in the rate of destruction of the disulfide bond under the influence of intracellular glutathione, as well as extracellular (systemic) reducing agents, resulting in increased difficulty in releasing the toxic agent both inside and outside the cell.

Thus, by carefully selecting the degree of steric hindrance on both sides of the disulfide bond, an optimal balance can be achieved between the stability of the disulfide bond in the bloodstream (which minimizes unwanted side effects of the toxin) and the efficiency of release in the intracellular environment (which maximizes the therapeutic effect).

Steric hindrance on both sides of the disulfide bond is controlled by introducing one or more methyl groups either on the targeting structural moiety (in this case, the bicyclic peptide) or on the toxic agent side of the molecular construct.

In one embodiment, the drug conjugate further includes a linker between said peptide ligand and said cytotoxic agents.

In one embodiment, the cytotoxic agent and linker are selected from any combination of cytotoxic agent and linker described in patent document WO 2016/067035 (the entire contents of which are incorporated by reference herein).

In one embodiment, the cytotoxic agent is MMAE.

In one embodiment, the linker between said cytotoxic agent and said bicyclic peptide includes one or more amino acid residues. Thus, in one embodiment, the cytotoxic agent is MMAE, and the linker is selected from -Val-Cit-, -Trp-Cit-, -Val-Lys-, -D-Trp-Cit-, -Ala-Ala-Asn- , D-Ala-Phe-Lys- or -Glu-Pro-Cit-Gly-hPhe-Tyr-Leu- (SEQ ID NO: 3). In yet another embodiment, the cytotoxic agent is MMAE and the linker is selected from -Val-Cit-, -Trp-Cit-, -Val-Lys- or -D-Trp-Cit-. In yet another embodiment, the cytotoxic agent is MMAE and the linker is -Val-Cit- or -ValLys-. And in yet another embodiment, the cytotoxic agent is MMAE and the linker is -Val-Cit-.

In an alternative embodiment, the linker between said cytotoxic agent includes a disulfide bond, such as a cleavable disulfide bond. Thus, in yet another embodiment, the cytotoxic agent is DM1, and the linker is selected from -SS-, -SS(SO3H)-, -SS-(Me)-, -(Me)-SS-(Me)-, - SS-(Me2)- or -SS-(Me)-SO3H-. In yet another embodiment, the cytotoxic agent is DM1, and the linker includes an -SS moiety, such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDB), or an -SS(SO ₃ H) moiety, such as SO3H-SPDB. In yet another embodiment, the cytotoxic agent is DM1, and the linker includes an -SS- moiety, such as -SS- or -SS-(SO ₃ H)-.

In one embodiment, the cytotoxic agent is DM1, and the drug conjugate includes a compound of formula (A):

- 8 044626

wherein said bicycle is BCY6099 as defined above. In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate includes a compound of formula (B):

wherein said bicycle is BCY6099 as defined above.

In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate includes a compound of formula (A), wherein said bicycle is selected from BCY6099 as defined above. This bicyclic drug conjugate (BDC) is designated in the invention as BCY6027. Presented in the invention in table. 4 and 8 data illustrate excellent competitive binding for BCY6027 in the EphA2 competitive binding assay.

In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate includes a compound of formula (B), wherein said bicycle is selected from BCY6099 as defined above. This bicyclic drug conjugate (BDC) is designated in the invention as BCY6028. Presented in the invention in table. 4 and 8 data illustrate excellent competitive binding for BCY6028 in the EphA2 competitive binding assay.

In yet another embodiment, the cytotoxic agent is MMAE or DM1, and the drug conjugate is selected from BCY6136 and BCY6173. Presented in the invention in table. 11 and 12 data show that these two bicyclic drug conjugates did not exhibit significant binding to the closely related human homologues EphA1, EphA3, EphA4, EphA5, EphA6, EphA7 and EphB4; mouse EphA3 and EphA4; and rat EphA3 and EphB1.

In yet another embodiment, the drug conjugate is selected from any one of BCY6135, BCY6136, BCY6173, BCY6174 and BCY6175:

- 9 044626

BCY6174

- 10 044626

BCY6175

And in yet another embodiment, the drug conjugate is BCY6136. The data presented in the invention in studies 7 and 8 show that BCY6136 exhibited significant and high antitumor activity in the PC-3 xenograft model of prostate cancer (see Figs. 5 and 6 and tables 16-19). The present data also show that BCY6136 exhibited strong antitumor activity in the NCI-H1975 xenograft model of lung cancer (non-small cell lung cancer) (see Fig. 8 and Table 2025). Data presented in the invention in studies 10 and 11 also show that BCY6136 exhibited high antitumor activity in LU-01-0251 PDX (non-small cell lung cancer) lung cancer models for both large tumor and small tumor (see FIG. 9 and 10 and tables 26-29), in which complete regression of the tumor was observed. The data presented in the invention in study 12 also show that BCY6136 exhibited a statistically significant antitumor effect in the LU-01-0046 PDX (non-small cell lung cancer) lung cancer model (see Fig. 11 and tables 30-31), in which complete tumor regression when using BCY6136. Data reported in Study 13 also show that BCY6136 exhibited dose-dependent antitumor activity in the LU-01-0046 PDX (non-small cell lung cancer) lung cancer model (see FIG. 12 and Tables 32 and 33). Data reported in Study 14 also show that use of BCY6136 resulted in complete tumor clearance in the LU-01-0046 PDX (non-small cell lung cancer) lung cancer model (see Figures 13-15 and Tables 34-37). The data presented in the invention in studies 15 and 16 also illustrate the effects of BCY6136 in two models using cell lines with low/negligible expression of EphA2 (namely Lu-01-0412 and Lu-01-0486). This data shown in Fig. 23 and 24 and table. 38-41 show that the use of BCY6136 did not lead to tumor regression in any of the cell lines, but the use of BCY8245 and BCY8781, which bind to a target highly expressed in the Lu-01-0412 cell line, led to complete tumor eradication . Data presented in Study 17 show that BCY6136 exhibited strong antitumor activity in the MDAMB-231 xenograft model of breast cancer (see FIG. 18 and Tables 42-45). Data presented herein in Study 18 illustrate the effects of BCY6136 in a breast cancer model using a cell line with low/negligible expression of EphA2 (namely EMT6). This data, presented in Fig. 19 and table. 46 and 47 show that the use of BCY6136 did not lead to tumor regression in this cell line. Data presented herein in Study 19 show that BCY6136 exhibited significant antitumor activity in the NCI-N87 gastric cancer xenograft model (see FIG. 20 and Tables 48 and 49). Data presented in the invention in study 20 show that BCY6136 exhibited significant antitumor activity in the SK-OV-3 xenograft model of ovarian cancer (see Fig. 21 and tables 50 and 51) compared to the ADC MEDI-547, which exhibited moderate antitumor activity. Data presented herein in Study 21 show that BCY6136 exhibited significant antitumor activity in the OE-21 xenograft model of esophageal cancer (see FIG. 22 and Tables 52 and 53). Data presented herein in Study 22 show that BCY6136 exhibited dose-dependent antitumor activity in the MOLP-8 xenograft model of multiple myeloma (see FIG. 23 and Tables 59 and 60). Data presented herein in Study 23 show that BCY6136 exhibited strong antitumor activity in the HT-1080 fibrosarcoma xenograft model (see FIGS. 24-28 and Tables 56 and 57).

Synthesis.

The peptides of the present invention can be synthesized using standard techniques and then reacted with a molecular scaffold in vitro. For this purpose standard chemical methods can be used. This approach allows for preparative synthesis of soluble material for subsequent experiments or validation. Such methods can

- 11 044626 be implemented using traditional chemical approaches, such as those disclosed in the publication of Timmerman et al (supra).

Accordingly, the invention also relates to the production of polypeptides or conjugates selected as specified in the invention, where the production optionally includes additional steps, which are explained below. In one embodiment, these steps are performed on a final polypeptide/conjugate product that has been produced by chemical synthesis.

When preparing a conjugate or complex, amino acid residues may optionally be replaced in the polypeptide of interest.

Peptides can also be extended to introduce, for example, another loop and thereby make the peptides polyspecific.

Extension of a peptide can be accomplished by simple chemical means at its N-terminus or C-terminus or within its loops using orthogonally protected lysines (and analogues) by standard solid-phase or liquid-phase chemical synthesis methods. Standard (bio)conjugation techniques can be used to introduce an activated or activated N- or C-terminus. Alternatively, additional extensions can be made by fragment condensation or native chemical ligation, for example, as described in Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779, or by enzymes, for example using subtiligase, as described in Chang et al Proc Natl Acad Sci USA. 1994 Dec 20; 91(26): 12544-8, or in Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).

Alternatively, the peptides can be extended or modified by subsequent conjugation using disulfide bonds. This approach has the additional advantage of allowing the first and second peptides to dissociate from each other when exposed to the reducing environment of the cell. In this case, a molecular scaffold (eg, TBMV) can be added during the chemical synthesis of the first peptide so that it reacts with the three cysteine groups; an additional cysteine or thiol can then be attached to the N- or C-terminus of the first peptide such that the cysteine or thiol reacts only with the free cysteine or thiol of the second peptide, forming a disulfide-linked bicyclic peptide peptide conjugate.

Similar methods are equally applicable to the synthesis/conjugation of two bicyclic and bispecific macrocycles, potentially allowing the creation of a tetraspecific molecule.

In addition, the addition of other functional groups or effector groups can be accomplished in the same manner using appropriate chemical methods, addition at the N or C termini or through side chains. In one embodiment, the coupling is carried out in such a way that it does not block the activity of any of the structural fragments.

Pharmaceutical compositions.

According to yet another aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand or drug conjugate as defined above in combination with one or more pharmaceutically acceptable excipients.

Typically, the provided peptide ligands will be used in purified form in conjunction with pharmacologically acceptable excipients or carriers. Typically, these excipients or carriers include aqueous or alcohol/aqueous solutions, emulsions or suspensions, including saline and/or buffered media. Media for parenteral administration include sodium chloride solution, dextrose Ringer's solution, dextrose and sodium chloride, and lactated Ringer's solution. Suitable physiologically acceptable adjuvants, if it is desired to maintain the polypeptide complex in suspension form, may be selected from thickening agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as dextrose Ringer's solution. In addition, preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents and inert gases may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

The peptide ligands of the present invention can be used in the form of separately administered compositions or in combination with other drugs. These drugs may include antibodies, antibody fragments and various immunotherapy drugs such as cyclosporine, methotrexate, adriamycin or cisplatin, and immunotoxins. Pharmaceutical compositions may include cocktails of various cytotoxic or other agents in combination with protein ligands of the present invention, or even combinations of selected polypeptides of the present invention having different specificities, such as polypeptides selected using different target ligands, regardless of whether their combined together before administration or will not.

The method of administration of the pharmaceutical compositions according to the invention may be any of the methods that are well known to those skilled in the art. For the purpose of therapy, the peptide ligands of the invention can be administered to any patient by standard methods. Administration may be by any suitable route, including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, inhalation, or direct infusion using a catheter, as appropriate. The dose and frequency of administration will depend on the age, gender and condition of the patient, on the simultaneous administration of other drugs, contraindications and other parameters that should be taken into account by the attending physician.

The peptide ligands of the present invention can be lyophilized for storage and then reconstituted in a suitable carrier before use. This method has been shown to be effective and known lyophilization and reconstitution techniques can be used in the invention. It will be apparent to those skilled in the art that lyophilization and reconstitution can result in varying degrees of loss of activity and that upward adjustments to the concentration of peptide ligands are necessary to compensate for these losses.

Compositions containing the peptide ligands or a cocktail thereof according to the invention can be administered for the purpose of prophylactic and/or therapeutic treatment. In particular therapeutic applications, a therapeutically effective dose is an appropriate amount that achieves at least partial inhibition, suppression, modulation, killing, or some other measurable parameter of a population of selected cells. The amounts required to achieve this dose will depend on the severity of the disease and the general condition of the patient's own immune system, but typically these amounts range from 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, with doses of 0 .05 to 2.0 mg/kg/dose. For prophylactic purposes, compositions comprising the peptide ligands or mixtures thereof of the invention may also be administered in similar or slightly lower doses.

A composition containing a peptide ligand of the present invention can be used for prophylactic and therapeutic purposes to promote the modification, inactivation, destruction or removal of a selected population of target cells in the body of a mammal. In addition, the peptide ligands described in the invention can be selectively used in vitro or in vitro to selectively kill, deplete, or otherwise effectively remove a population of target cells from a heterogeneous population of cells. Blood collected from a mammal can be combined extracorporeally with selected peptide ligands, resulting in unwanted cells being killed or otherwise removed from the blood, which is returned to the mammal using standard methods.

Therapeutic use.

The bicyclic peptides of the invention have the specific ability to bind EphA2.

Eph receptor tyrosine kinases (Eph) belong to a large group of receptor tyrosine kinases (RTKs), whose kinases phosphorylate proteins on tyrosine residues. Ephs and their membrane-bound ephrin ligands (ephrins) control cell positioning and tissue organization (Poliakov et al. (2004) Dev Cell 7, 465-80). Functional and biochemical Eph responses arise at higher states of ligand oligomerization (Stein et al. (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 leads to a lack of ability to remodel capillary beds into blood vessels (Poliakov et al., supra) and to embryonic lethality. Persistent expression of some Eph receptors and ephrins has also been observed in newly formed microvessels in adults (Brantley-Sieders et al. (2004) Curr Pharm Des 10, 343142; Adams (2003) J Anat 202, 105-12).

The dysregulated reappearance of certain ephrins and their receptors in adult humans has also been found to promote tumor invasion, metastasis and neoangiogenesis (Nakamoto et al. (2002) Microsc Res Tech 59, 58-67; Brantley-Sieders et al., supra). In addition, several members of the Eph family have been found to be overexpressed in tumor cells from various human tumors (Brantley-Sieders et al., supra); Marine (2002) Ann Hematol 81 Suppl 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).

EPH receptor A2 (ephrin type-A receptor 2) is a protein that in humans is encoded by the EPHHA2 gene.

EphA2 is upregulated in multiple human malignancies, often correlating with disease progression, metastasis and poor prognosis, such as in breast cancer (Zelinski et al (2001) Cancer Res. 61, 2301-2306; Zhuang et al (2010) Cancer Res. 70, 299-308; Brantley-Sieders et al (2011) PLoS One 6, e24426), lung cancer (Brannan et al (2009) Cancer Prev Res (Phila) 2, 1039-1049; Kinch et al (2003) Clin Cancer Res. 9, 613-618; Guo et al (2013) J Thorac Oncol. 8, 301-308), gastric cancer (Nakamura et al (2005) Cancer Sci. 96, 42-47; Yuan et al (2009 ) Dig Dis Sci 54, 2410-2417), pancreatic cancer (Mudali et al (2006) Clin Exp Metastasis 23, 357365), prostate cancer (Walker-Daniels et al (1999) Prostate 41, 275-280), cancer liver (Yang et

- 13 044626 al (2009) Hepatol Res. 39, 1169-1177) and glioblastoma (Wykosky et al (2005) Mol Cancer Res. 3, 541-551; Li et al (2010) Tumour Biol. 31, 477-488).

The full role of EphA2 in cancer progression is still unclear, although there is evidence of its influence at multiple stages of cancer progression, including tumor cell growth, survival, invasion and angiogenesis. Down-regulation of EphA2 expression suppresses tumor cell proliferation (Binda et al. (2012) Cancer Cell 22, 765-780), while blockade of EphA2 inhibits VEGF-induced cell migration (Hess et al. (2001) Cancer Res. 61, 32503255), germination and angiogenesis (Cheng et al. (2002) Mol Cancer Res. 1, 2-11; Lin et al. (2007) Cancer 109, 332-40), and progression of metastasis (Brantley-Sieders et al. ( 2005) FASEB J. 19, 1884-1886).

A drug conjugate with an anti-EphA2 antibody has been shown to significantly reduce tumor growth in rat and mouse xenograft models (Jackson et al (2008) Cancer Research 68, 9367-9374), and a similar approach was tried in a man, although treatment had to be interrupted due to the need to treat associated adverse side effects (Annunziata et al (2013) Invest New drugs 31, 77-84).

Polypeptide ligands selected in accordance with the method of the present invention can be used in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro research and as a reagent, and other similar purposes. Ligands with selected levels of specificity can be used in non-human animal testing where cross-reactivity is desired, or in diagnostics where cross-reactivity with homologs or paralogs needs to be carefully monitored. Some applications, such as vaccines, can take advantage of the ability of peptide ligands to induce an immune response to a predetermined series of antigens in order to tailor the vaccine to specific diseases and pathogens.

Substantially pure peptide ligands with a homogeneity of at least 90 to 95% are preferred for administration to a mammal, and with a homogeneity of 98 to 99% or more are most preferred for pharmaceutical use, particularly when the mammal is a human. After purification, partial or to a state of homogeneity, depending on the requirements, the selected polypeptides can be used in diagnostics or therapy (including in vitro), or in the development and implementation of analytical techniques, immunofluorescent stains and for other similar purposes (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).

In accordance with another aspect of the invention, there is provided a peptide ligand or drug conjugate as defined above for use in preventing, inhibiting or treating a disease or disorder characterized by overexpression of EphA2 in a diseased tissue (such as a tumor).

In accordance with another aspect of the invention, there is provided a method of preventing, inhibiting the development or treating a disease or disorder characterized by overexpression of EphA2 in a diseased tissue (such as a tumor), which includes administering to a patient in need thereof an effector moiety and a drug-drug conjugate as defined above. peptide ligand.

In one embodiment, EphA2 is mammalian EphA2. In yet another embodiment, the mammalian EphA2 is human EphA2.

In one embodiment, the disease or disorder characterized by overexpression of EphA2 in the affected tissue is selected from cancer.

Examples of cancers (and benign types thereof) that may be treated (or inhibited) include, but are not limited to, tumors of epithelial origin (adenomas and carcinomas of various types, including adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas and other carcinomas) , such as carcinomas of the bladder and urinary tract, breast, gastrointestinal tract (including carcinomas of the esophagus, stomach (gastric), small intestine, colon, rectum and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidney, lung (eg, adenocarcinomas, small cell lung carcinomas, non-small cell lung carcinomas, bronchoalveolar carcinomas, and mesotheliomas), head and neck carcinomas (eg, cancer of the tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity and paranasal sinuses), ovaries, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (eg, follicular thyroid carcinoma), adrenal glands, prostate, skin and adjacent organs (eg, melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevus); hematologic malignancies (eg, leukemias, lymphomas) and premalignant and borderline hematologic disorders, including hematologic malignancies and associated lymphoid cell conditions (eg, acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B -cell lymphomas, such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, lymphoma

- 14 044626 mantle cells, T-cell lymphomas and leukemias, natural killer [NK] cell lymphomas, Hodgkin lymphomas, hairy cell leukemia, monoclonal gammopathy of unknown origin, plasmacytoma, multiple myeloma and lymphoproliferative disorders after transplantation), as well as hematological malignancies and related myeloid cell conditions (eg, acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia and primary myelofibrosis, myeloproliferative syndrome, myelodysplasia syndrome and promyelocytic leukemia); tumors of mesenchymal origin, for example, sarcomas of soft tissue, bone or cartilage, such as osteosarcomas, fibrosarcomas, chondrosarcomas, rhabdomyosarcomas, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcomas, gastrointestinal stromal tumors, benign and malignant histocytomas and dermatofibrosarcoma protuberance; tumors of the central or peripheral nervous system (eg, astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumors and schwannomas); endocrine tumors (eg, pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors and medullary thyroid carcinoma); tumors of the eyes and their appendages (for example, retinoblastoma); germ cell and trophoblast tumors (eg, teratomas, seminomas, dysgerminomas, hydatidiform mole and choriocarcinoma); and pediatric and embryonal tumors (eg, medulloblastomas, neuroblastomas, Wilms tumor, and primitive neuroectodermal tumors); or syndromes, congenital or otherwise, that make the patient susceptible to the onset of malignancy (eg, xeroderma pigmentosum).

In yet another embodiment, the cancer is selected from breast cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, liver cancer, glioblastoma, and angiogenesis.

In yet another embodiment, the cancer is selected from prostate cancer, lung cancer (such as non-small cell lung carcinoma (NSCLC)), breast cancer (such as triple negative breast cancer), gastric cancer, ovarian cancer, esophageal cancer, multiple myeloma and fibrosarcomas.

In yet another embodiment, the cancer is prostate cancer. The data presented in the invention in studies 7 and 8 show that BCY6136 exhibited significant and high antitumor activity in the PC-3 xenograft model of prostate cancer (see Figs. 5 and 6 and tables 16-19).

In yet another embodiment, the drug conjugate is used to prevent, inhibit, or treat solid tumors such as fibrosarcomas and breast carcinomas, and non-small cell lung carcinomas.

In yet another embodiment, the cancer is selected from lung cancer, such as non-small cell lung carcinomas (NSCLC). The data presented in the invention in study 9 show that BCY6136 exhibited high antitumor activity in the NCI-H1975 xenograft model of lung cancer (NSCLC) (see Fig. 8 and tables 20-25). Data also presented herein in Studies 10 and 11 show that BCY6136 exhibited a strong antitumor effect in both large and small tumor sizes in the LU-010251 PDX lung cancer (NSCLC) model (see FIG. 9 and 10 and tables 26-29), in which complete regression of the tumor was observed. Data presented in the invention in study 12 show that BCY6136 exhibited a significant antitumor effect in the LU-01-0046 PDX lung cancer (NSCLC) model (see Fig. 11 and tables 30 and 31), in which complete tumor regression was observed for BCY6136. Data reported in Study 13 show that BCY6136 exhibited dose-dependent antitumor activity in the LU-01-0046 PDX lung cancer (NSCLC) model (see FIG. 12 and Tables 32 and 33). Data reported in Study 14 show that BCY6173 exhibited antitumor activity, and administration of BCY6136 and BCY6175 resulted in tumor clearance in the LU-01-0046 PDX lung cancer (NSCLC) model (see Figures 13-15 and Table 34 -37). Data also presented herein in Studies 15 and 16 illustrate the effects of BCY6136 in two models using cell lines with low/negligible EphA2 expression (namely Lu-01-0412 and Lu-01-0486). This data shown in Fig. 23 and 24 and in table. 38-41 show that BCY6136 had no effect on tumor regression in either cell line, but BCY8245 and BCY8781, which bind to a target highly expressed in the Lu-01-0412 cell line, resulted in complete destruction of the tumor. In yet another embodiment, the cancer is breast cancer. In yet another embodiment, the breast cancer is triple negative breast cancer. Data presented herein in Study 17 show that BCY6136 exhibited strong antitumor activity in the MDA-MB-231 xenograft model of breast cancer (see FIG. 18 and Tables 42-45). Data presented in the invention in study 18 illustrate the effects of BCY6136 in a breast cancer model using cell lines with low

- 15 044626 room/negligible expression of EphA2 (namely EMT6). This data, presented in Fig. 19 and in table. 46 and 47 show that BCY6136 had no effect on tumor regression in this cell line. In an alternative embodiment, the breast cancer is Herceptin-resistant breast cancer. Without relying on any theory as evidence, it is nevertheless suggested that EphA2 is directly involved in the emergence of Herceptin resistance, and therefore, an EphA2-targeting structural fragment has promise for use in patients who do not respond to Herceptin treatment .

In yet another embodiment, the cancer is gastric cancer. Data presented herein in Study 19 show that BCY6136 exhibited significant antitumor activity in the NCI-N87 gastric cancer xenograft model (see FIG. 20 and Tables 48 and 49).

In yet another embodiment, the cancer is ovarian cancer. Data presented in the invention in study 20 show that BCY6136 exhibited significant antitumor activity in the SK-OV-3 xenograft model of ovarian cancer (see Fig. 21 and tables 50 and 51) compared to the ADC MEDI-547, which exhibited moderate antitumor activity.

In yet another embodiment, the cancer is esophageal cancer. Data presented herein in Study 21 show that BCY6136 exhibited significant antitumor activity in the OE-21 xenograft model of esophageal cancer (see FIG. 22 and Tables 52 and 53).

In yet another embodiment, the cancer is multiple myeloma. Data presented herein in Study 22 show that BCY6136 exhibited dose-dependent antitumor activity in the MOLP-8 xenograft model of multiple myeloma, and BCY6082 exhibited significant antitumor activity (see FIG. 23 and Tables 59 and 60).

In yet another embodiment, the cancer is a fibrosarcoma. Data presented in the invention in study 23 show that BCY6173, BCY6135, BCY6174 and BCY6175 exhibited dose-dependent antitumor activity, and BCY6136 exhibited high antitumor activity in the HT-1080 fibrosarcoma xenograft model (see Figs. 24-28 and tables 56-57 ).

As used herein, the term prevention refers to administration of the protective composition before the onset of disease. Developmental inhibition refers to the administration of the composition after the onset of the disease, but before the clinical manifestation of the disease. Treatment involves the administration of a protective composition after the onset of symptoms of the disease.

Animal models are available in which studies can be conducted on the effectiveness of peptide ligands in preventing or treating disease. The use of animal models is facilitated by the present invention, which allows the development of polypeptide ligands capable of cross-reacting with human and animal targets, allowing the use of animal models with subsequent interpretation of the findings in humans.

In addition, the data presented herein in Study 3 illustrate the relationship between copy number variation (CNV) and gene expression for EphA2 across multiple tumor types. Thus, in accordance with another aspect of the invention, there is provided a method of preventing, inhibiting the development or treatment of cancer, which includes administering to a patient in need thereof an effector group and a drug-peptide ligand conjugate as defined above, wherein said patient is identified as having an increased number variation copies (CNV) of EphA2.

In one embodiment, the cancer is selected from those cancers identified in the invention as having an increased EphA2 copy number variation (CNV). In yet another embodiment, the cancer is breast cancer.

The invention is further described using the following examples.

- 16 044626

Examples

Conventional abbreviations Name Precursor name CAS precursor Company manufacturer β-Ala β-Alanine Rshosph-alanine 35737-10-1 Fluorochem D-Asp D-Aspartic acid Fmoc-Daspartic acid 4-tert-butyl ester 112883-39-3 Sigma aidrich Fl 5(6)-carboxyfluorescein Sigma HArg Homoarginine Fmoc-LHomoArg(Pbf)-OH 401915-53-5 Fluorochem NuR Hydroxyproline FmocΓ hydroxyproline (tBu)-OH 122996-47-8 Sigma Sar Sarcosine, so Sar _x represents x residues of Sar Fmoc-Sarcosine-OH 77128-70-2 Sigma

Materials and methods.

Peptide synthesis.

Peptides were synthesized by solid-phase synthesis. Rink Amide MBHA resin was used. DMF was added to a mixture containing Rink Amide MBHA resin (0.4-0.45 mmol/g) and Fmoc-Cys(Trt)-OH (3.0 eq), followed by DIC (3 eq) and HOAt (3 eq) ) and mixed for 1 hour. For unblocking, 20% piperidine in DMF was used. Each subsequent amino acid was coupled using 3 equiv of activating reagents, DIC (3.0 equiv) and HOAT (3.0 equiv) in DMF. The reaction was monitored using the ninhydrin color reaction or the tetrachlor color reaction. After completion of the synthesis, the peptide resin was washed with DMF x 3, MeOH x 3, and then dried by bubbling N ₂ overnight. The peptide resin was then treated with 92.5% TFA/2.5% TIS/2.5% EDT/2.5% H ₂ O for 3 hours. The peptide was precipitated with cold isopropyl ether and centrifuged (3 minutes at 3000 rpm). min). The precipitate was washed twice with isopropyl ether, and the crude peptide was dried under vacuum for 2 hours and then lyophilized. The lyophilized powder was dissolved in a mixture of ACN/H2O (50:50), and a solution of 100 mM TATA in ACN was added, then ammonium bicarbonate in _H2O (1M), and the solution was stirred for 1 hour. After completion of cyclization, the reaction was stopped with 1M aqueous solution of cysteine hydrochloride (10 equiv relative to TATA), then stirred and incubated for one hour. The solution was lyophilized to obtain the crude product. The crude peptide was purified by preparative high-performance liquid chromatography (HPLC) and lyophilized to obtain the product.

Unless otherwise stated, all amino acids were used in L configurations.

BCY6099

BCY6099

Sequence: (e-Ala)-Sar ₁₀ -(SEQ ID NO: 2)-CONH ₂ .

8.0 g of resin was used to generate 2.1 g of BCY6099 (99.2% purity; 16.3% yield) as a white solid.

- 17 044626

BCY6099 analysis data Mobile phase: A: 0.1% TFA in _H2O B: 0.1% TFA in ACN Consumption: 1.0 ml/min Column: Gemini-NX C18 5 microns 110A 150 x 4.6 mm Device: Agilent 1200 HPLC-BE(1-614) Method: 15-45% B for 20 minutes, then 3 minutes 95% B Retention time: 11.31 min LCMS (ESI): m/z 1061.8 [M+ZN] ³⁺ , 796.5 [M+4N] ⁴⁺ Peptide molecular weight 3183.68

Preparation of Bicyclic Peptide Drug Conjugates A general scheme for the preparation of bicyclic drug conjugates (BDCs) is shown in FIG. 1, and in table. And the targeting bicycle component and linker/toxic agent within each BDC are described.

Table A

BDC (BCY number) T Argent Bicycle (BCY number) Linker/toxic agent 6135 6099 DM1-SS- 6136 6099 ValCit-MMAE 6173 6099 DM1-SS(SO ₃ H)- 6174 6099 ValLys-MMAE 6175 6099 MMAE-D-Ala-Phe-Lys-

BCY6099 (114.1 mg, 35.84 μmol) was used as the bicyclic reagent. 22.4 mg of compound BCY6135 (5.30 µmol, 17.74% yield, 95.14% purity) was obtained as a white solid.

BCY6135 analysis data Mobile phase: A: 0.1% TFA in _H2O B: 0.1% TFA in ACN Consumption: 1.0 ml/min Column: Gemini-NX C18 5 microns 110A 150 x 4.6 mm Device: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B for 30 minutes, then 3 minutes 95% IN Retention time: 9.81 min LCMS (ESI): m/z 1341.5 [M+ZN] ³⁺ , 805.0 [M+5N] ⁵⁺ Peptide molecular weight 4021.08

BCY6136

BCY6099 (71.5 mg, 22.48 μmol) was used as the bicyclic reagent. Compound BCY6136 (40.9 mg, 9.05 µmol, 40.27% yield, 97.42% purity) was obtained as a white solid.

BCY6136 analysis data Mobile phase: A: 0.1% TFA in _H2O B: 0.1% TFA in ACN Consumption: 1.0 ml/min Column: Gemini-NX C18 5 microns 110A 150 x 4.6 mm Device: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B for 30 minutes, then 3 minutes 95% B Retention time: 11.35 min LCMS (ESI): m/z 1468.1 [M+ZN] ³⁺ , 1101.2 [M+4N] ⁴⁺ , 881.3 [M+5N] ⁵⁺ Peptide molecular weight 4404.2

BCY6173

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BCY6099 (200.15 mg, 62.89 µmol) was used as the bicyclic reagent. 57.1 mg of BCY6173 (3.40 µmol, 22.79% yield, 95.80% purity) was obtained as a white solid.

BCY6173 Analysis data Mobile phase: A: 0.1% TFA in _H2O B: 0.1% TFA in ACN Consumption: 1.0 ml/min Column: Gemini-NX C18 5 microns 110A 150 x 4.6 mm Device: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B for 30 minutes, then 3 minutes 95% B Retention time: 10.30 min LCMS (ESI): m/z 1361.9 [M+ZN-N ₂ O] ³⁺ , 1021.8 [M+4N-N ₂ O] ⁴⁺ Peptide molecular weight 4101.15 BCY6174

The bicyclic reagent used was BCY6099 (389.77 mg, 122.47 µmol, 1.2 equiv).

Dde-BCY6174 (0.250 g, 55.10 µmol, 53.99% yield) was obtained as a white solid.

| LCMS (ESI): | m/z 1513.0 [M+ZN] ³⁺ , 1135.0 [M+4H] ⁴⁺ , 908.2 [M+5H] ⁵⁺ | | Molecular weight | 4538.38 |

Deprotect Dde-BCY6174 (0.250 g, 55.10 µmol, 1.0 eq) using hydrazine according to the general procedure to give BCY6174 (0.1206 g, 27.45 µmol, 49.82% yield) in as a white solid.

BCY6174 analysis data Mobile phase: A: 0.1% TFA in _H2O B: 0.1% TFA in ACN Consumption: 1.0 ml/min Column: Gemini-NX C18 5 µm 110A 150x4.6 mm Device: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B for 30 minutes, then 3 minutes 95% B Retention time: 9.85 min LCMS (ESI): m/z 1458.5 [M+ZN] ³⁺ , 1094.1 [M+4N] ⁴⁺ , 875.4 [M+5N] ⁵⁺ Peptide molecular weight 4373.17

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BCY6175

General procedure for obtaining compound 10A.

To a solution of BCY6099 (195.15 mg, 61.32 μmol, 1.1 eq) in DMA (3 ml) was added DIEA (21.61 mg, 167.23 μmol, 29.13 μl, 3 eq) and compound 9 ( 0.085 g, 55.74 µmol, 1.0 equiv). The mixture was stirred at 25°C for 16 hours. The moment of complete consumption of compound 9 was monitored by LC-MS, and one main peak with the required m/z ratio was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent to leave a residue (light yellow oil). The reaction mixture was directly purified by preparative HPLC (neutral condition). Compound 10A (0.160 g, 34.84 µmol, 62.50% yield) was obtained as a white solid.

General procedure for obtaining BCY6175.

To a solution of compound 10A in DCM (4.5 ml) was added TFA (4.5 ml). The mixture was stirred at 0°C for 30 minutes. Using the LC-MS method, the moment of complete consumption of compound 10A was monitored, and one main peak with the required m/z ratio was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent to obtain a residue, which was purified by preparative HPLC (TFA condition). BCY6175 (61.40 mg, 13.56 µmol, 31.13% yield) was obtained as a white solid.

Biological activity data

Study 1: Fluorescence polarization measurements.

(a) Direct binding assay.

Fluorescently tagged peptides (either fluorescein from SIGMA or Alexa Fluor488™ from Fisher Scientific) were diluted to 2.5 nM in PBS with 0.01% tween 20 or in 50 mM HEPES with 100 mM NaCl and 0.01 % tween pH 7.4 (both called assay buffer). The protein was added to the same solution of the peptide in the assay buffer and titrated to obtain 1 nM of peptide in a total of 25 μl in 384-well plates with black walls and bottom with a low volume and low degree of binding, resulting in 5 μl of buffer for analysis, 10 μl of protein (Table 1), then 10 μl of fluorescent peptide. Serial one-in-two dilutions were used to obtain 12 different concentrations with highest concentrations ranging from 500 nM for known high affinity binding drugs to 10 µM for known low affinity binding drugs and to conduct selectivity studies . Measurements were performed on a BMG PHERAstar FS microplate reader equipped with an FP 485 520 520 optical module, which uses excitation radiation at 485 nm and detects parallel and perpendicular emission at 520 nm. The PHERAstar FS was used at 25°C with 200 flashes per well and a 0.1 s position delay, with each well measured at 5 to 10 min intervals for 60 min. The increment used for the analysis was determined for each labeled substance after 60 min, when there was no protein in the well. Data analysis was performed using Systat Sigmaplot version 12.0 software. The mP values were approximated using a user-defined second order equation to obtain the Kd value:

f^ymin+(ymax-ymin)/Lig*((x+Lig+Kd)/2-sqrt((((x+Lig+Kd)/2) ^A 2)-(Lig*x))).

Lig was the detectable value of the concentration of the labeled substance used

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(b) Competitive binding assay.

Peptides without a fluorescent label were tested for competitive binding with a peptide with a fluorescent label and a known Kd value (Table 2). Reference compound A has the sequence Fl-G-Sar5-ACPWGPAWCPVNRPGCA (Fl-G-SaMSEQ ID NO: 4)). Reference compound B has the sequence Fl-G-Sar ₅ -ACPWGPFWCPVNRPGCA (Fl-G-Sar ₅ -(SEQ ID NO: 5)). Reference compound C has the sequence Fl-G-Sar ₅ -ADVTCPWGPFWCPVNRPGCA (Fl-G-Sar ₅ (SEQ ID NO: 6). Reference compounds A, B and C each contain a TBMV molecular framework. The peptides were diluted to the appropriate concentration in buffer for assay described for the direct binding assay, with a maximum of 5% DMSO, then serially diluted 1 to 2. Five microliters of the diluted peptide was added to the plate, followed by 10 μl of human or mouse EphA2 (Table 1) at a fixed concentration, which depended on the used fluorescent peptide (Table 2), then 10 μl of fluorescent peptide was added. Measurements were carried out in the same way as in the case of direct binding assay, however, the increment was determined before the first measurement. Data analysis was performed using Systat Sigmaplot version 12.0 software. mP was approximated using a user-defined third order equation to obtain the value Ki:

f=ymin+(ymax-ymin)/Lig*((Lig*((2*((Klig+Kcomp+Lig+Comp-Prot*c) ^A 23*(Kcomp*(Lig-Prot*c)+Klig*( Comp-Prot*c)+Klig*Kcomp)) ^A 0.5*COS(ARCCOS((2 * (Klig+Kcomp+Lig+Comp-Prot* c) ^A 3+9 * (Klig+Kcomp+Lig+Comp-Prot * c) * (Kcomp * (LigProt*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)-27*(1 *Klig*Kcomp *Prot* c))/(2 * (((( Klig+Kcomp+Lig+Comp-Prot* c) ^A 2-3 * (Kcomp * (LigProt*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)) ^A 3) ^A 0.5)))/ 3 ))-(Klig+Kcomp+Lig+CompProt*c)))/((3*Klig)+((2*((Klig+Kcomp+Lig+Comp-Prot*c) ^A 2-3*(Kcomp *(LigProt*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)) ^A 0.5*COS(ARCCOS((2 * (Klig+Kcomp+Lig+Comp-Prot* c) ^A 3+9 * (Klig+Kcomp+Lig+Comp-Prot* c) * (Kcomp * (LigProt*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)-27*(1 *Klig*Kcomp *Prot* c ))/(2 * ((((Klig+Kcomp+Lig+Comp-Prot* c) ^A 2-3 * (Kcomp * (LigProt*c)+Klig*(Comp-Prot*c)+Klig*Kcomp) ) ^A 3) ^A 0.5)))/3 ))-(Klig+Kcomp+Lig+CompProt*c)))).

Lig, Klig, and Prot were detectable values related to fluorescent peptide concentration, fluorescent peptide Kd, and EphA2 concentration, respectively.

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Table 1

Ephrin receptors and source

Receptor (domain) View Format/label Supplier company Catalog no. EphAl (Ecto) Human Fc-fusion R&D systems 7146-Al EphA2 (Ecto) Human C-terminal PAULINE S R&D systems 3035-A2 EphA2 (Ecto) Human C-terminal PAULINE S In-house N/A EphA2 (Ecto) Mouse Fc-fusion R&D Systems 639-A2 EphA2 (Ecto) Mouse C-terminal PAULINE S Sino Biological 50586-M08H EphA2 (ligand binding) Rat C-terminal PAULINE S In-house N/A EphA2 (ligand binding) Dog C-terminal PAULINE S In-house N/A EphA3 (Ecto) Human Fc-fusion R&D systems 6444-A3 EphA3 (Ecto) Human N-terminal PAULINE S In-house N/A EphA3 (Ecto) Rat C-terminal PAULINE S Sino Biological 80465-R08H EphA4 (Ecto) Human Fc-fusion R&D systems 6827-A4 EphA4 (Ecto) Human C-terminal PAULINE S Sino Biological 11314-H08H EphA4 (Ecto) Rat C-terminal polyH S Sino Biological 80123-R08H EphA6 (Ecto) Human Fc-fusion R&D systems 5606-A6 EphA7 (Ecto) Human Fc-fusion R&D systems 6756-A7 EphBl (Ecto) Rat Fc-fusion R&D systems 1596-B1 EphB4 (Ecto) Human C-terminal PAULINE S R&D systems 3038-B4

table 2

Final concentrations of fluorescent peptide and EphA2 used in competitive binding assays

Fluorescent peptide Fluorescent peptide concentration (nM) Human EphA2 concentration (nM) Mouse EphA2 concentration (nM) Reference compound A 10 75 Reference connection B 1 thirty Reference connection C 0.8 (person) 1 (mouse) 2.4 50

Specific peptide ligands according to the invention were tested using the above-mentioned analytical methods, the results of which are shown in table. 3 and 4.

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Table 3

Biological test data for the peptide ligand of the invention (TATA peptides, competitive binding assay)

Bicyclic compound number Subsequence Frame Ki, nM ± 95% CI Human EphA2 Mouse EphA2 Fluorescent peptide, reference compound C BCY6099 (p-Ala)-San ₀ - A(HArg)DC(HyP)LVNPLCLHP( D-Asp)W(HArg)C (SEQ ID NO: 2) TATA 4.94±1.41 57.6±24.86

Table 4

Data from biological tests of peptide ligands according to the invention (data on competitive binding of BDCs to TATA scaffolds)

Connection number BDC Bicycle precursor General formula Frame Ki, nM, human EphA2 fluorescent peptide, reference compound C BCY6027 BCY6099 Formula (A) TATA 10.23 BCY6028 BCY6099 Formula (B) TATA 13.04

Study 2: Fluorescence polarization measurements (alternative protocol).

(a) Competitive binding.

Peptides without a fluorescent label were tested for competitive binding with a peptide with a fluorescent label and a known Kd value (Table 9).

Five microliters of the test compound were added to the plate with increasing concentrations (twofold), then 10 μl of EphA2 protein (Table 8) at a fixed concentration, which depended on the fluorescent peptide used (Table 9), then 10 μl of the fluorescent peptide was added. The buffer was assay buffer as described above with DMSO <1%. Measurements were performed on a BMG PHERAstar FS microplate reader equipped with an FP 485 520 520 optical module, which uses excitation radiation at 485 nm and detects parallel and perpendicular emission at 520 nm. The PHERAstar FS was used at 25°C with 200 flashes per well and a 0.1 s position delay, with each well measured at 5 to 10 min intervals for 60 min. Alternatively, measurements were performed at similar timings on a Perkin Elmer Envision plate analyzer equipped with a FITC FP Dual Mirror, FITC FP 480 excitation filter, and FITC FP P-pol 535 and FITC FP S-pol emission filters with 30 flashes and with a G-factor value of 1.2. Data analysis was performed using Systat Sigmaplot software version 12.0 or 13.0, where the mP values at 60 min were approximated using a user-defined third order equation to obtain the Ki value:

- 23 044626 f=ymin+(ymax-ymin)/Lig*((Lig ^:i: ((2*((Klig+Kcomp+Lig+Comp-Prot*c) ^A 23*(Kcomp*(Lig-Prot*c )+Klig*(Comp-Prot*c)+Klig*Kcomp)) ^A 0.5*COS(ARCCOS((2*(Klig+Kcomp+Lig+Comp-Prot*c) ^A 3+9*(Klig+Kcomp+ Lig+Comp-Prot*c)*(Kcomp*(LigProt*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)-27*(l*Klig*Kcomp*Prot*c))/(2 *((((Klig+Kcomp+Lig+Comp-Prot*c) ^A 2-3*(Kcomp*(LigProt*c)+Klig*(Comp-Prot* c)+Klig*Kcomp)) ^A 3) ^A 0.5)))/3 ))-(Klig+Kcomp+Lig+CompProt*c)))/((3 *Klig)+((2 * ((Klig+Kcomp+Lig+Comp-Prot* c) ^A 2 -3 * (Kcomp * (LigProt*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)) ^A 0.5*COS(ARCCOS((2*(Klig+Kcomp+Lig+Comp-Prot*c) ^A 3+9*(Klig+Kcomp+Lig+Comp-Prot*c)*(Kcomp*(LigProt*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)-27*(l*Klig* Kcomp*Prot*c))/(2*((((Klig+Kcomp+Lig+Comp-Prot*c) ^A 2-3*(Kcomp*(LigProt*c)+Klig*(Comp-Prot*c) +Klig*Kcomp)) ^A 3) ^A 0.5)))/3 ))-(Klig+Kcomp+Lig+CompProt*c)))).

Lig, Klig, and Prot were detectable values related to fluorescent peptide concentration, fluorescent peptide Kd, and EphA2 concentration, respectively.

Table 5

Eph receptors and source

Receptor (domain) View Format/label Supplier company Catalog number EphA2 (Ecto) Human C-terminal polyH S R&D systems 3035-A2 EphA2 (Ecto) Human C-terminal polyH S In-house N/A EphA2 (Ecto) Mouse C-terminal sex and His Sino Biological 50586-M08N EphA2 (ligand binding) Rat C-terminal POLINE18 In-house N/A

Table 6

Final concentrations of fluorescent peptide and EphA2 used in competitive binding assays

Fluorescent peptide Fluorescent peptide concentration (nM) Human EphA2 concentration (nM) Mouse EphA2 concentration (nM) Concentration of rat EphA2 (nM) Reference connection C 0.8 2.4 or 25 50 or 15 nM 25

Specific peptide ligands and bicyclic drug conjugates of the invention were tested in the above-mentioned competitive binding assay, and the results are presented in table. 7.

Table 7

Competitive binding with selected bicyclic peptides

Bike number Human Ki (nM) Mouse Ki (nM) Rat Ki (nM) BCY6099 2.7 4.5 1.9

The results of the competitive binding analysis are in table. 7 show that human EphA2-targeting bicyclic peptides (BCY6099) bind with high affinity to mouse and rat EphA2. These results indicate that the peptide of the invention can be used in in vivo experimental models in mice and rats to conduct studies assessing efficacy and toxic effects.

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Table 8

Competitive binding with selected bicyclic drug conjugates (BDCs)

Bicycle identification number Human Ki (nM) Mouse Ki (nM) Rat Ki (nM) BCY6027 10.2 BCY6028 13.0 BCY6135 2.4 5.0 2.9 BCY6136 1.9 5.5 3.2 BCY6173 1.7 4.3 2.5 BCY6174 1.7 3.9 h,o

The data given in table. 8 show that specific bicyclic drug conjugates of the invention exhibit excellent cross-reactivity between human, mouse and rodent EphA2. Therefore, the peptide of the invention can be used in in vivo experimental models in mice and rats to conduct studies assessing efficacy and toxic effects.

(b) Surface plasmon resonance (SPR) measurements.

Non-Fc-fusion proteins were biotinylated using EZ-Link™ Sulfo-NHS-LCBiotin for 1 h in 4 mM sodium acetate, 100 mM NaCl, pH 5.4 at a three-fold molar excess of biotin relative to the protein. The degree of labeling was determined using a Fluorescence Biotin Quantification Kit (Thermo) after dialysis of the reaction mixture in PBS. For peptide binding analysis, a Biacore T200 instrument with a XanTec CMD500D chip was used. Streptavidin was immobilized on the chip using a standard amine coupling chemistry at 25°C with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as running buffer. Briefly, the carboxymethyldextran surface was activated by injecting a 1:1 mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/0.1 M N-hydroxy-succinimide (NHS) over 7 min at a flow rate of 10 µl/min. For streptavidin capture, the protein was diluted to 0.2 mg/mL in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μL onto the activated chip surface. Residual activated groups were blocked by introducing a mixture of 1 M ethanolamine (pH 8.5):HBS-N (1:1) for 7 min. The buffer was changed to PBS/0.05% Tween 20, and biotinylated EphA2 was captured to 500–1500 resonance units using a 0.2 μM protein dilution in buffer. A series of peptide dilutions were prepared in this buffer with a final DMSO concentration of 0.5%, with the highest peptide concentration being 50 or 100 nM and 6 additional 2-fold dilutions. SPR analysis was carried out at 25°C at a flow rate of 90 μl/min with 60 s of association and 900-1200 s of dissociation. Data were adjusted to exclude volumetric effects of DMSO. All data were double-matched to blank injections and a reference surface using standard processing procedures, and data processing and kinetic data fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Data were fitted using a 1:1 simple coupling model incorporating mass transfer effects where appropriate.

A Biacore 3000 instrument was used to study the binding of bicyclic drug conjugates. For biotinylated proteins, immobilization levels were 1500 resonance units and the highest concentration was 100 nM. In all other respects, the method was the same as the method described above, using either the CMD500D chip or the CM5 chip (GE Healthcare). For Fc-tagged proteins, the CM5 chip was activated as described above, and then goat anti-human IgG (Thermo-Fisher H10500) was diluted to 20 μg/ml in 10 mM sodium acetate pH 5.0 and captured to approximately 3000 resonance units. The surface was then blocked as described above. Sequential capture of Fc-tagged proteins was carried out to obtain approximately 200-400 resonance units of the target protein. The proteins used are described below. All proteins were resuspended in manufacturer-recommended buffers and concentrations and captured using 5–10 μg/ml protein in PBS/0.05% Tween 20.

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Table 9

Receptor View Format/label Supplier company Catalog no. EphAl Human Fc-fusion Sino Biologies 15789-H02H EphA2 Human 0.95 mol biotin/monomer In house N/A EphA2 Mouse F c-merge R&D Systems 639-A2 EphA2 Rat 1.4 mol biotin/monomer In house N/A EphA3 Human Fc-fusion R&D Systems 6444-A3 EphA3 Mouse F c-merge Sino Biologies 51122-M02H EphA3 Rat Fc-fusion Sino Biologies 80465-R02H EphA4 Human F c-merge Sino Biologies 11314-H03H EphA4 Mouse Fc-fusion Sino Biologies 50575-M02H EphA4 Rat Fc-fusion Sino Biologies 80123-R02H EphA5 Human 3.1 mol biotin/monomer R&D Systems 3036-A5 EphA6 Human F c-merge R&D Systems 5606-A6 EphA7 Human Fc-fusion R&D Systems 6756-A7 EphBl Rat F c-merge R&D Systems 1596-B1 EphB4 Human F c-merge Sino Biologies 10235-H02H

Specific peptide ligands and bicyclic drug conjugates of the invention were tested in the above-mentioned competitive binding assay, and the results are presented in table. 10-12.

Table 10

SPR binding analysis of selected bicyclic peptides and bicyclic drug conjugates of the invention

Bicycle number/B DC Human Mouse Rat K _D (hM) K ₀ ff (s ¹ ) tl/2 (min) Kon (MY ¹ ) K _D (hM) K ₀ ff (s ¹ ) tl/2 (min) Kon (MY ¹ ) K _D (hM) K ₀ ff (s ¹ ) tl/2 (min) Kon (MY ¹ ) BCY6136 1D7 1D5E-03 10.0 9.86E+05 2.53 1D1E-03 10.4 4.37E+05 2.96 9D1E-04 12.6 3.07E+0 5 BCY6173 0.73 Ts24E-03 9.3 Ts69E+06 2.95 1D4E-03 10.1 3.86E+05 1.10 9.60E-04 12.0 8.81E+0 5

In table 10 provides detailed information on the binding affinities and kinetic parameters (K _off and K _op ) for the binding of selected bicyclic drug conjugates to human EphA2 obtained by SPR.

Table 11

SPR binding analysis of selected bicyclic drug conjugates of the invention to human Eph homologues

BDC number EphAl EphA3 EphA4 EphA5 EphA6 EphA7 EphB4 BCY61 36 no binding @ 5 µm no binding @ 5 µm no binding @ 5 µm no binding @25 µm no binding @ 20µM no binding @ 20µM no binding @ 20µM BCY61 73 no binding @ 5 µm no binding @ 5 µm no binding @ 5 µm no binding @25 µm no binding @ 20µM no binding @ 20µM no binding @ 20µM

In table 11 shows binding results for two bicyclic drug conjugates (BCY6136 and BCY6173) in SPR assays with closely related human ephrin homologs. The results show that the compounds of the invention do not exhibit significant binding to the closely related human homologs EphAl, EphA3, EphA4, EphA5, EphA6, EphA7 and EphB4.

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Table 12

SPR binding analysis of selected bicyclic drug conjugates of the invention to mouse and rat Eph orthologues

BDC number Mouse EphA3 Mouse EphA4 Rat EphA3 EphBl rats BCY61 absence absence absence absence binding binding binding linking @ 36 @ 20 µM @ 20 µM @ 20 µM 20 µM BCY61 absence absence absence absence binding binding binding binding 73 @ 20 µM @ 20 µM @ 20 µM @ 20 µM

Results in table. 12 show that the specific bicyclic drug conjugates of the invention (BCY6136 and BCY6173) are also selective for mouse and rat EphA2 and do not exhibit significant binding to the closely related homologues of mouse EphA3 and EphA4, and rat EphA3 and EphB1.

Studies 3 and 7-23.

In each of Studies 3 and 7-23, the following methodology was used for each study:

(a) Materials.

(i) Animals and conditions of their keeping.

Animals.

Species: house mouse.

Strain: Balb/c nude or CB17-SCID.

Age: 6-8 weeks.

Body weight: 18-22 g.

Number of animals: 9-90 mice.

Animal supplier: Shanghai Lingchang Biotechnology Experimental Animal Co. Limited.

Conditions of detention.

Mice were kept in individually ventilated cages at constant temperature and humidity with 3-5 animals in each cage.

Temperature: 20~26°C.

Humidity 40-70%.

The cages are made of polycarbonate. Size 300 mm x 180 mm x 150 mm. The bedding material is a corn cob, which is replaced twice a week.

Diet. Animals had free access to irradiation-sterilized dry pelleted food throughout the study period.

Water. Animals had free access to sterilized drinking water.

Cell identification. Identification labels for each cage contained the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and date of treatment.

Animal identification. Animals were marked by ear marking.

(ii) Test and positive control materials.

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Number Description of physical condition Molecular weight Purity Condition storage BCY6135 Liof silt powder 4021 95.14% Stored at -80°С BCY6136 Liof silt powder 4402.23 97.5-98.6% Stored at -80°С BCY6173 Liof silt powder 4101.15 95.80% Stored at -80°С BCY6174 Liof silt powder 4537 99.50% Stored at -80°С BCY6175 Liof silt powder 4492.29 96.20% Stored at -80°С BCY8245 Liof silt powder 4173.85 99.30% Stored at -80°С BCY8781 Liof silt powder 4173.83 99.00% Stored at -80°С ADC (MEDI547) ¹ Solution (concentration 10.47 mg/ml) - >99.00% Stored at -80°С

¹ Details of MEDI-547 (a fully human monoclonal antibody 1C1 (recognizing both human and murine EphA2) conjugated to MMAF via an MS linker) are given in Jackson et al (2008) Cancer Res 68, 9367-74.

(b) Experimental methods and techniques.

(i) Observations.

All procedures related to the housing, care and treatment of study animals were carried out in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the international pharmaceutical, biopharmaceutical and health care company WuXi AppTec, following the guidelines of the International Association for Laboratory Assessment and Accreditation of Laboratory Research on animals (AAALAC). During the routine monitoring program, animals were examined daily for any effects of tumor growth and treatment on normal behavior such as mobility, food and water intake (by visual inspection only), weight gain/loss, and eye dullness/ wool and any other abnormal influence specified in the protocol. The event of death and observed clinical manifestations were recorded taking into account the number of animals in each subgroup.

(ii) Tumor measurements and final clinical results.

The primary clinical endpoint was whether the mice were able to delay tumor growth or be cured. Tumor volume was measured three times a week in two directions using calipers, and the volume was expressed in mm ³ using the formula: V=0.5 a x b ² where a and b represent the largest and smallest diameters of the tumor, respectively. Tumor size was then used to calculate T/C values. The T/C value (in percent) is an indicator of antitumor activity; T and C represent the average volumes in the treated and control groups, respectively, on a given day.

TGI was calculated for each group using the formula:

TGI (%) = [1-(Ti-T0)/(Vi-Vc)] x 100;

Ti is the mean tumor volume in the treatment group on a given day, T ₀ is the mean tumor volume in the treatment group on the day of treatment, Vi is the mean tumor volume in the placebo control group on the same day as for Ti, and V ₀ represents the mean tumor volume in the placebo group on the day of treatment.

(iii) Sample collection.

At the end of the study, tumors in all groups were collected for formalin fixation and paraffin embedding (FFPE).

(iv) Statistical analysis.

Summary statistics including mean and standard error of the mean (SEM) are reported for tumor volume in each group at each time point.

Statistical analysis of differences in tumor volume between groups was performed on data obtained at the time point of achieving the best therapeutic result after the last dose.

To compare tumor volume between groups, one-way analysis of variance (ANOVA) was used, and then statistical significance was estimated with the Fisher F test (the ratio of treatment variance to error variance), comparisons between groups were performed using the Games-Howell test. -Howell). All data were analyzed using GraphPad Prism 5.0 software. A value of P < 0.05 was considered statistically significant.

Study 3: To determine if there is a relationship between copy number variation (CNV) and gene expression for EphA2 in multiple tumor types.

Methods.

1. Select all studies from cBioPortal (http://www.cbioportal.org/) and search for EHA2 studies.

(a) Remove preliminary studies.

(b) Deselect a study with overlapping samples to prevent sampling error (based on the cBioPortal warning), always select the PanCancer study if this selection can be used.

(c) Studies selected for analysis (Table 13).

Table 13 Studies analyzed in cBioPortal and subsections within the study

Study title Subsections Invasive breast carcinoma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Squamous cell carcinoma of the lung (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Papillary renal cell carcinoma (TCGA, PanCancer Atlas) mRNA Expression, RSEM (batch normalized from Illumina HiSeq_RNASeqV2) Clear cell carcinoma of the kidney (TCGA, PanCancer Atlas) mRNA Expression, RSEM (batch normalized from Illumina HiSeq_RNASeqV2) Colon adenocarcinoma (TCGA, PanCancer Atlas) RSEM (batch normalized from Illumina HiSeq_RNASeqV2) Squamous cell carcinoma of the head and neck (TCGA, PanCancer Atlas) mRNA Expression, RSEM (batch normalized from Illumina HiSeq_RNASeqV2) Urothelial carcinoma of the bladder (TCGA, PanCancer Atlas) RSEM (batch normalized from Illumina HiSeq_RNASeqV2) Uveal melanoma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Lung adenocarcinoma (TCGA, PanCancer Atlas) mRNA Expression, RSEM (batch normalized from Illumina HiSeq_RNASeqV2)

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Serous cystadenocarcinoma of the ovary (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Breast Cancer (METABRIC, Nature 2012 & Nat Commun 2016) mRNA expressions (microarray analysis) Mesothelioma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Colorectal adenocarcinoma (TCGA, Nature 2012) RNA sequence RPKM Squamous cell carcinoma of the cervix (TCGA, PanCancer Atlas) RSEM (batch normalized from Illumina HiSeq_RNASeqV2) Sarcoma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Encyclopedia of Cancer Cell Lines (Novartis/Broad, Nature 2012) mRNA expressions (microarray analysis) Colon adenocarcinoma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Hepatocellular carcinoma of the liver (TCGA, PanCancer Atlas) ERNA2: mRNA expression, RSEM (batch normalized from Illumina HiSeq_RNASeqV2) Adenocarcinoma of the stomach (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Endometrial carcinoma of the uterine body (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Cutaneous melanoma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Prostate adenocarcinoma (TCGA, PanCancer Atlas) mRNA Expression, RSEM (batch normalized from Illumina HiSeq_RNASeqV2)

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Chromophobe renal cell carcinoma (TCGA, PanCancer Atlas) mRNA Expression, RSEM (batch normalized from Illumina HiSeq_RNASeqV2) Wilms tumor in childhood (TARGET, 2018) Epha2: mRNA expression (RNA sequence RPKM) Pheochromocytoma and paraganglioma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Thyroid carcinoma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Esophageal adenocarcinoma (TCGA, PanCancer Atlas) RSEM (batch normalized from Illumina HiSeq_RNASeqV2) Cholangiocarcinoma (TCGA, PanCancer Atlas) RSEM (batch normalized from Illumina HiSeq_RNASeqV2) Low-grade brain glioma (TCGA, PanCancer Atlas) RSEM (batch normalized from Illumina HiSeq_RNASeqV2) Thymoma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Acute lymphocytic leukemia in childhood - phase II (TARGET, 2018) Epha2: mRNA expression (RNA sequence RPKM) Diffuse large B cell lymphoma (TCGA, PanCancer Atlas) mRNA Expression, RSEM (batch normalized from Illumina HiSeq_RNASeqV2) Glioblastoma multiforme (TCGA, PanCancer Atlas) mRNA Expression, RSEM (batch normalized from Illumina HiSeq_RNASeqV2) Metastatic Prostate Cancer, SU2C/PCF Dream Team (Robinson et al., Cell 2015) mRNA expression/capture (RNAseq RPKM) Acute myeloid leukemia (TCGA, PanCancer Atlas) mRNA Expression, RSEM (batch normalized from Illumina HiSeq_RNASeqV2)

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Testicular germ cell tumors (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Adrenocortical carcinoma (TCGA, PanCancer Atlas) RSEM (batch normalized from Illumina HiSeq_RNASeqV2) Uterine carcinosarcoma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Pancreatic adenocarcinoma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeq_RNASeqV2 syn4976369 Prostate adenocarcinoma (MSKCC, Cancer Cell 2010) mRNA expression Adenocarcinoma of the prostate (Fred Hutchinson CRC, Nat Med 2016) mRNA expression

2. Export copy number variations (CNVs) and RNA expression data from cBioPortal.

3. Test that copy number variations (CNVs) are statistically significantly associated with changes in mRNA expression for EphA2 (log2 is not used).

(a) Run the nonparametric Kruskal-Wallis test in GraphPad Prism (7.04) and R/R studio (significance threshold: p < 0.01).

(i) GraphPad Prism: Set a column in a table, run a nonparametric test without fitting or matching, and do not assume a Gaussian distribution.

(ii) Packages used in R:

1) XLConnect;

2) dplyr;

3) criterion for the Kruskal-Wallis rank sum: Kruskal.test.;

4) correct for multiple comparisons (include all possible comparisons, even if n=1 within group) in R/Rstudio using Dunn's test (threshold for significance: p<0.025).

(a) dunn.test with multiple comparison method = bonferroni.

Results.

The results are presented in table. 14 below. Across all 41 publicly available datasets collected in cBioPortal that report both copy number variation (CNV) and mRNA gene expression for EphA2, there are numerous cancer types in which cases of small EphA2 deletions (<2 copies) have been reported. . Although less common, in these same cancer types, a subpopulation of tumors harbored deep EphA2 deletions (>1 copy loss or biallelic loss), EphA2 gain (2-3 copies), or EphA2 amplifications (>3 copies). Cases in which >33% of tumors had either shallow deletions or deep deletions in EphA2 included: chromophobe renal cell carcinoma, cholangiocarcinoma, pheochromocytoma and paraganglioma, squamous cell carcinoma of the lung, breast, rectum, low-grade brain glioma, liver cancer , adrenocortical carcinoma, mesothelioma, esophageal adenocarcinoma and colon cancer. In contrast, there were no studies in which >33% of samples had either gains or amplifications in EphA2. Taken together, these results indicate that deletions in EphA2 DNA are found across a range of cancer types.

Approximately one-third of all samples analyzed in 41 studies harbored EphA2 copy number variations (CNVs). Based on this high percentage of copy number variations (CNVs) in all studies and the high percentage of small deletions in specific tumor types, a statistical test was performed to identify possible relationships between copy number changes and RNA expression. Tumors were classified into 1 of 5 classes:

a) deep deletion;

b) small deletion;

c) diploid;

d) increment; or

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e) amplification.

The Kruskal-Wallis test was then performed to determine whether there were differences between classes (P < 0.01) in the distribution of mRNA expression values across classes. For these TCGA datasets with P < 0.01 and to determine which classes differed from each other, post hoc testing was performed by calculating Z-statistics with adjusted calculated P-values (Bonferoni). For ease of interpretation, pairwise comparisons were considered against diploid by index (although all pairwise P-values were calculated). 19/41 of these studies had a Kruskal-Wallis p-value <0.01, indicating that copy number was statistically significantly associated with RNA expression. Of these 19 studies, 17 had a Bonferroni-corrected P < 0.025 for diploid versus small deletion, indicating that decreased EphA2 mRNA expression was associated with decreased EphA2 copy number. Only 2 of these 19 studies had a Bonferroni-corrected P < 0.025 for diploid versus increment, and both were breast cancer studies. In addition, one of these breast cancer studies (invasive breast carcinoma (TCGA, PanCancer Atlas)) had a Bonferroni adjusted P < 0.025 for both diploid relative to small deletion and diploid relative to gain, suggesting that copy number alterations may have a profound effect on EphA2 RNA expression in breast cancer.

The central dogma of genetics suggests that decreased copy number in EphA2 results in decreased RNA and protein expression. Therefore, the observed relationships between EphA2 copy number loss and decreased mRNA expression in various tumor types suggest that EphA2 protein expression may also be decreased. Likewise, EphA2 copy number gains in breast cancer, which were associated with increased mRNA expression, may also suggest increased EphA2 protein expression. Moreover, higher EphA2 protein expression (measured by fluorescence-activated cell sorting (FACS)) is associated with increased EphA2 efficacy of specific bicyclic drug conjugates of the invention (measured by tumor volume) in preclinical in vivo models. Based on the totality of the above facts, it can be assumed that if copy number alterations, which are interrelated with changes in mRNA expression, do indeed predict protein expression levels, then patients with tumors containing EphA2 copy number deletions may, with a certain degree of probability, respond less to treatment with bicyclic drug conjugates of the invention that are specific to EphA2. Likewise, if tumor patients have EphA2 copy number gain (eg, breast cancer), then it is possible that these patients are more likely to respond to treatment with the bicyclic EphA2 drug conjugates of the invention. Therefore, if patients were stratified by EphA2 copy number status, this information could be used to both exclude and select patients to improve the effectiveness of treatment with the EphA2-specific bicyclic drug conjugates of the invention.

Table 14

Results of a study of the relationship between copy number variations (CNVs) and gene expression for EphA2

Study title Subsections Number of samples/group (n=X) Kruskal-Wallis criterion Pairwise comparison, Z-statistic (r-value correction), Bonferroni Deep deletion Minor deletion Diploid Increments Amplification KruskalWallis Statistics p-value Deep deletion diploid Diploid small deletion Diploid increments Amplification pippipp Invasive breast carcinoma (TCGA, PanCancer Atlas) Batch of normalized/pooled mRNA expression from Illumina HiSeqRNASe qV2 syn4976369 5 415 511 61 2 80,816 <2.2E-16 0.176118 (1.0000) 6.460580 (0.0000)* 4.603180 (0.0000)* 0.713978 (1.0000) Squamous I lung carcinoma (TCGA, PanCancer Atlas) Batch of normalized/pooled mRNA expression from 3 207 201 55 0 52,942 1.89E-11 -1.584610 (0.3392) 6.786501 (0.0000)* 0.019607 (1.0000) N/A

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Illumina HiSeqRNASe qV2 syn4976369 Papillary renal cell carcinoma (TCGA, PanCancer Atlas) mRNA expression, rsem (batch normalized from Illumina HiSeqRNASe qV2) 1 48 224 0 1 42,161 3.71E-09 -1.586207 (0.3381) 6.097375 (0.0000)* N/A 1.549107 (0.3641) Clear cell I kidney carcinoma (TCGA, PanCancer Atlas) mRNA expression, RSEM (batch normalized from Illumina HiSeqRNASe qV2) 0 69 278 5 0 38,342 4.72E-09 N/A 6.133219 (0.0000)* 0.487059 (0.9393) N/A Colon adenocarcinoma (TCGA, PanCancer Atlas) RSEM (batch normalized from Illumina HiSeqRNASe qV2) 3 132 245 8 0 35,397 1.00E-07 -2.158194 (0.0927) 5.670600 (0.0000)* 0.781046 (1.0000) N/A Squamous cell carcinoma of the head and neck (TCGA, PanCancer Atlas) mRNA expression, RSEM (batch normalized from Illumina HiSeqRNASe qV2) 3 86 345 54 0 32.72 3.69E-07 .2.444914 (0.0435) 4.680789 (0.0000)* 1.530670 (0.3776) N/A Urothelial carcinoma of the bladder (TCGA, PanCancer Atlas) RSEM (batch normalized from Illumina HiSeqRNASe qV2) 0 73 245 80 4 28,906 2.34E-06 N/A 5.203251 (0.0000)* 0.211744 (1.0000) 0.581704 (1.0000) Uveal melanoma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeqRNASe qV2 syn4976369 0 24 56 0 0 21,051 4.47E-06 N/A 4.588095 (0.0000)* N/A N/A

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Lung adenocarcinoma (TCGA, PanCancer Atlas) Serous cystadenocarcinoma of the ovary (TCGA, PanCancer Atlas) mRNA expression, RSEM (batch normalized from Illumina HiSeqRNASe qV2) Batch of normalized/pooled mRNA expression from Illumina HiSeqRNASe qV2 syn4976369 1 0 115 59 263 78 121 60 3 4 28,874 25,349 8.29E-06 1.31E-05 -0.690460 (1.0000) N/A 4.280100 (0.0001)* 4.390097 (0.0000)* 0.626707 (1.0000) 0.239249 (1.0000) 2.276458 (0.1141) 0.240543 (1.0000) Breast cancer glands Expressions (METABRIC, Nature 2012 & mRNA (microarray 1 491 1349 25 0 23,875 2.65E-05 0.568937 (1.0000) 2.274564 (0.0688) 4.115288 (0.0001)* N/A Nat Commun nal analysis) 2016) The consignment normalized noah/ united Mesothelioma Noah (TCGA, PanCancer mRNA expression from 0 29 50 3 0 18,866 8.00E-05 N/A 4.319425 (0.0000)* 0.170478 (1.0000) N/A Atlas) Illumina HiSeqRNASe qV2 syn4976369 Colorectal adenocarcinoma a (TCGA, Nature 2012) RNA sequence RPKM 0 53 138 2 0 18,847 8.08E-05 N/A 4.298092 (0.0000)* 0.338975 (1.0000) N/A Squamous cell carcinoma of the cervix (TCGA, PanCancer Atlas) RSEM (batch normalized from Illumina HiSeqRNASe qV2) 1 31 167 76 0 19,435 2.22E-04 -1.618248 (0.3168) 3.429609 (0.0018)* 1.446339 (0.4442) N/A Sarcoma (TCGA, Party normalized/unified 0 43 FROM 70 4 19,389 2.27E-04 N/A 3.666949 (0.0007)* 0.852454 (1.0000) 0.953027 (1.0000)

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PanCancer Atlas) Encyclopedia of Cancer Cell Lines (Novartis/Broad, Nature 2012) Noah mRNA expression from Illumina HiSeqRNASe qV2 syn4976369 mRNA expressions (microarray analysis) 17 279 418 150 1 3 20,977 0.00032 -2.084879 (0.1854) -3.615935 (0.0015)* 2.007004 (0.2237) 0.108880 (1.0000) The consignment normalized Adenocarcinoma no/ united a rectum (TCGA, PanCancer Atlas) mRNA expression from Illumina HiSeqRNASe 1 54 78 3 0 18,215 0.0003971 -1.926519 (0.1621) 3.877166 (0.0003)* 1.167400 (0.7291) N/A qV2 syn4976369 EPHA2: Hepatocellulum mRNA expression, RSEM liver carcinoma (TCGA, PanCancer Atlas) (batch normalized from Illumina HiSeqRNASe 1 130 194 21 2 15,514 0.003745 0.302341 (1.0000) 3.697248 (0.0011)* 0.336659 (1.0000) 0.454454 (1.0000) qV2) The consignment normalized Adenocarcinoma ny/united a stomach (TCGA, PanCancer Atlas) mRNA expression from Illumina HiSeqRNASe 2 90 264 44 7 13,966 0.007404 -2.072978 (0.1909) 1.606072 (0.5413) 1.750466 (0.4002) 1.602806 (0.5449) qV2 syn4976369 Endometrial carcinoma of the uterine body (TCGA, Normalized/Pooled Expression Batch 3 61 395 43 5 12,916 0.0117 -1.905863 (0.2833) 1.039307 (1.0000) 1.597383 (0.5509) 2.268798 (0.1164)

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PanCancer Atlas) mRNA from Illumina HiSeqRNASe qV2 syn4976369 Cutaneous melanoma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeqRNASe qV2 syn4976369 2 70 216 72 3 12,242 0.01564 1.094526 (1.0000) 2.674493 (0.0374) 0.095966 (1.0000) 1.692628 (0.4526) Prostate adenocarcinoma (TCGA, PanCancer Atlas) mRNA expression, RSEM (batch normalized from Illumina HiSeqRNASe qV2) 0 44 438 4 1 10.112 0.01764 N/A 2.905502 (0.0110)* 1.374609 (0.5078) 0.082790 (1.0000) Chromophobe renal cell carcinoma (TCGA, PanCancer Atlas) mRNA expression, RSEM (batch normalized from Illumina HiSeqRNASe qV2) 0 52 12 1 0 7.8781 0.01947 N/A 2.498340 (0.0187)* 1.863169 (0.0937) N/A Wilms tumor in childhood (TARGET, 2018) Epha2: mRNA expression (RNA sequence RPKM) 0 22 74 5 0 7.4912 0.02362 N/A 2.690766 (0.0107)* 0.173274 (1.0000) N/A Pheochromocytoma and paraganglioma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeqRNASe qV2 syn4976369 4 96 60 1 0 8.8074 0.03196 -1.411567 (0.4742) 2.201344 (0.0831) 1.946134 (0.1549) N/A

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Thyroid carcinoma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeqRNASe qV2 syn4976369 0 4 474 2 0 5.1773 0.08 N/A 2.221884 (0.0394) 0.503577 (0.9218) N/A Esophageal adenocarcinoma (TCGA, PanCancer Atlas) RSEM (batch normalized from Illumina HiSeqRNASe qV2) 1 64 83 32 1 7.6886 0.1037 -1.462679 (0.7178) 0.910990 (1.0000) 1.682311 (0.4625) 0.362298 (1.0000) Cholangiocarcinoma (TCGA, PanCancer Atlas) RSEM (batch normalized from Illumina HiSeqRNASe qV2) 2 27 7 0 0 4.1691 0.1244 -2.037840 (0.0623) 0.972100 (0.4965) N/A N/A Low grade brain glioma RSEM (batch normalized from Illumina 0 191 303 13 0 4.0473 0.1322 N/A 0.722383 (0.7051) 1.771514 (0.1147) N/A malignancy (TCGA, PanCancer Atlas) HiSeqRNASe qV2) Thymoma (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeqRNASe qV2 syn4976369 0 8 110 1 0 4.0322 1,ЗЗЭ-01 N/A 1.982334 (0.0712) 0.369115 (1.0000) N/A Acute lymphocytic leukemia in childhood - phase II (TARGET, 2018) Epha2: mRNA expression (RNA sequence RPKM) 1 6 70 4 0 5.5309 0.1368 1.437404 (0.4518) -0.805100 (1.0000) 1.607586 (0.3238) N/A Diffuse large cell lymphoma mRNA expression, RSEM (batch, 0 4 33 0 0 1,744 0.1866 N/A 1.320613 (0.0933) N/A N/A

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(TCGA, PanCancer Atlas) normalized from Illumina HiSeqRNASe qV2) Glioblastoma multiforme (TCGA, PanCancer Atlas) mRNA expression, RSEM (batch normalized from Illumina HiSeqRNASe qV2) 0 13 104 28 0 2.9376 0.2302 N/A 1.428778 (0.2296) 0.716110 (0.7109) N/A Metastatic prostate cancer, SU2C/PCF Dream Team (Robinson et al., Cell 2015) mRNA expression/capture (RNA sequence RPKM) 2 21 87 7 0 4,069 0.254 -1.812613 (0.2097) 0.992571 (0.9628) 0.314089 (1.0000) N/A Acute myeloid leukemia (TCGA, mRNA expression, RSEM (batch, normalized 0 1 160 4 0 2.4016 0.301 N/A -1.539142 (0.1857) 0.199532 (1.0000) N/A PanCancer Atlas) from Illumina HiSeqRNASe qV2) Testicular germ cell tumors (TCGA, PanCancer Atlas) Normalized/pooled mRNA expression batch from Illumina HiSeqRNASe qV2 syn4976369 1 29 92 22 0 3.3144 0.3456 0.574846 (1.0000) -0.443110 (1.0000) 1.751161 (0.2398) N/A Adrenocortical linen carcinoma (TCGA, PanCancer Atlas) RSEM (batch normalized from Illumina HiSeqRNASe qV2) 0 28 47 1 0 2.0003 0.3678 N/A 1.346397 (0.2673) 0.550103 (0.8734) N/A Carcinosarcoma of the uterus (TCGA, PanCancer Atlas) Normalized/Unified Party 0 16 22 16 2 2.44 0.4862 N/A 0.476071 (1.0000) 0.550292 (1.0000) 1.215102 (0.6730)

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Adenocarcinoma of the pancreas mRNA expression from Illumina HiSeqRNASe qV2 syn4976369 Normalized/Pooled Expression Batch 2 50 106 9 1 3.3833 4.96E-01 -1.195082 0.159442 0.602558 1.217697 glands (TCGA, mRNA from (1.0000) (1.0000) (1.0000) (1.0000) PanCancer Illumina Atlas) HiSeqRNASe qV2 syn4976369 Adenocarcinoma A prostate - Expression -0.406579 glands 0 5 77 3 0 1.3139 0.5184 N/A 1.089948 N/A mRNA (1.0000) (MSKCC, Cancer Cell 2010) Adenocarcinoma of the prostate Expression 0.02835 (0.4136) 0.079785 0.160404 glands (Fred 0 39 84 10 0 0.9859 N/A N/A mRNA 1 (1.0000) (1.0000) Hutchinson CRC, Nat Med 2016)

Study 4: In vivo efficacy of BCY6136 in CDX xenograft models.

The study assessed the therapeutic efficacy of BCY6136 in three established cancer cell line (CDX) models: the fibrosarcoma line HT1080, the triple-negative breast cancer line MDA-MB-231, and the non-small cell lung cancer (NSCLC) line NCI-H1975.

(a) Experimental procedure.

Balb/c mice were inoculated subcutaneously with tumor cells into the right flank, and drug treatment was started when the average tumor volume reached 150 to 200 mm ³ . Tumor measurements and statistical analyzes were performed as described above. Tumor-bearing animals were treated once weekly with BCY6136 or placebo.

(b) Discussion.

In fig. 4-6 show that BCY6136 is effective in xenograft models of breast, lung, and fibrosarcoma cancers after once-weekly dosing.

Fibrosarcoma model HT1080

In the HT1080 model, complete regression of tumor growth was achieved by day 14 after dosing with BCY6136 once weekly on days 0 and 7 at 3 and 5 mg/kg (Figure 2). Dosing BCY6136 once weekly at 2 mg/kg on days 0 and 7 caused tumor growth to stop (partial regression) (Figure 2). Treatment with BCY6136 resulted in modest weight loss, and no adverse clinical events were observed in drug-treated mice throughout the study.

Model NCI-H1975 NSCLC.

Complete regression of tumor growth in the NCI-H1975 model was observed at approximately day 28 following once-weekly BCY6136 dosing at 2 and 3 mg/kg (Figure 3). After dosing was stopped on day 35, there was no resumption of tumor growth in the 3 mg/kg dosed animals from day 35 to day 72, when forepaw size measurements were completed in the 3 mg/kg dosed animals (Fig. .3). Dosing of BCY6136 at a dose rate of 2 mg/kg caused complete regression in this model from approximately day 28. After dosing was stopped on day 35, tumor regrowth was not detected until approximately day 51 at a dose rate of 2 mg/kg. At this dose level, modest tumor regrowth was observed from approximately day 51 until the end of the study on day 77. Treatment with BCY6136 at a dose of 1 mg/kg caused

- 40 044626 stopping tumor growth (partial regression) (Fig. 3). Treatment with BCY6136 caused little weight loss, and no adverse clinical events were observed in drug-treated mice throughout the study.

Breast cancer model MDA-MB-231.

Tumor growth arrest (partial regression) was observed in the MDA-MB231 model following once-weekly dosing at 2 and 3 mg/kg from day 0 to day 45 (Figure 4). Some loss of body weight (attributable to tumor weight) was observed in animals administered the 2 mg/kg dose.

These results show that BCY6136 causes profound tumor growth inhibition in mice implanted with fibrosarcoma, breast cancer, and lung cancer CDX xenografts after once-weekly dosing.

Study 5: Safety studies in rats.

Six (6) male rats were randomized into 3 groups of 2 rats per group to determine the toxicity of BCY6136, then given an intravenous bolus injection at 5, 7.5 and 10 mg/kg on days 1 and 8. The study ended on day 15 .

There were no significant effects on coagulation parameters (prothrombin time (sec), activated partial thromboplastin time (sec), or fibrinogen levels (g/L)) on days 2, 12, and 15 (data not shown). There were no intramortem cases of bleeding reported, and no evidence of internal bleeding was found after autopsy.

Study 6: Safety studies in cynomolgus monkeys.

Twenty-eight days of toxicology studies were conducted using BCY6136 in cynomolgus monkeys. BCY6136 was dosed at 1.0 and 2.0 mg/kg on days 1, 8, 15, and 22. Animals were euthanized and necropsied on day 29 (7 days after the last dose).

There were no significant effects on coagulation parameters compared with baseline on days 18, 22 and 25 (data not shown) and day 29 (Table 15). There were no intramortem cases of bleeding reported, and no evidence of internal bleeding was found after autopsy.

Table 15

Coagulation values on day 29 following dosing of BCY6136 to cynomolgus monkeys at 1.0 and 2.0 mg/kg

1.0 mg/kg x 4 2.0 mg/kg x 4 Original level Day 29 Original level Day 29 Prothrombin time (sec) 13.4 11.7 9.4 9.7 Prothrombin time (sec) AND 9.2 P,2 By Activated partial thromboplastin time (sec) 18.9 19.4 19.4 20.9 Activated partial thromboplastin time (sec) 16.1 15.7 18.7 18.2 Fibrinogen levels (g/l) 2.08 2.42 1.86 6.1 Fibrinogen levels (g/l) 2.28 2.35 1.82 3.1

Study 7. In vivo study of the effectiveness of BCY6136 and ADC in the treatment of PC-3 xenograft in Balb/c nude mice.

(a) Purpose of the study.

The purpose of the study is to evaluate the in vivo antitumor activity of BCY6136 in the treatment of PC-3 xenograft.

(b) Experimental design.

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Group Treatment η Dose (mg/kg) Dosing volume (µl/g) Dosing method Scheme 1 Placebo 3 - 10 intravenously once every a week 2 BCY6136 3 1 10 intravenously once every a week 3 BCY6136 3 2 10 intravenously once every a week 4 BCY6136 3 3 10 intravenously once every a week 5 ADC 3 3 10 intravenously once every a week

(c) Experimental methods and procedures.

(i) Cell culture.

PC-3 tumor cells should be maintained in F12K medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Tumor cells should be subcultured using the standard method twice a week. To inoculate a tumor, cells in the exponential growth phase should be collected and counted.

(ii) Tumor inoculation.

For tumor development, each mouse should be inoculated subcutaneously into the right lateral surface with PC-3 tumor cells (10x10 ⁶ ). Animals should be randomized and treatment should begin when the average tumor volume reaches approximately 150 mm ³ . The administration of the test drug and the number of animals in each group are presented in the following experimental design table.

(iii) Recipe for preparing the test drug.________________________________

Test drug Concentration (mg/ml) Recipe Placebo - 50 mM acetate/acetic acid pH 5 10% sucrose BCY6136 0.1 Dilute 90 µL of 1 mg/mL BCY6136 stock solution with 810 µL of placebo buffer 0.2 Dilute 180 µL of 1 mg/mL BCY6136 stock solution with 720 µL of placebo buffer o, s Dilute 270 µL of 1 mg/mL BCY6136 stock solution with 630 µL of placebo buffer ADC o, s Dilute 26 µL of 10.47 mg/mL ADC stock solution with 874 µL ADC buffer

(d) Results.

(i) Tumor growth curve.

The growth curve is shown in Fig. 5 and 6.

(ii) Volume of residual tumor.

The average tumor volume as a function of time in female Balb/c nude mice carrying the PC-3 xenograft is shown in Table. 16.

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Table 16

Residual tumor volume versus time

Group Treatment 0 2 4 7 9 AND 14 16 18 21 1 Placebo, once a week 149±9 235±9 377±9 718±30 1126±41 1431±79 1792±69 2070±152 2 BCY6136, 1 mg/kg, once weekly 150±C 185±25 228±31 201±17 183±23 153±38 137±33 107±32 64±28 45±23 3 BCY6136, 2 mg/kg, once weekly 149±18 179±28 158±22 137±16 122±15 114±20 101±16 79±20 57±19 42±17 4 BCY6136 3 mg/kg, once a week 149±2 155±8 144±16 132±20 107±28 94±23 83±22 70±27 38±16 35±17 5 ADC 3 mg/kg, once a week 151±27 203±10 210±12 189±C 185±16 190±37 158±36 124±35 103±27 74±14 Group Treatment 23 25 28 thirty 32 35 37 39 42 1 Placebo, once a week 2 BCY6136, 1 mg/kg, once weekly 35±18 28±14 37±19 34±17 42±21 42±23 43±21 28±14 18±9 3 BCY6136, 2 mg/kg, once weekly 21±11 22±12 22±12 24±12 33±16 22±11 26±14 22±12 16±9 4 BCY6136 21±10 23±12 27±14 22±11 24±12 20±11 27±14 12±6 12±6 3 mg/kg, once a week 5 ADC 3 mg/kg, once a week 53±16 50±22 46±23 70±35 78±39 53±27 60±30 53±27 40±22

(iii) Tumor growth inhibition assay.

The degree of tumor growth inhibition for test drugs in the PC-3 xenograft model was calculated based on tumor volume measurements at day 16 after the start of treatment.

Table 17

Tumor Growth Inhibition Assay

Group Treatment Tumor volume (mm ³ ) ^a T/S ^b (%) TGI (%) P-value compared with placebo 1 Placebo, once a week 2070±152 -- -- -- 2 BCY6136, 1 mg/kg, once weekly 107±32 5.2 102.2 p<0.001 3 BCY6136, 2 mg/kg, once weekly 79±20 3.8 103.6 p<0.001 4 BCY6136 3 mg/kg, once a week 70±27 3.4 104.1 p<0.001 5 ADC 3 mg/kg, once a week 124±35 6.0 101.4 p<0.001

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

(f) The results obtained and their discussion.

This study assessed the therapeutic efficacy of the test drugs in the PC-3 xenograft model. Measured tumor volumes in all treatment groups at various time points are presented in FIG. 5 and 6 and in table. 16 and 17.

The average tumor size in placebo-treated mice reached 2070 mm ³ on day 16. BCY6136 at a dose of 1 mg/kg, once a week (TV = 107 mm ³ , TGI = 102.2%, p < 0.001), BCY6136 at 2 mg/kg once weekly (TV=79 ^mm3 , TGI=103.6%, p<0.001) and BCY6136 at 3 mg/kg once weekly (TV=70 ^mm3 , TGI =104.1%, p<0.001) demonstrated a powerful antitumor effect. In this study, the body weight of the animals was regularly monitored. All mice maintained normal body weight.

Study 8. In vivo study of the effectiveness of BCY6136 in the treatment of Balb/c nude mice in the PC-3 xenograft model.

- 43 044626 (a) Purpose of the study.

The aim of the study is to evaluate the in vivo antitumor activity of BCY6136 in the treatment of PC-3 xenograft in Balb/c nude mice.

(b) Experimental design.__________________________________________________________

Group Treatment Dose (mg/kg) N ^a Dosing method Scheme 1 Placebo - 4 intravenously Once a week x 4 weeks 2 BCY6136 0.167 4 intravenously Once a week x 4 weeks Z ^b BCY6136 0.5 4 intravenously Once a week x 4 weeks 4 BCY6136 1.5 4 intravenously Once a week x 4 weeks 5 ^b BCY6136 0.5 4 intravenously Once every two weeks x 2 weeks 6 ^b BCY6136 1.5 4 intravenously Once every two weeks x 2 weeks 7 EphA2-ADC 0.33 4 intravenously Once a week x 4 weeks 8 EphA2-ADC 1 4 intravenously Once a week x 4 weeks 9 EphA2-ADC 3 4 intravenously Once a week x 4 weeks ^10th Docetaxel 15 4 intravenously Once a week x 4 weeks

^a N, number of animals in each group.

^b After 4 weeks of treatment shown in the experimental design table, mice in groups 3, 5 and 6 were treated with BCY6136 at a dose of 1.5 mg/kg once a week from day 52 under the ongoing control regimen.

^c Due to the very large body weight loss after the first dosing in mice treated with docetaxel, treatment was withheld for 2 weeks, followed by treatment at a lower dose (docetaxel, 10 mg/kg) on day 28. Mice were then treated with BCY6136 at 1.5 mg/kg once weekly from day 42 to day 70.

(c) Experimental methods and procedures.

(i) Cell culture.

Tumor cells were maintained in F12K medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Tumor cells were subcultured using the standard method twice a week. For tumor inoculation, cells in the exponential growth phase were collected and counted.

(ii) Tumor inoculation.

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface with PC-3 tumor cells (10x10 ⁶ ) in 0.2 ml of PBS. When the mean tumor volume reached 454 mm ³ , 52 animals were randomized. The administration of the test drug and the number of animals in each group are presented in the following experimental design table.

(iii) Formulation for preparation of the test drug.

- 44 044626

Test drug Purity Concentration (mg/ml) Recipe Placebo - - 25 mM histidine pH 7 10% sucrose BCY6136 98.6% - 50 mM acetate 10% sucrose pH 5 1 Dissolve 2.70 mg BCY6136 in 2.662 ml acetate buffer 0.3 Dilute 300 µl of 1 mg/ml BCY6136 stock solution with 700 µl acetate buffer ¹ 0.15 Dilute 600 µl of 0.3 mg/ml BCY6136 stock solution with 600 µl acetate buffer 0.05 Dilute 200 µl of 0.3 mg/ml BCY6136 stock solution with 1000 µl acetate buffer 0.0167 Dilute 66.7 µl of 0.3 mg/ml BCY6136 stock solution with 1133.3 µl acetate buffer EphA2-ADC - - 25 mM histidine pH 5.5 0.033 Dilute 9.3 µl of 4.24 mg/ml EphA2-ADC stock solution with 1191 µl histidine buffer 0.1 Dilute 28 µl of 4.24 mg/ml EphA2-ADC stock solution with 1172 µl histidine buffer 0.3 Dilute 84.9 µl of 4.24 mg/ml EphA2-ADC stock solution with 1115 µl histidine buffer Docetaxel - 10 Mix 0.5 ml of 20 mg docetaxel with 1.5 ml of buffer 1.5 Dilute 180 µl of 10 mg/ml docetaxel stock solution with 1020 µl saline buffer

¹ 50 mM acetate 10% sucrose pH 5.3. 25 mM histidine pH 5.5.

(c) Results.

(i) Tumor growth curve.

The tumor growth curve is shown in Fig. 7.

(ii) Volume of residual tumor.

The average tumor volume as a function of time in male Balb/c nude mice carrying the PC-3 xenograft is shown in Table. 18.

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Table 18

Residual tumor volume versus time (day 0 to day 20)

Group Treatment Days after starting treatment 0 2 4 6 8 10 13 15 17 20 1 Placebo, once a week 456±2 5 648±5 0 880±23 1022±29 1178±11 8 1327±13 3 1631±93 1868±90 2052±13 9 2364±10 2 2 BCY6136 0.167 mg/kg, once a week 450±3 3 631±5 5 695±78 739±39 850±68 904±73 975±47 1089±74 1124±92 1188±11 1 3 BCY6136 0.5 mg/kg, once a week 451±4 7 622±9 6 519 ±70 460±55 398±50 329±38 260±33 249±33 231±38 234±42 4 BCY6136 1.5 mg/kg, once a week 458±4 9 587±6 3 494±54 363±32 283±32 237±24 192±13 164±16 155±20 131±19 5 BCY6136 0.5 mg/kg, once every two weeks 454±3 7 643±2 5 531±37 458±33 411±32 382±49 430±88 522±124 560±129 530±147 6 BCY6136 1.5 mg/kg, once every two weeks 452±4 2 590±7 5 457±49 375±44 328±47 242±63 206±61 197±62 182±55 128±36 1.5 mg/kg, once a week 7 EphA2-ADC 0.33 mg/kg, once every two weeks 457±4 3 636±5 7 712±70 792±78 870±87 900±58 1049±66 1242±12 3 1443±12 9 1637±18 1 8 EphA2-ADC 1 mg/kg, once every two weeks 450±4 9 617±4 8 673±50 721±61 782±78 755±67 840±93 913±91 978±100 981±100 9 EphA2-ADC 3 mg/kg, once every two weeks 452±6 0 593±9 8 643±141 593±106 433±103 290±81 268±64 232±60 225±66 184±62 10 Docetaxel 453±6 2 584±7 2 632±56 636±48 568±50 408±31 374±26 388±36 361±25 419±31 15 mg/kg, once every two weeks

(iii) Tumor growth inhibition assay.

The degree of tumor growth inhibition for test drugs in the PC-3 xenograft model was calculated based on tumor volume measurements at day 20 after the start of treatment.

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Table 19

Tumor Growth Inhibition Assay

Group Treatment Tumor volume (mm ³ ) ^a t/s ^b (%) TGI (%) P-value compared with placebo 1 Placebo, once a week 2364±102 - - - 2 BCY6136, 0.167 mg/kg, once weekly 1188±111 50.2 61.4 p<0.001 3 BCY6136, 0.5 mg/kg, once weekly 234±42 9.9 111.4 p<0.001 4 BCY6136, 1.5 mg/kg, once weekly 131±19 5.5 117.2 p<0.001 5 BCY6136, 0.5 mg/kg, once every two weeks 530±147 22.4 96.0 p<0.001 6 BCY6136, 1.5 mg/kg, once every two weeks 128±36 5.4 117.0 p<0.001 7 EphA2-ADC, 0.33 mg/kg, once a week 1637±181 69.2 38.1 p<0.001 8 EphA2-ADC, 1 mg/kg, once a week 981±100 41.5 72.2 p<0.001 9 EphA2-ADC, 3 mg/kg, once a week 184±62 7.8 114.0 p<0.001 10 Docetaxel, 15 mg/kg, once a week 419±31 17.7 101.8 p<0.001

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

(d) The results obtained and their discussion.

This study assessed the therapeutic efficacy of the test drugs in the PC-3 xenograft model. Measured tumor volumes in all treatment groups at various time points are presented in FIG. 7 and in table. 18 and 19.

The mean tumor size in placebo-treated mice reached 2364 mm ³ on day 20. BCY6136 at a dose of 0.167 mg/kg once weekly (TV=1188 mm ³ , TGI=61.4%, p<0.001), with and a dose of 0.5 mg/kg once every two weeks (TV=530 ^mm3 , TGI=96.0%, p<0.001), at a dose of 0.5 mg/kg once a week (TV=234 ^mm3 , TGI=111.4%, p<0.001) and at a dose of 1.5 mg/kg once a week (TV=131 mm ³ , TGI=117.2%, p<0.001) showed significant antitumor activity, dependent on dose or dose frequency, on day 20. BCY6136 at a dose of 1.5 mg/kg once every two weeks (TV=128 mm ³ , TGI=117.0%, p<0.001) showed comparable antitumor activity to the drug BCY6136 at a dose of 1.5 mg/kg once weekly. Of these, mice treated with BCY6136 at a dose of 0.5 mg/kg once a week or with BCY6136 at a dose of 0.5 mg/kg once every two weeks showed obvious tumor recurrence after stopping treatment, further treated with BCY6136 at a dose of 1.5 mg/kg once a week from day 52 gave a good result in tumor regression. Mice treated with BCY6136 at a dose of 1.5 mg/kg once every two weeks also showed tumor recurrence after treatment was stopped, but further dosing did not result in complete tumor regression. Mice treated with BCY6136 at 1.5 mg/kg once weekly showed no tumor recurrence until day 48.

EphA2-ADC at a dose of 0.33 mg/kg once a week (TV=1637 ^mm3 , TGI=38.1%, p<0.001), at a dose of 1 mg/kg once a week (TV=981 mm ³ , TGI=72.2%, p<0.001) and at a dose of 3 mg/kg, once a week (TV=184 mm ³ , TGI=114.0%, p<0.001) showed significant antitumor activity, depending on dose values, on day 20. Mice treated with EphA2-ADC at a dose of 3 mg/kg once a week did not show tumor recurrence until day 59.

- 47 044626

Docetaxel at a dose of 15 mg/kg once a week (TV=419 mm ³ , TGI=101.8%, p<0.001) showed significant antitumor activity, but caused very large body weight losses in animals.

After treatment was stopped, the mice showed obvious tumor recurrence. Treatment with the drug

BCY6136 at a dose of 1.5 mg/kg once a week from day 42 produced good tumor regression in these mice.

Study 9. In vivo testing of the effectiveness of BCY6136 in the treatment of NCHIH1975 xenograft in Balb/c nude mice.

(a) Purpose of the study.

The purpose of the study was to evaluate the in vivo antitumor activity of BCY6136 in the treatment of NCI-H1975 xenograft in Balb/c nude mice.

(b) Experimental design. _____________________________________________________

Group Treatment η Dose (mg/kg) Dispensed volume (µl/g) Dosing method Scheme 1 Placebo 3 - 10 intravenously once a week 2 BCY6136 3 1 10 intravenously once a week 3 BCY6136 h 2 10 intravenously once a week 4 BCY6136 3 3 10 intravenously once a week

(c) Experimental methods and procedures.

(i) Cell culture.

For tumor inoculation, cells in the exponential growth phase were collected and counted.

(j) Tumor inoculation.

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface with NCI-H1975 tumor cells (10x10 ⁶ ) in 0.2 ml PBS. When the mean tumor volume reached 149 mm ³ , 36 animals were randomized. The administration of the test drug and the number of animals in each group are presented in the following experimental design table.

(iii) Formulation for preparation of the test drug.

Treatment Dose (mg/ml) Recipe Placebo 50 mM acetate, 10% sucrose pH 5 BCY6136 1 Dissolve 3.79 MrBCY6136 in 3.695 ml buffer preparations 0.3 Dilute 270 µl 1 mg/ml BCY6136 with 630 µl preparation buffer 0.2 Dilute 180 µl 1 mg/ml BCY6136 with 720 µl preparation buffer 0.1 Dilute 90 µl 1 mg/ml BCY6136 with 810 µl preparation buffer

(iv) Sample collection.

On PG-D44, formalin-fixed and paraffin-embedded (FFPE) group 2 tumors.

At the end of the study, group 3 tumors were formalin-fixed and paraffin-embedded (FFPE).

(d) Results.

(i) Tumor growth curve.

The tumor growth curve is shown in Figure 8.

(i) Volume of residual tumor.

The average tumor volume as a function of time in male Balb/c nude mice carrying the NCI-H1975 xenograft is shown in Table. 20-24.

-48044626

Table 20

Residual tumor volume (PG-D0-PG-D17)

Group Treatment Days after starting treatment 0 2 4 7 9 AND 14 17 1 Placeb o, once a week 148±4 195±1 1 297±3 3 466±6 4 732±10 7 1028±1 92 1278±2 52 1543± 298 2 BCY61 36 1 mg/kg, once a week 150±6 178±2 0 232±4 9 336±4 3 400±24 407±42 299±11 3 261±1 27 3 BCY61 36 2 mg/kg, one 150±1 4 181±2 6 237±2 7 277±3 6 297±37 306±55 256±53 218±4 9 once a week 4 BCY61 36 3 mg/kg, once a week 148±9 168±1 0 231±6 365±1 6 390±13 423±42 319±26 228±1 6

Table 21

Residual tumor volume (PG-D18-PG-D35)

Group Treatment Days after starting treatment 18 21 23 25 28 thirty 33 35 1 Placeb o, once a week 1864±3 95 2371±4 70 — — — — — — 2 BCY61 36 1 mg/kg, once a week 215±11 3 205±11 7 197±11 3 200±1 05 202±1 12 202±1 17 230±1 42 241±1 27 3 BCY61 36 2 mg/kg, one 149±31 99±30 69±22 42±13 30±10 16±8 20±9 4±2 once a week 4 BCY61 36 3 mg/kg, once a week 149±17 94±30 50±15 41±21 21±8 6±6 10±6 3±1

-49044626

Table 22

Residual tumor volume (PG-D37~PG-D53)

Group a Treatment Days after starting treatment 37 39 42 44 46 49 51 53 2 BCY613 6 1 mg/kg, once a week 277±149 294±159 351±188 - - - - - 3 BCY613 6 2 mg/kg, once a week 7±4 2±1 1±0 3±1 2±1 3±2 6±3 14±10 4 BCY613 6 3 mg/kg, once a week 3±3 2±1 1±0 0±0 0±0 0±0 1±0 1±0

Table 23

Residual tumor volume (PG-D56~PG-D74)

Group Treatment Days after starting treatment 56 58 60 63 65 67 70 72 74 3 BCY6136 2 mg/kg, once a week 16±11 27±1 8 34±2 3 45±3 1 63±4 0 71±47 95±7 0 111±7 3 122±7 5 4 BCY6136 3 mg/kg, once a week 1±0 1±0 1±0 0±0 0±0 0±0 0±0 0±0 -

Table 24

Residual tumor volume (PG-D77~PG-D98)

Group Treatment Days after starting treatment 77 81 84 88 91 95 98 3 BCY6136 2 mg/kg, once a week 208±C 2 337±12 3 501±17 2 626±18 2 856±24 5 1035±1 69 1266±3 9

(iii) Tumor growth inhibition assay.

The degree of tumor growth inhibition for BCY6136 in the NCI-H1975 xenograft model was calculated based on tumor volume measurements at day 21 after initiation of treatment.

Table 25

Tumor Growth Inhibition Assay

Group Treatment Tumor volume (mm ³ ) ^a t/s ^b (%) TGI (%) P-value 1 Placebo, once a week 2371±470 - - - 2 BCY6136, 1 mg/kg, once weekly 205±117 8.6 97.5 p<0.001 3 BCY6136, 2 mg/kg, once weekly 99±30 4.2 102.3 p<0.001 4 BCY6136, 3 mg/kg, once weekly 94±30 4.0 102.4 p<0.001

a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

(f) The results obtained and their discussion.

- 50 044626

This study assessed the therapeutic efficacy of BCY6136 in the NCI-H1975 xenograft model. Measured tumor volumes in all treatment groups at various time points are presented in FIG. 8 and in table. 20-25.

The mean tumor size in placebo-treated mice reached 2371 ^mm3 on day 21. BCY6136 at 1 mg/kg (TV=205 ^mm3 , TGI=97.5%, p<0.001), at 2 mg/kg (TV=99 mm ³ , TGI=102.3%, p<0.001) and at a dose of 3 mg/kg (TV=94 mm ³ , TGI=102.4%, p<0.001) showed high antitumor activity. BCY6136 at a dose of 2 mg/kg and 3 mg/kg destroyed tumors or caused tumor regression to a small size. Treatment was suspended from day 35, and tumors in the 3 mg/kg dose group showed no obvious regrowth during the next 5-6 weeks of continuous observation, but tumors in the 2 mg/kg dose group showed obvious regrowth, and resumption of dosing did not led to significant tumor inhibition. In this study, the body weight of the mice remained virtually unchanged.

Study 10. In vivo study of the effectiveness of BCY6136 in the LU-01-0251 PDX model in Balb/c nude mice.

(a) Purpose of the study.

The purpose of the study is to evaluate the in vivo antitumor activity of BCY6136 in the LU-010251 PDX model in Balb/c nude mice.

(b) Experimental design. _______________________________________________

Group Treatment η Dose (mg/kg) Dispensed volume (µl/g) Dosing method Scheme 1 Placebo 5 — 10 intravenously once a week 2 BCY6136 5 1 10 intravenously once a week 3 BCY6136 5 2 10 intravenously once a week 4 BCY6136 5 3 10 intravenously once a week 5 ADC 5 3 10 intravenously once a week

(c) Experimental methods and procedures.

(i) Tumor inoculation

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface with a tumor fragment LU-01-0251 (~30 mm ³ ). For the efficacy study, treatment began when the average tumor volume reached 174 mm ³ . The administration of the test drug and the number of animals in each group are presented in the following experimental design table.

(ii) Recipe for the preparation of the test drug.________________________________

Test drug Concentration (mg/ml) Recipe Placebo — 50 mM acetate, 10% sucrose pH 5 BCY6136 0.3 Dissolve 6.11 mg BCY6136 in 20 ml acetate buffer ¹ 0.2 Dilute 940 µl of stock solution 0.3 mg/ml BCY6136 using 470 µl acetate buffer 0.1 Dilute 470 µl of stock solution 0.3 mg/ml BCY6136 using 940 µl acetate buffer ADC o, s Dilute 43 µl of 10.47 mg/ml ADC stock solution with 1457 µl ADC buffer ²

¹ Acetate buffer: 50 mM acetate 10% sucrose pH 5.

² Buffer for ADC: 20 mM histidine pH 5.5.

- 51 044626 (d) Results.

(i) Tumor growth curve.

The tumor growth curve is shown in Fig. 9.

(ii) Volume of residual tumor.

The average tumor volume on day 28 after the start of treatment in female Balb/c nude mice carrying the LU-01-0251 xenograft is shown in Table. 26.

Table 26

Residual tumor volume versus time

Day Group 1 Group 2 Group 3 Group 4 Group 5 Placebo BCY6136 1 mg/kg, once a week BCY6136 2 mg/kg, once a week BCY6136 3 mg/kg, once a week ADC, 3 mg/kg, once weekly 0 174±17 175±15 174±17 175±14 174±16 3 264±33 230±29 205±21 187±19 227±12 7 403±68 281±55 154±21 118±13 239±42 10 562±83 370±104 111±19 72±12 241±46 14 777±163 362±104 62±17 30±5 191±47 17 1021±246 437±136 46±13 17±3 139±39 21 1472±342 526±167 30±18 4±3 101±31 24 1790±417 491±132 32±24 1±1 70±23 28 2208±512 499±128 32±30 0±0 39±14

(iii) Tumor growth inhibition assay.

The degree of tumor growth inhibition for BCY6136 and ADC in the LU-01-0251 PDX model was calculated based on tumor volume measurements at day 28 after the start of treatment.

Table 27

Tumor Growth Inhibition Assay

Group Treatment Tumor volume (mm ³ ) ^a t/s ^b (%) TGI (%) P-value 1 Placebo, once a week 2208±512 — — — 2 BCY6136, 1 mg/kg, once weekly 499±128 22.6 84.0 P<0.001 3 BCY6136, 2 mg/kg, once weekly 32±30 1.4 107.0 P<0.001 4 BCY6136, 3 mg/kg, once weekly 0±0 0.0 108.6 P<0.001 5 ADC, 3 mg/kg, once every 39±14 1.8 106.6 p <0.001

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

(f) The results obtained and their discussion

This study evaluated the therapeutic efficacy of BCY6136 and ADC in the LU-01025 1 PDX model. Measured tumor volumes in all treatment groups at various time points are presented in FIG. 9 and in table. 26 and 27.

In this study, the average tumor size in mice treated with placebo reached 2208 mm ³ on day 28 after the start of treatment. BCY6136 at a dose of 1 mg/kg once a week (TV=499 ^mm3 , TGI=84.0%, p<0.001), at a dose of 2 mg/kg once a week (TV=32 ^mm3 , TGI =107.0%, p<0.001) and at a dose of 3 mg/kg once a week (TV=0 ^mm3 , TGI=108.6%, p<0.001) exhibited dose-dependent antitumor activity. The ADC drug at a dose of 3 mg/kg once a week (TV=39 ^mm3 , TGI=106.6%, p<0.001) showed significant antitumor activity.

Study 11. In vivo study of the effectiveness of BCY6136 in the LU-01-0251 PDX model in Balb/c nude mice.

(a) Purpose of the study.

The purpose of the study is to evaluate the in vivo antitumor activity of BCY6136 in the LU-01-52 044626 model

0251 PDX in Balb/c nude mice.

(b) Experimental design. _______

Group Treatment η Dose (mg/kg) Dispensed volume (µl/g) Dosing method Scheme 1 Placebo 5 — 10 intravenously once a week x 21 weeks 2 BCY6136 5 1 10 intravenously once a week x 28 weeks ^Behind BCY6136 5 2 10 intravenously once a week x 70 weeks 4 ^b BCY6136 5 3 10 intravenously once a week x 56 weeks ^5th ADC 5 3 10 intravenously once a week x 70 weeks

a The dosing schedule was maintained from day 0 to day 70 for all mice in this group, then mouse 3-2 and mouse 3-4 were additionally dosed with BCY6136 at 3 mg/kg once weekly from day 77 while treatment other mice from group 3 were temporarily stopped.

^b The dosing schedule was maintained from day 0 to day 56 for all mice in this group.

^c The dosing schedule was maintained from day 0 to day 70 for all mice in this group.

(c) Experimental methods and procedures.

(i) Tumor inoculation.

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface with a tumor fragment LU-01-0251 (~30 mm ³ ). For the efficacy study, treatment began when the average tumor volume reached 960 mm ³ . The administration of the test drug and the number of animals in each group are presented in the experimental design table.

(ii) Recipe for preparing the test drug.______________________________

Test drug Concentration (mg/ml) Recipe Placebo — 25 mM histidine 10% sucrose pH 7 BCY6136 0.3 0.3 mg/ml BCY6136 was prepared as described above in Study 10 0.2 Dilute 940 µl of 0.3 mg/ml BCY6136 stock solution with 470 µl histidine buffer ¹ 0.1 Dilute 470 µl of 0.3 mg/ml BCY6136 stock solution with 940 µl histidine buffer ADC o, s Dilute 43 µl of 10.47 mg/ml ADC stock solution with 1457 µl ADC buffer ²

¹ Histidine buffer: 25 mM histidine 10% sucrose pH 7.

² Buffer for ADC: 20 mM histidine pH 5.5.

(iii) Sample collection.

Mouse #3-2 tumor was harvested for formalin fixation and paraffin embedding (FFPE) for a day

- 53 044626

94. Tumors from mice #5-2 and 5-3 were harvested and placed in the same formalin-fixed-paraffin-embedded (FFPE) block on day 140.

(d) Results.

(i) Tumor growth curve.

The tumor growth curve is shown in Fig. 10.

(ii) Volume of residual tumor.

The average tumor volume in the period from day 0 to day 28 after the start of treatment in female Balb/c nude mice carrying the LU-01-0251 xenograft is shown in Table. 28.

Table 28

Residual tumor volume versus time

Day Group 1 Group 2 Group 3 Group 4 Group 5 Placebo BCY6136 1 mg/kg, once a week BCY6136 2 mg/kg, once a week BCY6136 3 mg/kg, once a week ADC, 3 mg/kg, once weekly 0 962±102 963±97 962±137 960±103 959±124 3 1176±У8 1003±121 973±105 989±128 1043±158 7 135ы142 1056±151 873±125 890±98 1100±156 10 159ы179 1122±139 722±157 674±96 1172±188 14 195ы225 1417±191 503±151 342±64 1228±174 17 2301±344 1672±262 398±160 216±43 1143±186 21 1794±328 307±169 94±26 996±187 24 1867±408 261±168 62±14 867±178 28 2120±483 217±167 45±16 713±178

(iii) Tumor growth inhibition assay.

The degree of tumor growth inhibition for BCY6136 and ADC in the LU-01-0251 PDX model was calculated based on tumor volume measurements at day 17 after the start of treatment.

Table 29

Tumor Growth Inhibition Assay

Group Treatment Tumor volume (mm ³ ) ^a T/S ^b (%) TGI (%) P-value 1 Placebo, once a week 2301±344 — — — 2 BCY6136, 1 mg/kg, once weekly 1672±262 72.7 47.0 p>0.05 3 BCY6136, 2 mg/kg, once weekly 398±160 17.3 142.1 P<0.001 4 BCY6136, 3 mg/kg, once weekly 216±43 9.4 155.6 P<0.001 5 ADC, 3 mg/kg, once weekly 1143±186 49.7 86.3 P<0.01

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

(f) The results obtained and their discussion.

This study evaluated the therapeutic efficacy of BCY6136 and ADC in the LU-010251 PDX model. Measured tumor volumes in all treatment groups at various time points are presented in FIG. 10 and in table. 28 and 29.

In this study, treatment began when the average tumor volume reached 960 mm ³ . At day 17 after initiation of treatment, the mean tumor volume of placebo-treated mice reached 2301 mm ³ . BCY6136 at a dose of 1 mg/kg once a week (TV=1672 mm ³ , TGI=47.0%, p>0.05) did not exhibit obvious antitumor activity; BCY6136 at a dose of 2 mg/kg once a week (TV=398 ^mm3 , TGI=142.1%, p<0.001) and at a dose of 3 mg/kg once a week (TV=216 ^mm3 ,

- 54 044626

TGI=155.6%, p<0.001) exhibited dose-dependent antitumor activity on day 17.

After treatment for 70 days with BCY6136 at a dose of 2 mg/kg once a week, 3 of 5 of these mice showed complete tumor regression, the remaining 2 mice showed apparent tumor relapse from day 42 to day 77. Subsequent treatment of two tumor relapses with BCY6136 at a dose of 3 mg/kg once a week from day 7, one of the tumors achieved obvious tumor regression, while the other tumor showed resistance to treatment.

After treatment for 56 days with BCY6136 at a dose of 3 mg/kg once a week, all mice in this group showed complete tumor regression.

ADC at a dose of 3 mg/kg once weekly (TV=1143 ^mm3 , TGI=86.3%, p<0.01) showed obvious antitumor activity on day 17, after treatment for an additional 53 days, in these mice showed additional, but not complete, tumor regression.

In this study, some mice exhibited dramatic weight loss, which may be associated with prolonged feeding periods in immunodeficient mice.

Study 12. In vivo study of the effectiveness of BCY6136 in the LU-01-0046 NSCLC PDX model in Balb/c nude mice.

(a) Purpose of the study.

The purpose of the study is to evaluate the in vivo antitumor efficacy of BCY6136 in large LU-01-0046 PDX tumors from Balb/c nude mice.

(b) Experimental design. _______________________________________________

Group Treatment η Dose (mg/kg) Dispensed volume (µl/g) Dosing method Scheme 1 Placebo 5 - 10 intravenously once a week 2 BCY6136 5 1 10 intravenously once a week 3 BCY6136 5 3 10 intravenously once a week 4 ADC 5 3 10 intravenously once a week

(c) Experimental methods and procedures.

(i) Tumor inoculation.

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface with a tumor fragment LU-01-0046 (~30 mm ³ ). Treatment began when the average tumor volume reached 1039 mm ³ . The administration of the test drug and the number of animals in each group are presented in the experimental design table.

(ii) Formulation for preparation of the test drug.

Test drug Concentration (mg/ml) Recipe Placebo - 50 mM acetate 10% sucrose pH 5 BCY6136 OD Dilute 150 µl of 1 mg/ml BCY6136 stock solution with 1350 µl acetate buffer o, s Dilute 450 µl of 1 mg/ml BCY6136 stock solution with 1050 µl acetate buffer ADC o, s Dilute 43 µl of 10.47 mg/ml ADC stock solution in 1457 µl buffer ²

¹ Acetate buffer: 50 mM acetate 10% sucrose pH 5.

² Dissolve 0.419 g histidine hydrochloride in 100 ml water, use 1 M HCl to adjust pH to 5.5.

(d) Results.

(i) Tumor growth curve.

The tumor growth curve is shown in Fig. eleven.

(ii) Volume of residual tumor.

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The average tumor volume as a function of time in female Balb/c nude mice carrying LU-01-0046 is presented in Table. thirty.

Table 30

Residual tumor volume versus time (bicyclic cross-sectional study (BCY))

Group Treatment Days after starting treatment 0 4 8 eleven 15 18 22 1 Placebo, once a week 1044±115 1762±178 2404±262 - - - - 2 BCY6136, 1 mg/kg, once a week 1037±130 1163±146 1927±283 2483±530 - - - 3 BCY6136, 3 mg/kg, once weekly 1036±100 784±146 548±107 362±110 325±122 275±152 233±187 4 A.D.C. 3 mg/kg, once a week 1033±114 1155±230 2200±505 - - - -

Note: No residual tumor volume was detected after day 22 for groups 2 and 4.

(iii) Tumor growth inhibition assay.

The degree of tumor growth inhibition for test drugs in the LU-01-0046 PDX model was calculated based on tumor volume measurements at day 22 and day 28, respectively, for two cross-sectional studies after the start of treatment.

Table 31

Tumor Growth Inhibition Assay (BCYs cross-sectional studies at day 22)

Group Treatment Tumor volume (mm ³ ) ^a T/S ^b (%) TGI (%) P-value 1 Placebo, once a week 6186±596* — — — 2 BCY6136, 1 mg/kg, once weekly 4564±981* 73.8 31.4 p>0.05 3 BCY6136, 3 mg/kg, once weekly 233±187 3.8 115.6 p<0.001 4 ADC, 3 mg/kg, once weekly 5446±1250* 88.0 14.2 p>0.05

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

*In some groups, studies were stopped before day 22 and tumor volume was calculated using the exponential growth equation presented below.

Placebo group: Y=995.4 x exp(0.1134 x X).

Group BCY6136, 1 mg/kg: Y=855.0 x exp(0.0974 x X).

ADC group, 3 mg/kg: Y=757.4 x exp(0.1312 x X).

(f) The results obtained and their discussion.

This study evaluated the therapeutic efficacy of the test drugs against large tumors LU-01-0046. Measured tumor volumes for all treatment groups at various time points are presented in FIG. 11 and in table. 30 and 31.

In this study, the mean tumor volume of placebo-treated mice was 6186 mm ³ on day 22. BCY6136 at a dose of 1 mg/kg and ADC at a dose of 3 mg/kg did not exhibit obvious antitumor activity when tumor size was measured before treatment. was 1000 mm ³ .

The drug BCY6136 (TV=233 mm ³ , TGI=115.6%, p<0.001) at a dose of 3 mg/kg exhibited significant antitumor activity. In particular, the use of the drug BCY6136 led to complete

-56044626 destruction of 2/5 and 4/5 tumors.

Study 13. In vivo efficacy of BCY6136 in the Balb/c nude mouse model. carriers LU-01-0046 NSCLC PDX (a) Purpose of the study.

The aim of the study was to evaluate the in vivo therapeutic efficacy of BCY6136 in a Balb/c nude mouse model carrying LU-01-0046 NSCLC PDX.

(b) Experimental design.

Group Treatment η Dose (mg/kg) Dosing method Scheme 1 Placebo 5 — intravenously once a week x 2 weeks 2 BCY6136 5 1 intravenously once a week x 3 weeks 3 BCY6136 5 2 intravenously once a week x 4 weeks 4 BCY6136 5 3 intravenously once a week x 4 weeks 5 ADC 5 3 intravenously once a week x 3 weeks 6 ADC 5 5 intravenously once a week x 3 weeks

Note: The study groups were stopped when the mean tumor volume exceeded 2000 mm ³ and the tumors were collected for formalin fixation and paraffin embedding (FFPE): group 1 on PGD14, group 5 on PG-D18, groups 2 and 6 on PG-D21, and group 3 and 4 on PG-D31.

(c) Experimental methods and procedures.

(i) Tumor inoculation.

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface with a tumor fragment of a specific type (~30 mm ³ ). Treatment was started when the average tumor volume reached approximately 198 mm ³ . The administration of the test drug and the number of animals in each group are presented in the experimental design table.

(ii) Formulation for preparation of the test drug.

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Group Connections Dose (mg/kg) Concentration (mg/ml) Recipe 1 Placebo - - 50 mM acetate, 10% sucrose pH 5 (no DMSO) 2 BCY6136 1 0.1 Dissolve 10.93 mg BCY6136 in 10.766 ml media using sonication to obtain a stock solution of 1 mg/ml BCY6136. Dilute 150 µl of 1 mg/ml BCY6136 stock solution with 1350 µl of media 3 BCY6136 2 0.2 Dilute 300 µl of 1 mg/ml BCY6136 stock solution with 1200 µl of media 4 BCY6136 3 o, s 1 mg/ml BCY6136 stock solution using 1050 µl media Buffer 2: dissolve 0.419 g histidine hydrochloride in 100 ml water, use 1 M H O to adjust pH to 5.5 5 ADC 3 o, s Dilute 43 µl of 10.47 mg/ml ADC stock solution with 1457 µl buffer 2 6 ADC 5 0.5 Dilute 71.6 µl of 10.47 mg/ml ADC stock solution with 1428.4 µl of buffer 2

Note: The drug being dosed is usually freshly prepared.

(iii) Sample collection.

Study groups were stopped when the mean tumor volume reached more than 2000 mm ³ and, after the last measurement, tumors were collected for formalin fixation and paraffin embedding (FFPE): group 1 on PG-D14, group 5 on PG-D18, group 2 and 6 on the PG-D21, and group 3 and 4 on the PG-D31.

(d) Results.

(i) Tumor growth curve.

The tumor growth curve is shown in Fig. 12.

(ii) Volume of residual tumor.

The average tumor volume as a function of time in the model of female Balb/c nude mice carrying LU-01-0046 NSCLC PDX is presented in Table. 32.

Table 32

Volume of residual tumor (mm ³ ) depending on time

Group 1 2 3 4 5 6 Treatment Placebo once a week BCY6136 1 mg/kg, once a week BCY6136 2 mg/kg, once a week BCY6136 3 mg/kg, once a week ADC 3 mg/kg, once a week ADC 5 mg/kg, once every a week 0 201 ±37 198 ±39 201 ±40 200 ± 46 195 ±28 195 ±40 3 441 ±82 310 ± 59 283 ± 77 155 ±40 418 ±99 389 ±68 7 927 ± 171 547 ±88 423 ±132 74 ± 19 643 ± 159 596 ± 116 10 1546 ±377 747 ± 121 321±108 31 ±8 938 ±230 882± 134 14 2307 ±594 1058±140 264 ± 95 26 ± I 1475 ±466 1215 ±193 17 - 1390 ±205 127 ±41 26 ± 13 2281±556 1576 ±228 21 - 2138±301 118 ± 34 64 ±42 - 2049 ±242 24 - - 101 ±40 99 ±63 - - 28 - - 255 ± 140 276±176 - - 31 — - 582 ±346 477 ±283 - -

(iii) Tumor growth inhibition assay.

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The degree of tumor growth inhibition for the tested drugs in the Balb/c nude mouse model carrying LU-01-0046 PDX was calculated based on the tumor volume measured on

PG-D14.

Table 33

Tumor Growth Inhibition Assay

Group 1 2 3 Treatment Placebo once a week BCY6136 1 mg/kg, once a week BCY6136 2 mg/kg, once a week Tumor volume (mm ³ ) ^a 2307 ± 594 1058 ± 140 264 ± 95 T/S (%) ^b 45.9 I.4 TGI (%) ^from 59.1 97.0 P-value compared with placebo p<0.05 p<0.001 4 BCY6136 3 mg/kg, once a week 26 ± I 1.1 108.3 p<0.001 5 ADC 3 mg/kg, once a week 1475 ±466 63.9 39.2 p>0.05 6 ADC 5 mg/kg, once a week 1215±193 52.7 51.6 p>0.05

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition was calculated by dividing the mean tumor volume per group for the treated group by the mean tumor volume per group for the control group (T/C).

^c TGI was calculated for each group using the formula: TGI (%) = [1-(T _i -T ₀ )/(V _i -V ₀ )] x 100.

(f) The results obtained and their discussion.

The present study evaluated the therapeutic efficacy of the test drugs in the LU-01-0046 PDX model. Measured tumor volumes in all treated groups at various time points are presented in FIG. 12 and in table. 32 and 33.

The mean tumor volume of placebo-treated mice reached 2307 mm ³ at PG-D14. Drug BCY6136 at a dose of 1 mg/kg (TV=1058 ^mm3 , TGI=59.1%, p<0.05), at a dose of 2 mg/kg (TV=264 ^mm3 , TGI=97.0%, p <0.001) and at a dose of 3 mg/kg (TV=26 mm ³ , TGI=108.3%, p<0.001) exhibited dose-dependent antitumor activity. ADC at a dose of 3 mg/kg and 5 mg/kg did not exhibit obvious antitumor activity (p>0.05).

In this study, the body weight of animals in all groups remained almost constant.

Study 14. In vivo study of the effectiveness of BCY6136, BCY6173 and BCY6175 in the LU-01-0046 NSCLC PDX model in Balb/c nude mice.

(a) Purpose of the study.

The purpose of the study is to evaluate in vivo the antitumor efficacy of the tested drugs in the LU-01-0046 NSCLC PDX model in athymic Balb/c mice.

(b) Experimental design.

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Group Treatment η Dose (mg/kg) Dosing volume (µl/g) Dosing method Scheme Part 1 1 Placebo 5 - 10 intravenously once a week 2 BCY6136 5 1/2 10 intravenously once a week 3 BCY6136 5 3 10 intravenously once a week Part 2 4 Placebo 5 - 10 intravenously once a week 5 BCY6173 5 1 10 intravenously once a week 6 BCY6173 5 3 10 intravenously once a week 7 BCY6175 5 3 10 intravenously once a week

(c) Experimental methods and procedures.

(i) Tumor inoculation.

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface with a tumor fragment LU-01-0046 (~30 mm ³ ). Treatment began when the average tumor volume reached 200 mm ³ for part 1 of the study and 192 mm ³ for part 2 of the study. The administration of the test drug and the number of animals in each group are presented in the experimental design table.

(i) Recipe for preparing the test drug._________________________________

Test drug Concentration (mg/ml) Recipe Placebo - 50 mM acetate 10% sucrose pH 5 BCY6136 OD Dilute 150 µl of 1 mg/ml BCY6136 stock solution with 1350 µl acetate buffer o, s Dilute 450 µl of 1 mg/ml BCY6136 stock solution with 1050 µl acetate buffer BCY6173 OD Dissolve 3.65 mg VSU6173 in 3.5 ml acetate buffer to obtain 1 mg/ml stock solution. Dilute 150 µl with 1350 µl acetate buffer o, s Dilute 450 µl of 1 mg/ml BCY6173 stock solution with 1050 µl acetate buffer BCY6175 o, s Dissolve 3.02 mgVSU6175 in 2.9 mL acetate buffer to obtain a 1 mg/mL stock solution. Dilute 450 µl of 1 mg/ml BCY6175 stock solution with 1050 µl acetate buffer

¹ Acetate buffer: 50 mM acetate 10% sucrose pH 5.

-60044626 (d) Results.

(i) Tumor growth curve.

The tumor growth curve is shown in Fig. 13-15.

(ii) Volume of residual tumor.

The average tumor volume on day 21 after the start of treatment in female Balb/c nude mice carrying LU-01-0046 is presented in Table. 34 and 35.

Table 34

Residual tumor volume versus time (part 1)

Group Treatment Days after starting treatment 0 3 6 10 14 17 21 1 Placebo, once a week 202±2 6 328±4 8 536±6 8 953±10 7 1386±97 1833±13 2 2551±24 2 2 BCY6136, 1 mg/kg, once a week 200±3 3 293±5 6 426±9 1 682±15 1 964±194 976±258 1285±23 4 3 BCY6136, 3 mg/kg, once weekly 201±3 3 194±3 1 135±2 7 52±18 13±9 4±4 0±0

Table 35

Residual tumor volume versus time (part 2)

Gru ppa Treatment Days after starting treatment 0 3 7 10 14 17 21 4 Placebo, once a week 192±30 311±8 3 562±14 6 830±23 0 1320±44 4 1652±52 8 2342±65 1 5 BCY6173, 1 mg/kg, once a week 191±33 318±5 8 553±88 817±16 5 1314±27 6 1546±27 6 2151±26 2 6 BCY6173, 3 mg/kg, once weekly 192±37 259±5 1 400±53 455±28 636±92 646±138 890±260 7 BCY6175, 3 mg/kg, once weekly 192±42 186±5 7 92±38 19±11 0±0 0±0 0±0

(iii) Tumor growth inhibition assay.

The degree of tumor growth inhibition for test drugs in the LU-01-0046 PDX model was calculated based on tumor volume measurements at day 21 after the start of treatment.

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Table 36

Tumor Growth Inhibition Assay (Part 1)

Group Treatment Tumor volume (mm ³ ) ^a T/S ^b (%) TGI (%) P-value 1 Placebo, once a week 255ы242 — — — 2 BCY6136, 1 mg/kg, once weekly 1285±234 50.4 53.9 P<0.001 3 BCY6136, 3 mg/kg, once weekly 0±0 0.0 108.5 P<0.001

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

Table 37

Tumor Growth Inhibition Assay (Part 2)

Group Treatment Tumor volume (mm ³ ) ^a T/S ^b (%) TGI (%) P-value 4 Placebo, once a week 2342±651 — — — 5 BCY6173, 1 mg/kg, once a week 215ы262 91.8 8.9 p>0.05 6 BCY6173, 3 mg/kg, once weekly 890±260 38.0 67.5 P<0.05 7 BCY6175, 3 mg/kg, once weekly 0±0 oh oh 108.9 P<0.001

a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

(f) The results obtained and their discussion.

This study evaluated the therapeutic efficacy of test drugs in the LU-01-0046 PDX model. Measured tumor volumes for all treatment groups at various time points are presented in FIG. 13-15 and in table. 34-37.

In Part 1 of the study, the average tumor volume of placebo-treated mice reached 2551 mm ³ on day 21 after the start of treatment.

BCY6136 at a dose of 1/2 mg/kg once a week (TV=1285 ^mm3 , TGI=53.9%, p<0.001) exhibited significant antitumor activity, but it did not lead to tumor regression. BCY6136 at a dose of 3 mg/kg once a week (TV=0 mm ³ , TGI=108.5%, p<0.001) completely destroyed tumors, 1 out of 5 tumors, respectively, in the BCY6136 3 mg/kg dosing groups showed regrowth after temporary cessation of dosing, and tumors were resistant to treatment with BICY6136 when dosing was resumed. The remaining tumors in the BCY6136 dosing groups (4/5 in each group) did not show regrowth after 80 days of temporary dosing cessation.

In Part 2 of the study, the average tumor volume of placebo-treated mice reached 2342 mm ³ on day 21 after the start of treatment. BCY6136 at a dose of 1 mg/kg once a week (TV=2151

- 62 044626 mm ³ , TGI=8.9%, p>0.05) did not exhibit antitumor activity. BCY6173 at a dose of 3 mg/kg once a week (TV=890 mm ³ , TGI=67.5%, p<0.05) showed obvious antitumor activity.

BCY6175 at a dose of 3 mg/kg once a week (TV=0 ^mm3 , TGI=108.9%, p<0.001) completely destroyed 4/5 tumors on day 14.

Study 15. In vivo study of the effectiveness of BCY6136 in the LU-01-0412 NSCLC PDX model in Balb/c nude mice.

(a) Purpose of the study.

The purpose of the study is to evaluate the in vivo therapeutic efficacy of BCY6136 in the LU-01-0412 NSCLC PDX model in Balb/c nude mice.

(b) Experimental design. _______________________________________________________

Group Treatment η Dose (mg/kg) Dosing volume (µl/g) Dosing method Scheme 1 Placebo 6 — 10 intravenously Once a week, 4 weeks 2 BCY613 6 6 1 10 intravenously Once a week, 4 weeks 3 BCY613 6 6 3 10 intravenously Once a week, 4 weeks 4 BCY824 5 6 3 10 intravenously Once a week, 4 weeks 5 BCY878 1 6 3 10 intravenously Once a week, 4 weeks

(c) Experimental methods and procedures.

(i) Tumor inoculation.

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface with a tumor fragment LU-01-0412 (~30 mm ³ ). Animals were randomized when the average tumor volume reached 159 mm ³ . The administration of the test drug and the number of animals in each group are presented in the experimental design table.

(ii) Recipe for preparing the test drug._________________________________

Test drug Concentration (mg/ml) Recipe

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Placebo - 25 mM histidine 10% sucrose pH 7 BCY6136 1 Dissolve 6.06 mg BCY6136 in 5.969 ml 50 mM acetate/acetic acid pH 5 10% sucrose 0.1 Dilute 180 µl of 1 mg/ml BT5528 solution with 1620 µl 50 mM acetate/acetic acid pH 5 10% sucrose 0.3 Dilute 540 µl of 1 mg/ml BT5528 solution with 1260 µl 50 mM acetate/acetic acid pH 5 10% sucrose BCY8245 1 Dissolve 4.15 mg BCY8245 powder in 4.121 ml in placebo buffer 0.3 Dilute 540 µl of 1 mg/ml BCY8245 solution with 1260 µl of placebo buffer BCY8781 1 Dissolve 4.08 mg BCY8781 powder in 80.8 µl DMSO, then dilute to 1 mg/ml with 3958 µl placebo buffer 0.3 Dilute 540 µl of 1 mg/ml BCY8781 solution with 1260 µl of placebo buffer

(iii) Sample collection.

Plasma from placebo-treated mice treated with BCY6136, BCY8245, and BCY8781 was collected at 30 min and 24 h post-dosing. Tumors were collected from placebo-treated and BCY6136-, BCY8245-, and BCY8781-treated mice 24 hours after dosing.

(d) Results.

(i) Tumor growth curve.

The tumor growth curve is shown in Fig. 16.

(ii) Volume of residual tumor.

The average tumor volume as a function of time in female BALB/c nude mice carrying the LU-01-0412 xenograft is presented in Table. 38.

Table 38

Residual tumor volume versus time

Days Group 1 Group 2 Group 3 Group 4 Group 5 Placebo once a week x 4 weeks BCY6136 1 mg/kg, once a week x 4 weeks BCY6136 3 mg/kg, once a week x 4 weeks BCY8245 3 mg/kg, once a week x 4 weeks BCY8781 3 mg/kg, once a week x 4 weeks 0 159±11 159±13 159±11 159±12 159±11 4 255±12 214±16 197±16 168±18 176±21 7 309±20 237±16 195±16 132±10 167±13 AND 395±31 246±19 156±18 78±4 107±15 14 464±31 300±18 177±29 45±5 72±12 18 521±26 369±32 210±32 21±2 44±8 21 611±33 470±46 225±32 11±1 31±6 25 737±68 632±47 252±37 6±1 20±6 28 788±80 664±52 299±37 2±1 14±5 32 1104±142 758±70 416±52 1±1 12±5

(iii) Tumor growth inhibition assay.

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The degree of tumor inhibition for BCY6136, BCY8245 and BCY8781 in the LU-01-0412 xenograft model was calculated based on tumor volume measurements at day 32 after the start of treatment.

Table 39

Tumor Growth Inhibition Assay

Group Treatment Tumor volume (mm ³ ) ^a T/S ^b (%) TGI (%) P-value 1 Placebo, once a week x 4 weeks 1104+142 — — — 2 BCY6136, 1 mg/kg, once a week x 4 weeks 758+70 68.6 36.7 P<0.05 3 BCY6136, 3 mg/kg, once a week x 4 weeks 416+52 37.6 72.9 P<0.001 4 BCY8245, 3 mg/kg, once a week x 4 weeks 1+1 0.1 116.8 P<0.001 5 BCY8781, 3 mg/kg, once a week x 4 weeks 12+5 1.0 115.6 P<0.001

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

(f) The results obtained and their discussion.

This study evaluated the therapeutic efficacy of BCY6136, BCY8245, and BCY8781 in the LU-01-0412 xenograft model. Measured tumor volumes in all treatment groups at various time points are presented in FIG. 16 and in table. 38 and 39.

The average tumor volume of mice treated with placebo reached 1104 mm ³ on day 32 after the start of treatment. BCY6136 at a dose of 1 mg/kg once a week x 4 weeks (TV=758 ^mm3 , TGI=36.7%, p<0.05) and at a dose of 3 mg/kg once a week x 4 weeks ( TV=416 mm ³ , TGI=72.9%, p<0.001) exhibited dose-dependent antitumor activity, but its use did not lead to tumor regression. BCY8245 at a dose of 3 mg/kg once a week x 4 weeks (TV=1 ^mm3 , TGI=116.8%, p<0.001) and BCY8781 at a dose of 3 mg/kg once a week x 4 weeks ( TV=12 mm ³ , TGI=115.6%, p<0.001) caused obvious tumor regression. Of these, 5 of 6 tumors treated with BCY8245 at 3 mg/kg and 2 of 6 tumors treated with BCY8781 at 3 mg/kg were completely eradicated at day 32.

In this study, body weight of animals in all groups remained almost constant.

Study 16. In vivo study of the effectiveness of BCY6136 in the treatment of athymic Balb/c mice in the LU-01-0486 PDX model.

(a) Purpose of the study.

The purpose of the study is to evaluate the in vivo antitumor efficacy of BCY6136 in the LU-01-0486 PDX model in Balb/c nude mice.

(b) Experimental design.

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Group Treatment η Dose (mg/kg) Dosing volume (µl/g) Dosing method Scheme 1 Placebo 5 — 10 intravenously once a week 2 BCY6136 5 1 10 intravenously once a week 3 BCY6136 5 2 10 intravenously once a week 4 BCY6136 5 3 10 intravenously once a week

(c) Experimental methods and procedures.

(i) Tumor inoculation.

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface with a tumor fragment LU-01-0486 (~30 mm ³ ). For the efficacy study, treatment began when the average tumor volume reached 180 mm ³ . The administration of the test drug and the number of animals in each group are presented in the experimental design table.

(i) Recipe for preparing the test drug.

Test drug Concentration (mg/ml) Recipe Placebo — 50 mM acetate 10% sucrose pH 5 BCY6136 0.3 A 0.3 mg/mL BCY6136 solution was prepared as described in Study 10 0.2 Dilute 940 µl of 0.3 mg/ml BCY6136 solution with 470 µl acetate buffer ¹ OD Dilute 470 µl of 0.3 mg/ml BCY6136 solution with 940 µl acetate buffer

¹ Acetate buffer: 50 mM acetate 10% sucrose pH 5.

(d) Result.

(i) Tumor growth curve.

The tumor growth curve is shown in Fig. 17.

(i) Volume of residual tumor.

The average tumor volume on day 14 after the start of treatment in female Balb/c nude mice carrying the LU-01-0486 xenograft is shown in Table. 40.

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Table 40

Residual tumor volume versus time

Group Treatment Days after starting treatment 0 3 7 10 14 1 Placebo, once a week 179±20 232±30 358±45 450±47 651±112 2 BCY6136, 1 mg/kg, once weekly 180±23 221±20 326±34 420±34 638±71 3 BCY6136, 179±27 222±26 365±44 459±82 645±105 2 mg/kg, once a week 4 BCY6136, 3 mg/kg, once weekly 180±25 209±37 304±51 348±77 449±P5

(iii) Tumor growth inhibition assay.

The degree of tumor inhibition for BCY6136 in the LU-01-0486 PDX model was calculated based on tumor volume measurements at day 14 after initiation of treatment.

Table 41

Tumor Growth Inhibition Assay

Group Treatment Tumor volume (mm ³ ) ^a T/S ^b (%) TGI (%) P-value 1 Placebo, once a week 651±112 — — — 2 BCY6136, 1 mg/kg, once weekly 638±71 98.0 3.0 p>0.05 3 BCY6136, 2 mg/kg, once weekly 645±105 99.1 1.2 p>0.05 4 BCY6136, 3 mg/kg, once weekly 449±P5 68.9 43.1 p>0.05

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

(f) The results obtained and their discussion.

This study evaluated the therapeutic efficacy of BCY6136 in the LU-01-0486 PDX model. Measured tumor volumes in all treatment groups at various time points are presented in FIG. 17 and in table. 40 and 41.

In this study, the mean tumor volume of placebo-treated mice reached 651 mm ³ on day 14 after the start of treatment. BCY6136 at a dose of 1 mg/kg once a week (TV=638 ^mm3 , TGI=3.0%, p>0.05) and at a dose of 2 mg/kg once a week (TV=645 ^mm3 , TGI=1.2%, p>0.05) did not exhibit antitumor activity. BCY6136 at a dose of 3 mg/kg once a week (TV=449 mm ³ , TGI=43.1%, p>0.05) showed a small amount of antitumor activity without statistical significance.

Study 17: In Vivo Test of the Efficacy of BCY6136 in the Treatment of Xenograft

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MDA-MB-231-luc in Balb/c nude mice.

(a) Purpose of the study.

The aim of the study was to evaluate the in vivo antitumor activity of BCY6136 in the treatment of Balb/c nude mice in the MDA-MB-231-luc xenograft model.

(b) Experimental design. _____________________________________________________

Group Treatment η Dose (mg/kg) Dosing volume (µl/g) Dosing method Scheme 1 Placebo 3 — 10 intravenously once in Week 2 BCY6136 3 1 10 intravenously once in Week 3 BCY6136 3 2 10 intravenously once in Week 4 BCY6136 3 2 10 intravenously once in Week

(c) Experimental methods and procedures.

(i) Cell culture.

For inoculation, cells at the exponential growth stage were collected and counted.

(i) Tumor inoculation.

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface of a tumor cell with MDA-MB-231-luc (10x10 ⁶ ) in 0.1 ml PBS with 0.1 ml Matrigel. When the average tumor volume reached 159 mm ³ , 36 animals were randomized. The administration of the test drug and the number of animals in each group are presented in the experimental design table.

(iii) Formulation for preparation of the test drug.

Treatment Dose (mg/ml) Recipe Placebo 50 mM acetate, 10% sucrose pH=5 BCY6136 1 Dissolve 3.79 mg BCY6136 in 3.695 ml in buffer preparations o, s Dilute 270 µl of 1 mg/ml BCY6136 solution in 630 µl of preparation buffer 0.2 Dilute 180 µl of 1 mg/ml BCY6136 solution in 720 µl of preparation buffer 0.1 Dilute 90 µl of 1 mg/ml BCY6136 solution in 810 µl of preparation buffer

(iv) Sample collection.

At PG-D33, group 2 tumors were collected and fixed for formalin-fixation and paraffin-embedded (FFPE).

At the end of the study, group 3 and 4 tumors were collected and fixed for formalin fixation and paraffin embedding (FFPE).

(d) Results.

(i) Tumor growth curve.

The growth of the tumor is shown in Fig. 18.

(i) Volume of residual tumor.

The average tumor volume as a function of time in female Balb/c nude mice carrying the MDA-MB-231-luc xenograft is presented in Table. 42-44.

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Table 42

Residual tumor volume (PG-D0~PG-D17)

Group Treatment Days after starting treatment 0 2 4 7 9 AND 14 17 1 Placebo, once a week 159±14 269±8 306±1 9 425±5 2 688±5 4 908±5 4 1064±9 8 1315±9 5 2 BCY6136, 1 mg/kg, once weekly 159±10 226±3 6 221±5 4 310±7 2 416±8 9 526±7 7 636±92 809±13 5 3 BCY6136, 2 mg/kg, once weekly 159±16 218±1 7 182±2 2 182±2 6 101±2 0 77±24 36±4 41±10 4 BCY6136, 3 mg/kg, once weekly 158±5 241±1 2 259±6 325±1 4 258±1 2 246±1 5 162±19 178±10

Table 43

Residual tumor volume (PG-D19~PG-D33)

Treatment Days after starting treatment Group 19 21 24 26 28 31 33 1 Placebo, once a week 1453±1 28 1661±1 73 — — — — — 2 BCY6136 1 mg/kg, once a week 879±19 0 994±21 3 1253±3 13 1431±3 53 1507±2 53 2181±6 09 — 3 BCY6136 2 2 mg/kg, once a week 35±9 33±9 31±17 41±32 59±45 82±59 87±71 4 BCY6136 2 3 mg/kg, once a week 171±21 132±19 108±19 85±15 81±8 87±14 92±18

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Table 44

Residual tumor volume (PG-D35~PG-D47)

Group Treatment Days after starting treatment 35 38 40 42 45 47 3 BCY6136, 2 mg/kg, once weekly 124±106 156±120 179±142 239±19 7 285±23 9 350±298 4 BCY6136, 3 mg/kg, once weekly 129±38 173±65 181±65 269±11 3 293±11 4 371±128

(iii) Tumor growth inhibition assay.

The degree of tumor inhibition for BCY6136 in the MDA-MB-231-luc xenograft model was calculated based on tumor volume measurements at day 21 after initiation of treatment.

Table 45

Tumor Growth Inhibition Assay

Group Treatment Tumor volume (mm ³ ) ^a T/S ^b (%) TGI (%) P-value 1 Placebo, once a week 166ы173 — — — 2 BCY6136, 1 mg/kg, once weekly 994±213 59.8 44.4 p<0.01 3 BCY6136, 2 mg/kg, once weekly 33±9 2.0 108.4 p<0.001 4 BCY6136, 3 mg/kg, once weekly 132±19 8.0 101.7 p<0.001

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

(f) The results obtained and their discussion.

This study evaluated the therapeutic efficacy of BCY6136 in the MDA-MB-231-luc xenograft model. Measured tumor volumes in all treatment groups at various time points are presented in FIG. 18 and in table. 42-45.

The mean tumor volume of placebo-treated mice reached 1661 ^mm3 on day 21. BCY6136 at a dose of 1 mg/kg (TV=994 ^mm3 , TGI=44.4%, p<0.01) exhibited moderate antitumor activity, drug BCY6136 at a dose of 2 mg/kg (TV=33 mm ³ , TGI=108.4%, p<0.001) and at a dose of 3 mg/kg (TV=132 mm ³ , TGI=101.1%, p<0.001 ) exhibited strong antitumor activity, but tumors showed no obvious regrowth from day 28. In this study, one mouse treated with BCY6136 at a dose of 2 mg/kg lost more than 15% of body weight during the course of treatment, while another mouse maintained almost constant body weight.

Study 18: In vivo testing of the efficacy of BCY6136 in the treatment of BALB/c mice in a syngeneic EMT-6 model.

(a) Purpose of the study.

The purpose of the study was to evaluate in vivo the antitumor activity of the drug BCY6136 in the treatment of BALB/c mice in the syngeneic EMT-6 model.

(b) Experimental design.

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Group Treatment Dose (mg/kg) N Dosing method Scheme Sample collection 1 Placebo — 5 intravenously once a week x 4 weeks Tumors from spare mice will be collected for fluorescence-activated cell sorting (FACS) 2 BCY6136 3 5 intravenously once a week x 4 weeks 3 BCY6136 1/5 ^b 5 intravenously once a week x 4 weeks 4 BCY6136 o, z/z ^b 5 intravenously once a week x 4 weeks

^a The injected volume per mouse is 10 ml/kg.

^b The dose in group 3 and group 4 was changed to 5 mg/kg and 3 mg/kg from day 14.

(c) Experimental methods and procedures.

(i) Cell culture.

EMT-6 tumor cells were maintained in vitro as a monolayer culture in EMEM medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Tumor cells were routinely passaged twice a week by treatment with trypsin-EDTA. For inoculation, cells were collected at the exponential growth stage and counted.

(ii) Tumor inoculation.

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface of an EMT-6 tumor cell (5x 106) in 0.1 ml PBS. When the average tumor volume reached 75 mm ³ , 44 animals were randomized. The administration of the test drug and the number of animals in each group are presented in the experimental design table.

(iii) Recipe for preparing the test drug._________________________________

Preparation of BCY6136 Treatment Concentration (mg/ml) Recipe Placebo/buffer — 50 mM acetate, 10% sucrose pH 5 BCY6136 1 Dissolve 6.2 mg BCY6136 with 6113 µl buffer

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BCY6136 0.3 Dilute 450 µl of 1 mg/ml BCY6136 stock solution with 1050 µl buffer BCY6136 0.1 Dilute 150 µl of 1 mg/ml BCY6136 stock solution with 1350 µl buffer BCY6136 0.03 Dilute 45 µl of 1 mg/ml BCY6136 stock solution with 1455 µl buffer

Preparation of BCY6136 Treatment Concentration (mg/ml) Recipe Placebo/buffer — 50 mM acetate, 10% sucrose pH 5 BCY6136 1 Stock solution BCY6136 0.3 Dilute 420 µl of 1 mg/ml BCY6136 stock solution with 980 µl of buffer BCY6136 o, s Dilute 420 µl of 1 mg/ml BCY6136 stock solution with 980 µl of buffer BCY6136 0.5 Dilute 700 µl of 1 mg/ml BCY6136 stock solution with 700 µl of buffer

(iv) Sample collection.

3 tumors were collected from reserve mice for fluorescence-activated cell sorting (FACS) on day 11. Data were presented by a team of biologists.

(d) Results.

(i) Tumor growth curve.

The tumor growth curve is shown in Fig. 19.

(i) Volume of residual tumor.

The average tumor volume as a function of time in female BALB/c mice carrying syngeneic EMT-6 is presented in Table. 46.

Table 46

Residual tumor volume versus time

Group Treatment Days after starting treatment 0 3 5 7 10 12 14 17 19 21 1 Placebo, once a week 82±4 141±11 260±24 443±90 o o -Η 703±119 812±139 948±191 1129±24 1499±34 2 BCY6136, 3 mg/kg, once weekly 82±4 -n OO 59±2 125±18 240±23 322±23 374±22 431±37 486±50 561±61 3 BCY6136, 1/5 ^a mg/kg, once a week 82±4 108±18 204±27 350±57 426±49 -n OO OO -n oh oh 850±98 ШТ8Ш 1272±140 4 BCY6136, 0.3/3 ^mg /kg, once a week 82±4 130±16 255±35 358±34 450±67 O -n Oh oh 731± 872±119 1082± 1394±161

Group 3 and Group 4 dosing was changed to 5 mg/kg and 3 mg/kg from day 14.

-72044626 (iii) Tumor growth inhibition assay.

The degree of tumor inhibition for BCY6136 in the syngeneic EMT-6 model was calculated based on tumor volume measurements at day 21 after initiation of treatment.

Table 47

Tumor Growth Inhibition Assay

Group Treatment Tumor volume (mm ³ ) ^a t/s ^b (%) TGI (%) P-value compared with placebo 1 Placebo, once a week 1499±340 — — — 2 BCY6136, 3 mg/kg, once weekly 561±61 37.4 66.2 p<0.05 3 BCY6136, 1/5 ^s mg/kg, once weekly 1272±140 84.8 16.1 ns 4 BCY6136, O.3/3 ^C mg/kg, once a week 1394±161 93.0 7.4 ns

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

^c The dosing of group 3 and group 4 was changed to 5 mg/kg and 3 mg/kg from day 14.

(f) The results obtained and their discussion.

This study assessed the therapeutic efficacy of BCY6136 in a syngeneic EMT-6 model. Measured tumor volumes in all treatment groups at various time points are presented in FIG. 19 and in table. 46 and 47.

The mean tumor volume of placebo-treated mice reached 1499 ^mm3 on day 21. BCY6136 at a dose of 3 mg/kg once weekly (TV=561 ^mm3 , TGI=66.2%, p<0.05) showed obvious antitumor activity. BCY6136 at a dose of 1/5 mg/kg once a week (TV=1272 ^mm3 , TGI=16.1%, p>0.05) and BCY6136 at a dose of 0.3/3 mg/kg once a week week (TV=1394 mm ³ , TGI=7.4%, p>0.05) did not exhibit antitumor activity.

The dosage of group 3 and group 4 was changed to 5 mg/kg and 3 mg/kg from day 1414. Tumor ulceration was detected in mice 3-5 on day 14, and the mice were treated with antibiotic cream. In this study, all mice maintained almost constant body weight.

Study 19. In vivo study of the effectiveness of BCY6136 in the treatment of NCI-N87 xenograft in Balb/c nude mice.

(a) Purpose of the study.

The purpose of the study is to evaluate the in vivo antitumor efficacy of BCY6136 in the treatment of NCI-N87 xenograft in Balb/c nude mice.

(b) Experimental design.

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Group Treatment η Dose (mg/kg) Dosing volume (µl/g) Dosing method Scheme 1 Placebo 3 — 10 intravenously once a week 2 BCY6136 3 1 10 intravenously once a week 3 BCY6136 3 2 10 intravenously once a week 4 BCY6136 3 3 10 intravenously once a week

(c) Experimental methods and procedures.

(i) Cell culture.

NCI-N87 tumor cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Tumor cells were routinely passaged twice a week. For inoculation, cells at the exponential growth stage were collected and counted.

(ii) Tumor inoculation.

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface of an NCI-N87 tumor cell (10χ 10 ⁶ ) with Matrigel (1:1) in 0.2 ml PBS. When the average tumor volume reached approximately 176 mm ³ , animals were randomized and treatment began. The administration of the test drug and the number of animals in each group are presented in the experimental design table.

(iii) Formulation for preparation of the test drug.

Test drug Concentration (mg/ml) Recipe Placebo - 50 mM acetate 10% sucrose pH 5 BCY6136 1 Dissolve 4.295 MrBCY6136 in 4.214 ml acetate buffer ¹ OD Dilute 90 µl of 1 mg/ml stock solution BCY6136 using 810 µl acetate buffer 0.2 Dilute 180 µl of 1 mg/ml stock solution BCY6136 using 720 µl acetate buffer o, s Dilute 270 µl of 1 mg/ml BCY6136 stock solution with 630 µl acetate buffer

¹ Acetate buffer: 50 mM acetate 10% sucrose pH 5.

(d) Results.

(i) Tumor growth curve.

The tumor growth curve is shown in Fig. 20.

(ii) Volume of residual tumor.

The average tumor volume as a function of time in female Balb/c nude mice carrying the NCI-N87 xenograft is presented in Table. 48.

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Table 48

Residual tumor volume versus time

Group Treatment Days after starting treatment 0 2 4 7 9 eleven 14 16 18 21 23 25 28 thirty 1 Placebo once a week 174±7 213±5 266±6 421±1 0 537±1 7 598±3 0 734±4 6 821±5 5 918±9 1 1024±8 3 1151±6 8 1305±5 7 1407±6 4 1465± 90 2 BCY613 6.1 mg/kg, once a week 176±7 200±8 210±1 4 224±2 7 238±2 1 184±1 8 244±2 3 276±3 5 308±4 4 343±37 390±43 406±48 422±42 425±4 7 3 BCY613 6.2 mg/kg, once in Week 176±1 8 197±2 5 168±2 5 170±2 6 165±3 4 96±27 133±3 5 150±5 2 160±4 9 190±63 203±65 218±66 201±53 210±6 0 4 BCY613 6.3 mg/kg, once a week 177±8 197±9 169±7 158±3 148±8 95±16 141±1 2 145±2 4 164±2 8 202±28 205±30 201±16 196±21 201±2 2

(iii) Tumor growth inhibition assay.

The degree of tumor inhibition by BCY6136 in the NCI-N87 xenograft was calculated based on tumor volume measurements at day 30 after initiation of treatment.

Table 49

Tumor Growth Inhibition Assay

Group Treatment Tumor volume (mm ³ ) ^a T/S ^ъ (%) TGI (%) P-value 1 Placebo, once a week 1465±90 — - - 2 BCY6136, 1 mg/kg, once weekly 425±47 29.0 80.7 P<0.001 3 BCY6136, 2 mg/kg, once a week 210±60 14.3 97.4 P<0.001 4 BCY6136, 3 mg/kg, once weekly 201±22 13.7 98.1 P<0.001

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume of the treatment group by the mean tumor volume of the control group (T/C).

(f) The results obtained and their discussion.

This study assessed the therapeutic efficacy of BCY6136 in the NCI-N87 model. Measured tumor volumes in all treatment groups at various time points are presented in FIG. 20 and in table. 48 and 49.

The mean tumor volume of placebo-treated mice reached 1465 ^mm3 on day 30. BCY6136 at 1 mg/kg once weekly (TV=425 ^mm3 , TGI=80.7%, p<0.001) and at dose 2 mg/kg once weekly (TV=210 ^mm3 , TGI=97.4%, p<0.001) exhibited significant dose-dependent antitumor activity, BCY6136 at a dose of 3 mg/kg once weekly (TV=201 ^mm3 , TGI= 98.1%, p<0.001) exhibited comparable antitumor activity to that of BCY6136 at a dose of 2 mg/kg.

In this study, mice in all groups showed no apparent weight loss during treatment.

Study 20. In vivo study of the effectiveness of BCY6136 in the treatment of xenografts

SK-OV-3 in Balb/c nude mice.

(a) Purpose of the study.

The purpose of the study is to evaluate the in vivo antitumor efficacy of BCY6136 in the treatment of SK-OV-3 xenografts in Balb/c nude mice.

(b) Experimental design. ___________________________________________________

Group Treatment η Dose (mg/kg) Dosing volume (µl/g) Dosing method Scheme 1 Placebo 3 — 10 intravenously once a week 2 ADC 3 3 10 intravenously once a week 3 BCY6136 3 1 10 intravenously once a week 4 BCY6136 3 2 10 intravenously once a week 5 BCY6136 3 3 10 intravenously once a week

(c) Experimental methods and procedures.

(i) Cell culture.

SK-OV-3 tumor cells were maintained in McCoy 5a medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Tumor cells were routinely passaged twice a week. For inoculation, cells at the exponential growth stage were collected and counted.

(ii) Tumor inoculation.

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface of SK-OV-3 tumor cells (10x10 ⁶ ) with Matrigel (1:1) in 0.2 ml PBS. Animals were randomized and treatment began when the mean tumor volume reached approximately 186 mm ³ . The administration of the test drug and the number of animals in each group are presented in the experimental design table.

(iii) Formulation for preparation of the test drug.

Test drug Purity Concentration (mg/ml) Recipe Placebo - - 50 mM acetate 10% sucrose pH5

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BCY6136 98.5% 1 Dissolve 3.65 mg VSU6136 in 3.60 ml 50 mM acetate buffer ¹ 0.1 Dilute 90 µl of 1 mg/ml BCY6136 stock solution with 810 µl acetate buffer ¹ 0.2 Dilute 180 µl of 1 mg/ml BCY6136 stock solution with 720 µl acetate buffer ¹ o, s Dilute 270 µl of 1 mg/ml BCY6136 stock solution with 630 µl acetate buffer ¹ LAN LAN 0.3 Dilute 69 µl of 10.47 mg/ml ADC stock solution with 2331 µl ADC buffer ²

¹ Acetate buffer: 50 mM acetate 10% sucrose pH 5.

² Buffer for ADC: 25 mM histidine 10% sucrose pH 5.5.

(d) Results.

(i) Tumor growth curve.

The tumor growth curve is shown in Fig. 21.

(ii) Volume of residual tumor.

The average tumor volume as a function of time in female Balb/c nude mice carrying the SK-OV-3 xenograft is presented in Table. 50.

Table 50

Residual tumor volume versus time

Group Treatment Days after starting treatment 0 2 5 7 9 12 14 16 19 21 23 26 28 1 Placebo, once a week 187±1 6 243±2 4 313±2 8 399±3 7 470±2 3 606±6 1 742±10 3 891±13 3 1076±1 85 1173±2 14 1340±2 36 1490±2 73 1560± 305 2 ADC, 3 mg/kg, once weekly 187±1 6 181±1 5 212±1 6 263±3 5 268±1 4 335±2 3 353±18 392±63 449±4 481±27 573±33 647±26 684±1 eleven 3 BCY6136 2 mg/kg, once a week 186±2 3 222±1 9 293±3 4 331±2 1 356±2 3 440±8 503±28 587±33 702±43 752±26 893±34 1002±6 8 1035± 67 4 BCY6136 2 mg/kg, once a week 186±2 3 170±1 8 164±2 8 188±3 3 180±3 4 202±2 9 200±29 230±46 229±48 231±58 236±49 240±48 277±5 8 5 BCY6136 184±2 4 168±1 8 150±1 2 164±1 2 158±8 180±8 187±4 212±17 208±29 204±12 205±17 227±31 254±4 8 3 mg/kg, once a week

(iii) Tumor growth inhibition assay.

The degree of tumor inhibition for BCY6136 in the SK-OV-3 xenograft was calculated based on tumor volume measurements at day 28 after the start of treatment.

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Table 51

Tumor Growth Inhibition Assay

Group Treatment Tumor volume (mm ³ ) ^a T/S ^b (%) TGI (%) P-value 1 Placebo, once a week 1560±305 -- -- -- 2 ADC, 3 mg/kg, once weekly 684±111 43.9 63.8 P<0.01 3 BCY6136, 1 mg/kg, once weekly 1035±67 66.4 38.1 p>0.05 4 BCY6136, 2 mg/kg, once weekly 277±58 17.8 93.3 P<0.001 5 BCY6136, 3 mg/kg, once weekly 254±48 16.3 95.0 P<0.001

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

(f) The results obtained and their discussion.

This study evaluated the therapeutic efficacy of BCY6136 in the SK-OV-3 model. Measured tumor volumes in all treatment groups at various time points are presented in FIG. 21 and in table. 50 and 51.

The mean tumor volume of placebo-treated mice reached 1560 mm ³ on day 28. ADC at a dose of 3 mg/kg once weekly (TV=684 mm ³ , TGI=63.8%, p<0.01) showed moderate antitumor activity. BCY6136 at a dose of 1 mg/kg once weekly (TV=1035 mm ³ , TGI=38.1%, p>0.05) did not exhibit obvious antitumor activity. BCY6136 at 2 mg/kg once weekly (TV=277 ^mm3 , TGI=93.3%, p<0.001) and at 3 mg/kg once weekly (TV=254 ^mm3 , TGI=95 .0%, p<0.001) exhibited significant antitumor activity.

In this study, there was no obvious loss of body weight in animals in all groups during the course of treatment.

Study 21. In vivo study of the effectiveness of BCY6136 in the treatment of OE21 xenograft in Balb/c nude mice.

(a) Purpose of the study.

The purpose of the study is to evaluate the in vivo antitumor efficacy of BCY6136 in the treatment of OE21 xenograft in Balb/c nude mice.

(b) Experimental design. ________________________________________________

Group Treatment η Dose (mg/kg) Dosing volume (µl/g) Dosing method Scheme 1 Placebo 3 - 10 intravenously once a week 2 BCY6136 3 1 10 intravenously one once every a week 3 BCY6136 3 2 10 intravenously once a week 4 BCY6136 3 3 10 intravenously once a week

(c) Experimental methods and procedures.

(i) Cell culture.

OE21 tumor cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO ₂ in air. Tumor cells were routinely passaged twice a week. For inoculation, cells at the exponential growth stage were collected and counted.

(ii) Tumor inoculation.

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface of an OE21 tumor cell (5x10 ⁶ ) with Matrigel (1:1) in 0.2 ml of PBS. Animals were randomized and treatment began when the mean tumor volume reached approximately 157 mm ³ . The administration of the test drug and the number of animals in each group are presented in the experimental design table.

(iii) Formulation for preparation of the test drug.

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Test drug Concentration (mg/ml) Recipe Placebo - 50 mM acetate 10% sucrose pH 5 BCY6136 1 Dissolve 4.295 mg BCY6136 in 4.214 ml acetate buffer ¹ 0.1 Dilute 90 µl of 1 mg/ml BCY6136 stock solution with 810 µl acetate buffer 0.2 Dilute 180 µl of 1 mg/ml BCY6136 stock solution with 720 µl acetate buffer 0.3 Dilute 270 µl of 1 mg/ml stock solution BCY6136 using 630 µl acetate buffer

¹ Acetate buffer: 50 mM acetate 10% sucrose pH 5.

(d) Results.

(i) Tumor growth curve

The tumor growth curve is shown in Fig. 22.

(j) Volume of residual tumor.

The average tumor volume as a function of time in female Balb/c nude mice carrying the OE21 xenograft is presented in Table. 52.

Table 52

Residual tumor volume versus time

Group Treatment Days after starting treatment 0 2 4 7 9 AND 14 16 18 21 23 1 Placebo, once every a week 155±9 211±1 6 291±1 6 379±1 4 456±3 2 539±1 3 828±4 2 955±4 0 1035±5 8 1250±4 6 1586± 57 2 BCY6136, 1 mg/kg, once weekly 159±1 4 202±2 8 251±2 9 282±6 331±1 9 392±3 5 609±5 6 694±4 4 777±68 1083±8 5 1155± 98 3 BCY6136, 2 mg/kg, once weekly 157±1 9 197±1 3 219±6 235±2 7 268±3 5 243±3 7 346±7 8 371±9 8 396±10 9 515±94 537±1 22 4 BCY6136, 3 mg/kg, once weekly 155±1 9 200±1 6 197±7 209±1 1 229±2 6 211±1 4 289±3 8 318±5 3 330±40 474±42 489±5 1

(iii) Tumor growth inhibition assay.

The degree of tumor inhibition for BCY6136 in the OE21 xenograft was calculated based on tumor volume measurements at day 23 after the start of treatment.

Table 53

Inhibi analysis yuvaniya tumor growth Group Treatment Tumor volume (mm ³ ) ^a T/S ^b (%) TGI (%) P-value 1 Placebo, once a week 1586±57 - - -- 2 BCY6136, 1 mg/kg, once weekly 1155±98 72.8 30.4 P<0.05 3 BCY6136, 2 mg/kg, once weekly 537±122 33.9 73.4 P<0.001 4 BCY6136, 3 mg/kg, once weekly 489±51 30.8 76.7 P<0.001

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume of the treatment group by the mean tumor volume of the control group (T/C).

(f) The results obtained and their discussion.

This study evaluated the therapeutic efficacy of BCY6136 in the OE21 model. Measured tumor volumes in all treatment groups at various time points are presented in FIG. 22 and in table. 52 and 53.

The mean tumor volume of placebo-treated mice reached 1586 ^mm3 on day 23. BCY6136 at a dose of 1 mg/kg once a week (TV=1155 ^mm3 , TG1-30.4% p<0.05) showed antitumor activity in weak degree. BCY6136 at 2 mg/kg once weekly (TV=537 ^mm3 , TGI=73.4%, p<0.001) and at 3 mg/kg once weekly (TV=489 ^mm3 , TGI=76 .7%, p<0.001) exhibited significant antitumor activity.

In this study, there was no obvious loss of body weight in animals in all groups during the course of treatment.

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Study 22: In Vivo Test of the Efficacy of BCY6136 in the Treatment of Xenografts

MOLP-8 in CB17-SCID mice.

(a) Purpose of the study.

The purpose of the study is to evaluate the in vivo antitumor efficacy of BCY6136 in the treatment of MOLP-8 xenografts in CB17-SCID mice.

(b) Experimental design._____ ________________________________________________

Group Treatment η Dose (mg/kg) Dosing volume (µl/g) Dosing method Scheme 1 Placebo 3 - 10 intravenously once a week 2 BCY6136 3 1 10 intravenously once a week 3 BCY6136 3 2 10 intravenously once a week 4 BCY6136 3 3 10 intravenously once a week

(c) Experimental methods and procedures.

(i) Cell culture.

MOLP-8 tumor cells were maintained in vitro as a monolayer culture in RMPI-1640 medium supplemented with 20% heat-inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO ₂ in air. Tumor cells were routinely passaged by treatment with trypsin-EDTA. For inoculation, cells at the exponential growth stage were collected and counted.

(ii) Tumor inoculation.

For tumor development, each mouse was inoculated subcutaneously into the right lateral surface of a MOLP-8 tumor cell (10x106) in 0.2 ml PBS with 50% Matrigel. When the average tumor volume reached 141 mm ³ , 36 animals were randomized. The administration of the test drug and the number of animals in each group are presented in the experimental design table.

(iii) Formulation for preparation of the test drug.

Treatment Concentration (mg/ml) Recipe Placebo — 50 mM acetate, 10% sucrose pH 5 BCY6136 OD Dilute 90 µl stock solutions* of 1 mg/ml BCY6136 with 810 µl buffer** 0.2 Dilute 180 µl stock solutions* with 1 mg/ml BCY6136 with 720 µl buffer** o, s Dilute 270 µl stock solutions* with 1 mg/ml BCY6136 with 630 µl buffer**

*BCY6136 stock solutions: 10.93 mg BCY6136 was dissolved in 10.93 ml 50 mM acetate, 10% sucrose, pH 5 and dispensed into separate tubes and stored at -80°C.

** Buffer: 50 mM acetate, 10% sucrose pH 5.

(d) Results.

(i) Tumor growth curve.

The tumor growth curve is shown in Fig. 23.

(ii) Volume of residual tumor.

The average tumor volume over time in female CB17-SCID mice bearing the MOLP-8 xenograft is presented in Table 1. 54.

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Table 54

Residual tumor volume as a function of time______________

Group pa Treatment Days after starting treatment 0 2 4 7 9 AND 14 1 Placebo, once a week 139±2 375±3 6 604±2 8 984±8 8 1451±1 33 1981±1 96 2528±2 95 2 BCY6136, 1 mg/kg, once a week 143±1 3 299±6 444±4 9 576±3 1 806±85 1132±1 70 1446±2 34 3 BCY6136, 2 mg/kg, once weekly 140±1 5 271±4 3 250±2 509±2 3 662±78 873±49 1218±1 44 4 BCY6136, 3 mg/kg, once weekly 142±1 9 239±6 7 197±2 0 342±7 8 425±90 693±13 3 938±15 5

(iii) Tumor growth inhibition assay.

The degree of tumor inhibition for BCY6136 in the MOLP-8 xenograft model was calculated based on tumor volume measurements at day 14 after initiation of treatment.

Table 55

Tumor Growth Inhibition Assay

Group Treatment Tumor volume (mm ³ ) ^a t/s ^b (%) TGI (%) P-value compared with placebo 1 Placebo, once a week 2528±295 - - - 2 BCY6136, 1 mg/kg, once a week 1446±234 57.2 45.5 p>0.05 3 BCY6136, 2 mg/kg, once weekly 1218±144 48.2 54.9 p<0.05 4 BCY6136, 3 mg/kg, once weekly 938±155 37.1 66.7 p<0.01

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

(f) The results obtained and their discussion.

This study evaluated the therapeutic efficacy of BCY6136 in the MOLP-8 xenograft model. Measured tumor volumes in all treatment groups at various time points are presented in FIG. 23 and in table. 54 and 55.

The mean tumor volume of placebo-treated mice reached 2528 ^mm3 on day 14. BCY6136 at a dose of 1 mg/kg (TV=1146 ^mm3 , TGI=45.5%, p>0.05), at a dose of 2 mg/kg kg (TV=1218 ^mm3 , TGI=54.9%, p<0.05) and at a dose of 3 mg/kg (TV=938 ^mm3 , TGI=66.7%, p<0.01) showed dose-dependent antitumor activity, but tumor regression in MOLP-8 xenografts was not achieved at all dose levels. IN

- 81 044626 in this study, all mice maintained body weight at an almost constant level.

Study 23. In vivo testing of the effectiveness of BCY drugs in the treatment of HT1080 xenograft in BALB/c nude mice.

(a) Purpose of the study.

The aim of the study was to evaluate in vivo the antitumor activity of BCY drugs in the treatment of the HT1080 xenograft model in BALB/c nude mice.

(b) Experimental design_____ ___________________________________________

Group Treatment η Dose (mg/kg) Dosing volume (µl/g) Dosing method Scheme 1 Placebo 3 - 10 intravenously once in Week 2 BCY6173 3 1 10 intravenously once in Week 3 BCY6173 3 2 10 intravenously once in Week 4 BCY6173 3 3 10 intravenously once in Week 5 BCY6135 3 1 10 intravenously once in Week 6 BCY6135 3 2 10 intravenously once in Week

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7 BCY6135 3 3 10 intravenously once in Week 8 BCY6136 3 2 10 intravenously once in Week 9 BCY6136 3 3 10 intravenously once in Week 10 BCY6136 3 5 10 intravenously once in Week AND BCY6174 3 1 10 intravenously once in Week 12 BCY6174 3 2 10 intravenously once in Week 13 BCY6174 3 3 10 intravenously once in Week 14 BCY6175 3 1 10 intravenously once in Week 15 BCY6175 3 2 10 intravenously once in Week 16 BCY6175 3 3 10 intravenously once in Week 17 ADC 3 3 10 intravenously once in Week

Note: n: number of animals; dosing volume: adjusted dosing volume 10 µl/g based on body weight.

(c) Experimental methods and procedures.

(i) Cell culture.

HT1080 tumor cells should be maintained in a medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Tumor cells should be routinely passaged twice a week. For tumor inoculation, cells should be collected in the exponential growth phase and their number counted.

(ii) Tumor inoculation.

For tumor development, each mouse should be inoculated subcutaneously into the right lateral surface of an HT1080 tumor cell (5x10 ⁶ ). Animals should be randomized and treatment should begin when the average tumor volume reaches approximately 150-200 mm ³ . The administration of the test drug and the number of animals in each group are given in the experimental design table.

(iii) Formulation for preparation of the test drug.

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Treatment Dose (mg/ml) Recipe Placebo — 50 mM acetate/acetic acid pH 5 10% sucrose BCY6173 1 Dissolve 2.13 mtBCY6173 in 2.04 ml buffer OD Dilute 90 µL of 1 mg/mL BCY6173 stock solution with 810 µL buffer 0.2 Dilute 180 µl of 1 mg/ml BCY6173 stock solution with 720 µl of buffer o, s Dilute 270 µl of 1 mg/ml BCY6173 stock solution with 630 µl of buffer BCY6135 1 Dissolve 2 mtBCY6135 in 1.9 ml buffer OD Dilute 90 µl of 1 mg/ml BCY6135 stock solution with 810 µl of buffer 0.2 Dilute 180 µl of 1 mg/ml BCY6135 stock solution with 720 µl of buffer o, s Dilute 270 µl of 1 mg/ml BCY6135 stock solution with 630 µl of buffer BCY6136 0.2 Dilute 200 µl of 1 mg/ml BCY6136 stock solution with 800 µl of buffer o, s Dilute 300 µl of 1 mg/ml BCY6136 stock solution with 700 µl of buffer 0.5 Dilute 500 µl of 1 mg/ml BCY6136 stock solution with 500 µl of buffer BCY6174 1 Dissolve 2.69 mg BCY6174 in 2.677 ml buffer OD Dilute 90 µl of 1 mg/ml BCY6174 stock solution with 810 µl of buffer 0.2 Dilute 180 µl of 1 mg/ml BCY6174 stock solution with 720 µl of buffer o, s Dilute 270 µl of 1 mg/ml BCY6174 stock solution with 630 µl of buffer BCY6175 1 Dissolve 2 mtBCY6175 in 1.924 ml buffer OD Dilute 90 µl of 1 mg/ml BCY6175 stock solution with 810 µl buffer 0.2 Dilute 180 1 mg/mL BCY6175 stock solution with 720 µL buffer o, s Dilute 270 µl of 1 mg/ml BCY6175 stock solution with 630 µl of buffer ADC o, s Dilute 25.78 µl of 10.47 mg/ml ADC stock solution with 874.22 µl 25 mM histidine pH 7 10% sucrose

(d) Results.

(i) Tumor growth curve.

The tumor growth curve is shown in Fig. 24-29.

(ii) Volume of residual tumor.

The average tumor volume as a function of time in female Balb/c nude mice carrying the HT1080 xenograft is shown in Table. 56.

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Table 56

Residual tumor volume versus time

Group Treatment Days after starting treatment 0 2 4 7 9 AND 14 1 Placebo, once a week 179±2 2 312± 84 529±1 35 886±2 07 1185±1 72 1467±2 24 1737±2 58 2 BCY6173 1 mg/kg, once every a week 178±2 6 276± 8 328±7 3 594±6 2 745±22 960±53 1074±1 50 3 BCY6173, 2 mg/kg, 178±2 8 277± 61 262±1 25 309±2 38 425±33 4 436±32 3 480±34 7 once a week 4 BCY6173, 3 mg/kg, once weekly 179±4 3 182± 71 133±8 8 87±68 77±65 60±54 47±42 5 BCY6135 1 mg/kg, once a week 178±2 2 267± 66 262±5 8 436±6 7 599±89 703±36 871±28 6 BCY6135 2 mg/kg, once a week 178±2 3 176± 48 117±4 3 70±23 67±23 52±21 62±7 7 BCY6135 3 mg/kg, once a week 177±3 9 178± 79 92±67 62±46 62±51 57±51 44±40 8 BCY6136 2mg/kg, once a week 178±1 9 249± 22 115±8 126±5 3 158±71 140±89 245±11 6 9 BCY6136 3 mg/kg, once a week 178±3 6 168± 21 72±18 22±7 21±15 8±6 3±2 10 BCY6136 5 mg/kg, once a week 178±2 6 165± 33 52±10 18±7 9±4 5±2 2±1 eleven BCY6174 1 mg/kg, once a week 180±3 5 231± 19 226±2 9 432±3 7 602±63 742±62 1066±1 thirty

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12 BCY6174 2 mg/kg, once a week 178±3 1 203± 50 123±2 9 216±4 7 291±40 326±68 532±91 13 BCY6174 3 mg/kg, once a week 178±3 3 195± 13 110±3 9 58±23 34±17 21±11 11±7 14 BCY6175 1 mg/kg, once a week 178±2 7 248± 62 244±7 4 347±1 8 435±18 558±38 769±26 15 BCY6175 2 mg/kg, once a week 178±2 2 223± 42 158±5 9 116±3 5 156±52 166±51 295±88 16 BCY6175 3 mg/kg, once a week 179±3 9 189± 48 116±5 0 43±18 33±18 25±13 11±9 17 ADC 3 mg/kg, once every a week 180±2 6 158± thirty 58±8 18±2 7±1 2±2 0±0

(iii) Tumor growth inhibition assay

The degree of tumor inhibition for BCY drugs in the HT 1080 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment.

Table 57

Tumor Growth Inhibition Assay

Group Treatment Tumor volume (mm ³ ) ^a T/S ^b (%) TGI (%) P-value when compared with placebo 1 Placebo, once a week 1737±258 — — —

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2 BCY6173, 1 mg/kg, once a week 1074±150 61.8 42.5 p>0.05 3 BCY6173, 2 mg/kg, once weekly 480±347 27.6 80.6 p<0.05 4 BCY6173, 3 mg/kg, once weekly 47±42 2.7 108.4 p<0.01 5 BCY6135, 1 mg/kg, once a week 871±28 50.1 55.5 p<0.01 6 BCY6135, 2 mg/kg, once weekly 62±7 3.5 107.5 p<0.001 7 BCY6135, 3 mg/kg, once weekly 44±40 2.5 108.6 p<0.001 8 BCY6136, 2 mg/kg, once weekly 245±116 14.1 95.7 p<0.001 9 BCY6136, 3 mg/kg, once weekly 3±2 0.2 111.2 p<0.001 10 BCY6136, 5 mg/kg, once weekly 2±1 θ,ι 111.3 p<0.001 AND BCY6174, 1 mg/kg, once a week 1066±130 61.4 43.1 p<0.05 12 BCY6174, 2 mg/kg, once weekly 532±91 30.6 77.3 p<0.01 13 BCY6174, 3 mg/kg, once weekly 11±7 0.6 110.7 p<0.001 14 BCY6175, 1 mg/kg, once a week 769±26 44.3 62.1 p<0.01 15 BCY6175, 2 mg/kg, once weekly 295±88 17.0 92.5 p<0.001 16 BCY6175, 3 mg/kg, once weekly 11±9 0.6 110.8 p<0.001 17 ADC, 3 mg/kg, once weekly 0±0 oh oh 111.5 -

^a Mean ± standard error of the mean (SEM).

^b Tumor growth inhibition is calculated by dividing the mean tumor volume in the treated group by the mean tumor volume in the control group (T/C).

(f) The results obtained and their discussion.

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Claims (21)

This study evaluated the therapeutic efficacy of BCY drugs in the HT1080 xenograft model. Measured tumor volumes in all treatment groups at various time points are presented in FIG. 24-29 and in table. 56 and 57. The mean tumor volume of placebo-treated mice reached 1737 ^mm3 on day 14. The drug BCY6173 at a dose of 1 mg/kg once a week (TV=1074 ^mm3 , TGI=42.5%, p>0.05), at a dose of 2 mg/kg once a week (TV=480 ^mm3 , TGI=80.6%, p<0.05) and at a dose of 3 mg/kg once a week (TV=7 mm ³ , TGI=108.4%, p<0.01) exhibited dose-dependent antitumor activity. BCY6135 at a dose of 1 mg/kg once a week (TV=871 ^mm3 , TGI=55.5%, p<0.01), at a dose of 2 mg/kg once a week (TV=62 ^mm3 , TGI=107.5%, p<0.001) and a dose of 3 mg/kg once a week (TV=44 ^mm3 , TGI=108.6%, p<0.001) exhibited dose-dependent antitumor activity. BCY6136 at a dose of 2 mg/kg once a week (TV=345 ^mm3 , TGI=95.7%, p<0.001), at a dose of 3 mg/kg once a week (TV=3 ^mm3 , TGI= 111.2%, p<0.001) and at a dose of 5 mg/kg once a week (TV=2 mm ³ , TGI=111.3%, p<0.001) showed powerful antitumor activity. BCY6174 at a dose of 1 mg/kg once a week (TV=1066 ^mm3 , TGI=43.1%, p<0.05), at a dose of 2 mg/kg once a week (TV=532 ^mm3 , TGI=77.3%, p<0.01) and at a dose of 3 mg/kg once a week (TV=11 mm ³ , TGI=110.7%, p<0.001) exhibited dose-dependent antitumor activity. BCY6175 at a dose of 1 mg/kg once a week (TV=769 ^mm3 , TGI=62.1%, p<0.01), at a dose of 2 mg/kg once a week (TV=295 ^mm3 , TGI=92.5%, p<0.001) and at a dose of 3 mg/kg once a week (TV=11 mm ³ , TGI=110.8%, p<0.001) exhibited dose-dependent antitumor activity. The ADC drug at a dose of 3 mg/kg once a week (TV=0 ^mm3 , TGI=111.5%) completely destroyed tumors by day 14. CLAIM 1. A peptide ligand having specificity for EphA2, comprising a polypeptide containing at least three cysteine residues separated by at least two loop sequences, and a non-aromatic molecular framework that forms covalent bonds with the cysteine residues of the polypeptide such that At least two polypeptide loops are formed on the molecular framework, where the peptide ligand includes the amino acid sequence: Ci(HyP)LVNPLCiiLHP(D-Asp)W(HArg)Ciii (SEQ ID NO: 1); wherein HyP represents hydroxyproline, HArg represents homoarginine, and Ci, Cii and Cl represent the first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof. 2. The peptide ligand according to claim 1, where the molecular framework is 1,1',1''-(1,3,5-triazinan1,3,5-triyl)-triprop-2-en-1-one (TATA ). 3. Peptide ligand according to claim 1 or 2, where the peptide ligand includes the amino acid sequence: (3-Ala)-Sar ₁₀ -A(HArg)D-Ci(HyP)LVNPLCiiLHP(D-Asp)W(HAr _g )C _ii i (SEQ ID NO: 2) (BCY6099); where Sar is sarcosine, HArg is homoarginine, and HuP is hydroxyproline. 4. The peptide ligand according to any one of claims 1 to 3, wherein the pharmaceutically acceptable salt is selected from a free acid salt or a sodium, potassium, calcium, ammonium salt. 5. The peptide ligand according to any one of claims 1 to 4, wherein EphA2 is human EphA2. 6. A drug conjugate comprising a peptide ligand according to any one of claims 15, conjugated to one or more effector and/or functional groups, such as a cytotoxic agent. 7. The drug conjugate of claim 6, wherein said cytotoxic agent is selected from DM1 or MMAE. 8. The drug conjugate of claim 7, wherein said cytotoxic agent is selected from MMAE. 9. A drug conjugate according to any one of claims 6 to 8, which further includes a linker between said peptide ligand and said cytotoxic agents. 10. The drug conjugate of claim 9, wherein said cytotoxic agent is MMAE and the linker is selected from -Val-Cit-, -Trp-Cit-, -Val-Lys-, -D-Trp-Cit-, -Ala -Ala-Asn-, D-AlaPhe-Lys- or -Glu-Pro-Cit-Gly-hPhe-Tyr-Leu- (SEQ ID NO: 3). 11. The drug conjugate of claim 10, wherein said cytotoxic agent is MMAE and the linker is -Val-Cit-. 12. The drug conjugate of claim 9, wherein said cytotoxic agent is DM1 and the linker is selected from -SS-, -SS(SO ₃ H)-, -SS-(Me)-, -(Me)-SS- (Me)-, -SS-(Me ₂ )- or -SS(Me)-SO3H-. 13. A drug conjugate according to claim 12, wherein said cytotoxic agent is - 88 044626 is DM1, and the linker is selected from -SS- and -SS(SO ₃ H)-. 14. The drug conjugate according to any one of claims 6 to 13, which is selected from any one of BCY6027: where the specified bicycle is BCY6099, BCY6028 defined above: where the specified bicycle is BCY6099, BCY6135 defined above: Molecular spacer (SarlO) Bicycle (BCY6099} Cleavable linker (S-S) Toxic (DM1) BCY6136: Bicycle (BCY6099) Molecular spacer (Sari0) Cleavable linker (Val-Cit) Toxic agent (MIDE) BCY6173: - 89 044626 Cleavable linker (S-S(SO3H)) Molecular spacer (SarlO) Bicycle (BCY6099) Toxic agent (DMI) BCY6174: Toxic Cleavable Linker Agent (MMAE) (Val-Lys) Molecular spacer (SarlO) Bicycle (BCY6099) and BCY6175: NHj Toxic agent (MMAE) Cleavable linker (Val-Lys) Molecular spacer (SarlO) Bicycle (BCY6099) 15. The drug conjugate of claim 14, which is selected from any one of BCY6135, BCY6136, BCY6173, BCY6174 and BCY6175. 16. The drug conjugate of claim 14 or 15, which is BCY6136. 17. A pharmaceutical composition which comprises a peptide ligand according to any one of claims 1 to 5 or a drug conjugate according to any one of claims 6 to 16 in combination with one or more pharmaceutically acceptable excipients. 18. Use of a drug conjugate according to any one of claims 6 to 16 for the purpose of preventing, inhibiting the development or treating a disease or disorder characterized by overexpression of EphA2 in the affected tissue. 19. Use of a drug conjugate according to any one of claims 6 to 16 for the purpose of preventing, inhibiting the development or treating cancer. 20. Use according to claim 19, wherein the cancer is selected from prostate cancer, lung cancer (such as non-small cell lung carcinomas (NSCLC)), breast cancer (such as triple negative breast cancer), gastric cancer, ovarian cancer, esophageal cancer and fibrosarcomas. 21. A method of preventing, inhibiting the development or treatment of cancer, which includes administering to a patient in need thereof a drug conjugate according to any one of claims 6 to 16, wherein the patient is found to have an increased copy number variation (CNV) of EphA2. -