BICYCLIC PEPTIDE LIGANDS SPECIFIC FOR EphA2

FIELD OF THE INVENTION

The present invention relates to polypeptides which are covalently bound to non-aromatic molecular scaffolds such that two or more peptide loops are subtended between attachment points to the scaffold. In particular, the invention describes peptides which are high affinity binders of the Eph receptor tyrosine kinase A2 (EphA2). The invention also includes drug conjugates comprising said peptides, conjugated to one or more effector and/or functional groups, to pharmaceutical compositions comprising said peptide ligands and drug conjugates and to the use of said peptide ligands and drug conjugates in preventing, suppressing or treating a disease or disorder characterised by overexpression of EphA2 in diseased tissue (such as a tumour).

BACKGROUND OF THE INVENTION

Cyclic peptides are able to bind with high affinity and target specificity to protein targets and hence are an attractive molecule class for the development of therapeutics. In fact, several cyclic peptides are already successfully used in the clinic, as for example the antibacterial peptide vancomycin, the immunosuppressant drug cyclosporine or the anti-cancer drug octreotide (Driggers et ai (2008), Nat Rev Drug Discov 7 (7), 608-24). Good binding properties result from a relatively large interaction surface formed between the peptide and the target as well as the reduced conformational flexibility of the cyclic structures. Typically, macrocycles bind to surfaces of several hundred square angstrom, as for example the cyclic peptide CXCR4 antagonist CVX15 (400 A ² ; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with the Arg-Gly-Asp motif binding to integrin aVb3 (355 A ² ) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1 binding to urokinase-type plasminogen activator (603 A ² ; Zhao et al. (2007), J Struct Biol 160 (1), 1-10).

Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, leading to a smaller loss of entropy upon binding to targets and resulting in a higher binding affinity. The reduced flexibility also leads to locking target-specific conformations, increasing binding specificity compared to linear peptides. This effect has been exemplified by a potent and selective inhibitor of matrix metalloproteinase 8, (MMP-8) which lost its selectivity over other MMPs when its ring was opened (Cherney et al. (1998), J Med Chem 41 (11), 1749- SI). The favorable binding properties achieved through macrocyclization are even more pronounced in multicyclic peptides having more than one peptide ring as for example in vancomycin, nisin and actinomycin. Different research teams have previously tethered polypeptides with cysteine residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and co-workers had used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclisation of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for the generation of candidate drug compounds wherein said compounds are generated by linking cysteine containing polypeptides to a molecular scaffold as for example TATA (1 , T,1 "-(1 ,3,5-triazinane-1 ,3,5-triyl)triprop-2-en-1-one, Heinis et al. Angew Chem, Int Ed. 2014; 53: 1602-1606).

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

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a peptide ligand specific for EphA2 comprising a polypeptide comprising at least three cysteine residues, separated by at least two loop sequences, and a non-aromatic molecular scaffold which forms covalent bonds with the cysteine residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold, wherein the peptide ligand comprises the amino acid sequence:

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

wherein HyP is hydroxyproline, HArg is homoarginine and C,, C _M and C _M , represent first, second and third cysteine residues, respectively or a pharmaceutically acceptable salt thereof.

According to a further aspect of the invention, there is provided a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effector and/or functional groups.

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand or a drug conjugate as defined herein in combination with one or more pharmaceutically acceptable excipients. According to a further aspect of the invention, there is provided a peptide ligand or drug conjugate as defined herein for use in preventing, suppressing or treating a disease or disorder characterised by overexpression of EphA2 in diseased tissue (such as a tumour).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : General schematic demonstrating the concept of preparing Bicycle drug conjugates (BDCs).

Figure 2: Plot of mean tumour volume versus time for BCY6136 in HT1080 xenograft mice. Doses (2, 3 and 5 mg/kg) were administered on days 0 and 7.

Figure 3: Plot of mean tumour volume versus time for BCY6136 in NCI-H1975 xenograft mice. Doses (1 , 2 and 3 mg/kg) were administered on days 0, 7, 14, 21 , 28 and 35.

Figure 4: Plot of mean tumour volume versus time for BCY6136 in MDA-MB-

231 xenograft mice. Doses (1 , 2 and 3 mg/kg) were administered on day 0, 7, 14, 21 , 28, 35 and 45.

Figures 5 and 6: Tumor volume traces after administering BCY6136 (Figure 5) and ADC (Figure 6) to female BALB/c nude mice bearing PC-3 xenograft.

Figure 7: Tumor volume traces after administering BCY6136, EphA2-ADC or Docetaxel to male Balb/c nude mice bearing PC-3 xenograft.

Figure 8: Tumor volume trace after administering BCY6136 to female Balb/c nude mice bearing NCI-H1975 xenograft. Data points represent group mean tumor volume.

Figures 9 and 10: Tumor volume traces after administering BCY6136 and ADC to female Balb/c nude mice bearing LU-01-0251 xenograft.

Figure 11 : Tumor volume traces after administering BCY6136 to female Balb/c nude mice bearing LU-01-0046.

Figure 12: T umor volume traces after administering BCY6136 or ADC to female Balb/c nude mice bearing LU-01-0046 NSCLC PDX model. Figures 13 to 15: Tumor volume traces after administering BCY6136 (Figure 13), BCY6173 (Figure 14) and BCY6175 (Figure 15) to female Balb/c nude mice bearing LU-01- 0046.

Figure 16: Tumor volume traces after administering BCY6136 (referred to in Figure 16 as BT5528), BCY8245 or BCY8781 to female BALB/c nude mice bearing LU-01-0412 xenograft. Data points represent group mean tumor volume.

Figure 17: Tumor volume traces after administering BCY6136 to female Balb/c nude mice bearing LU-01 -0486 xenograft.

Figure 18: Tumor volume trace after administering BCY6136 to female Balb/c nude mice bearing MDA-MB-231-luc xenograft. Data points represent group mean tumor volume.

Figure 19: Tumor volume traces after administering BCY6136 to female BALB/c mice bearing EMT-6 syngeneic. The dosage of group 3 and group 4 was changed to 5 mpk and 3 mpk from Day 14.

Figure 20: Tumor volume traces after administering BCY6136 to female Balb/c nude mice bearing NCI-N87 xenograft.

Figure 21 : Tumor volume traces after administering BCY6136 to female Balb/c nude mice bearing SK-OV-3 xenograft.

Figure 22: Tumor volume traces after administering BCY6136 to female Balb/c nude mice bearing OE21 xenograft.

Figure 23: Tumor volume traces after administering BCY6136 to female CB17-SCID mice bearing MOLP-8 xenograft.

Figures 24 to 29: Tumor volume traces after administering BCY6173 (Figure 24), BCY6135 (Figure 25), BCY6136 (Figure 26), BCY6174 (Figure 27), BCY6175 (Figure 28) and ADC (Figure 29) to female BALB/c nude mice bearing HT1080 xenograft.

Where error bars are present in the above Figures, these represent standard error of the mean

DETAILED DESCRIPTION OF THE INVENTION In one embodiment, the peptide ligand comprises the amino acid sequence:

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

wherein Sar is sarcosine, HArg is homoarginine and HyP is hydroxyproline.

In one embodiment, the molecular scaffold is 1 ,1',1"-(1 ,3,5-triazinane-1 ,3,5-triyl)triprop-2-en-

1-one (TATA).

In a further embodiment, the molecular scaffold is 1 , 1 ', 1 "-(1 ,3,5-triazinane-1 ,3,5-triyl)triprop-

2-en-1-one (TATA) and the peptide ligand comprises the amino acid sequence:

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

wherein Sar is sarcosine, HArg is homoarginine and HyP is hydroxyproline.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as in the arts of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (see Sam brook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001 , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999) 4 ^th ed., John Wiley & Sons, Inc.), which are incorporated herein by reference.

Nomenclature

Numbering

When referring to amino acid residue positions within the peptides of the invention, cysteine residues (C,, C _M and C _m ) are omitted from the numbering as they are invariant, therefore, the numbering of amino acid residues within the peptides of the invention is referred to as below:

-Ci-HyP ₁ -L ₂ -V ₃ -N ₄ -P ₅ -L ₆ -Cii-L ₇ -H ₈ -P ₉ -(D-Asp) ₁ o-W ₁₁ -(HArg) ₁₂ -Ciii- (SEQ ID NO: 1).

For the purpose of this description, all bicyclic peptides are assumed to be cyclised with 1 ,1',1"-(1 ,3,5-triazinane-1 ,3,5-triyl)triprop-2-en-1-one (TATA) yielding a tri-substituted 1 ,1',1"- (1 ,3,5-triazinane-1 ,3,5-triyl)tripropan-1-one structure. Cyclisation with TATA occurs on C,, C _M , and Ciii.

Molecular Format N- or C-terminal extensions to the bicycle core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal ^-Ala)-Sario-Ala tail would be denoted as:

^-Ala)-Sar ₁₀ -A-(SEQ ID NO: X).

Inversed Peptide Sequences

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

Peptide Ligands

A peptide ligand, as referred to herein, refers to a peptide, peptidic or peptidomimetic covalently bound to a molecular scaffold. Typically, such peptides, peptidics or peptidomimetics comprise a peptide having natural or non-natural amino acids, two or more reactive groups (i.e. cysteine residues) which are capable of forming covalent bonds to the scaffold, and a sequence subtended between said reactive groups which is referred to as the loop sequence, since it forms a loop when the peptide, peptidic or peptidomimetic is bound to the scaffold. In the present case, the peptides, peptidics or peptidomimetics comprise at least three cysteine residues (referred to herein as C,, CM and C _M ,), and form at least two loops on the scaffold.

Advantages of the Peptide Ligands

Certain bicyclic peptides of the present invention have a number of advantageous properties which enable them to be considered as suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include:

Species cross-reactivity. This is a typical requirement for preclinical pharmacodynamics and pharmacokinetic evaluation;

Protease stability. Bicyclic peptide ligands should in most circumstances demonstrate stability to plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases and the like. Protease stability should be maintained between different species such that a bicyclic peptide lead candidate can be developed in animal models as well as administered with confidence to humans; Desirable solubility profile. This is a function of the proportion of charged and hydrophilic versus hydrophobic residues and intra/inter-molecular H-bonding, which is important for formulation and absorption purposes;

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

Selectivity. Certain peptide ligands of the invention demonstrate good selectivity over 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 may be seen in Tables 1 1 and 12). It should also be noted that selected peptide ligands of the invention exhibit cross reactivity with other species (eg mouse and rat) to permit testing in animal models (Tables 3, 7-8, 10 and 12); and

Safety. Bleeding events have been reported in pre-clinical in vivo models and clinical trials with EphA2 Antibody Drug Conjugates. For example, a phase 1 , open-label study with MEDI-547 was halted due to bleeding and coagulation events that occurred in 5 of 6 patients (Annunziata et ai, Invest New Drugs (2013) 31 :77-84). The bleeding events observed in patients were consistent with effects on the coagulation system observed in rat and monkey pre-clinical studies: increased activated partial thromboplastin time and increased

fibrinogen/fibrin degradation product (Annunziata et al IBID). Overt bleeding events were reportedly seen in toxicology studies in monkeys (Annunziata et ai, IBID). Taken together these results imply that MEDI-547 causes Disseminated Intravascular Coagulation (DIC) in both preclinical species and patients. The BDCs reported here have short in vivo half lives (< 30 minutes) and are therefore intrinsically less likely to give rise to DIC in patients. Results shown here (see BIOLOGICAL DATA sections 5 and 6 and Table 15) demonstrate that selected Bicycle Drug Conjugates of the invention have no effect on coagulation parameters and gave rise to no bleeding events in pre-clinical studies.

Pharmaceutically Acceptable Salts

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

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

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

If the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO ), then a salt may be formed with an organic or inorganic base, generating a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li ⁺ , Na ⁺ and K ⁺ , alkaline earth metal cations such as Ca ²⁺ and Mg ²⁺ , and other cations such as Al ³⁺ or Zn ⁺ . Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. , NH ₄ ⁺ ) and substituted ammonium ions (e.g., NHsR ⁺ , NH2R2 ⁺ , NHR3 ⁺ , NR ₄ ⁺ ). Examples of some suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 ⁺ .

Where the peptides of the invention contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the peptides of the invention.

Isotopic variations

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

Examples of isotopes suitable for inclusion in the peptide ligands of the invention comprise isotopes of hydrogen, such as ² H (D) and ³ H (T), carbon, such as ¹¹ C, ¹³ C and ¹⁴ C, chlorine, such as ³⁶ CI, fluorine, such as ¹⁸ F, iodine, such as ¹²³ l, ¹²⁵ l and ¹³¹ l, nitrogen, such as ¹³ N and ¹⁵ N, oxygen, such as ¹⁵ 0, ¹⁷ 0 and ¹⁸ 0, 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.

Certain isotopically-labelled peptide ligands of the invention, for example, those

incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies, and to clinically assess the presence and/or absence of the EphA2 target on diseased tissues. The peptide ligands of the invention can further have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes or receptors. The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The radioactive isotopes tritium, i.e. ³ H (T), and carbon-14, i.e. ¹⁴ C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Substitution with heavier isotopes such as deuterium, i.e. ² H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹ C, ¹⁸ F, ¹⁵ 0 and ¹³ N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.

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

Non-Aromatic Molecular scaffold

References herein to the term“non-aromatic molecular scaffold” refer to any molecular scaffold as defined herein which does not contain an aromatic (i.e. unsaturated) carbocyclic or heterocyclic ring system.

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

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

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

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

The molecular scaffold may comprise chemical groups which form the linkage with a peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides. An example of an a unsaturated carbonyl containing compound is 1 , T, 1 "-(1 ,3,5-triazinane- 1 ,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606).

Effector and Functional Groups

According to a further aspect of the invention, there is provided a drug conjugate comprising a peptide ligand as defined herein 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 within the polypeptide, or to the molecular scaffold.

Appropriate effector groups include antibodies and parts or fragments thereof. For instance, an effector group can include an antibody light chain constant region (CL), an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof, in addition to the one or more constant region domains. An effector group may also comprise a hinge region of an antibody (such a region normally being found between the CH1 and CH2 domains of an IgG molecule).

In a further embodiment of this aspect of the invention, an effector group according to the present invention is an Fc region of an IgG molecule. Advantageously, a peptide ligand- effector group according to the present invention comprises or consists of a peptide ligand Fc fusion having a ΐb half-life of a 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. Most advantageously, the peptide ligand according to the present invention comprises or consists of a peptide ligand Fc fusion having a ΐb half-life of a day or more.

Functional groups include, in general, binding groups, drugs, reactive groups for the attachment of other entities, functional groups which aid uptake of the macrocyclic peptides into cells, and the like.

The ability of peptides to penetrate into cells will allow peptides against intracellular targets to be effective. Targets that can be accessed by peptides with the ability to penetrate into cells include transcription factors, intracellular signalling molecules such as tyrosine kinases and molecules involved in the apoptotic pathway. Functional groups which enable the penetration of cells include peptides or chemical groups which have been added either to the peptide or the molecular scaffold. Peptides such as those derived from such as VP22, HIV- Tat, a homeobox protein of Drosophila (Antennapedia), e.g. as described 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 which have been shown to be efficient at translocation through plasma membranes include the 16 amino acid penetratin peptide from Drosophila Antennapedia protein (Derossi et al (1994) J Biol. Chem. Volume 269 p10444), the 18 amino acid‘model amphipathic peptide’ (Oehlke et al (1998) Biochim Biophys Acts Volume 1414 p127) and arginine rich regions of the HIV TAT protein. Non peptidic approaches include the use of small molecule mimics or SMOCs that can be easily attached to biomolecules (Okuyama et al (2007) Nature Methods Volume 4 p153). Other chemical strategies to add guanidinium groups to molecules also enhance cell penetration (Elson-Scwab et al (2007) J Biol Chem Volume 282 p13585).

Small molecular weight molecules such as steroids may be added to the molecular scaffold to enhance uptake into cells.

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

In one embodiment, a peptide ligand-effector group according to the invention has a ίb 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. Advantageously a peptide ligand-effector group or composition according to the invention will have a ΐb range 12 to 60 hours. In a further embodiment, it will have a ΐb half-life of a day or more. In a further embodiment still, it will be in the range 12 to 26 hours.

In one particular embodiment of the invention, the functional group is selected from a metal chelator, which is suitable for complexing metal radioisotopes of medicinal relevance.

Possible effector groups also include enzymes, for instance such as carboxy peptidase G2 for use in enzyme/prodrug therapy, where the peptide ligand replaces antibodies in ADEPT.

In one particular embodiment of the invention, the functional group is selected from a drug, such as a cytotoxic agent for cancer therapy. Suitable examples include: alkylating agents such as cisplatin and carboplatin, as well as oxaliplatin, mechlorethamine,

cyclophosphamide, chlorambucil, ifosfamide; Anti-metabolites including purine analogs azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids including vinca alkaloids such as Vincristine, Vinblastine, Vinorelbine and Vindesine;

Podophyllotoxin and its derivatives etoposide and teniposide; Taxanes, including paclitaxel, originally known as Taxol; topoisomerase inhibitors including camptothecins: irinotecan and topotecan, and type II inhibitors including amsacrine, etoposide, etoposide phosphate, and teniposide. Further agents can include antitumour antibiotics which include the

immunosuppressant dactinomycin (which is used in kidney transplantations), doxorubicin, epirubicin, bleomycin, calicheamycins, and others. In one further particular embodiment of the invention, the cytotoxic agent is selected from maytansinoids (such as DM1) or monomethyl auristatins (such as MMAE).

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

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

In one embodiment, the cytotoxic agent is linked to the bicyclic peptide by a cleavable bond, such as a disulphide bond or a protease sensitive bond. In a further embodiment, the groups adjacent to the disulphide bond are modified to control the hindrance of the disulphide bond, and by this the rate of cleavage and concomitant release of cytotoxic agent.

Published work established the potential for modifying the susceptibility of the disulphide bond to reduction by introducing steric hindrance on either side of the disulphide bond (Kellogg et al (2011) Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrance reduces the rate of reduction by intracellular glutathione and also extracellular (systemic) reducing agents, consequentially reducing the ease by which toxin is released, both inside and outside the cell. Thus, selection of the optimum in disulphide stability in the circulation (which minimises undesirable side effects of the toxin) versus efficient release in the intracellular milieu (which maximises the therapeutic effect) can be achieved by careful selection of the degree of hindrance on either side of the disulphide bond.

The hindrance on either side of the disulphide bond is modulated through introducing one or more methyl groups on either the targeting entity (here, the bicyclic peptide) or toxin side of the molecular construct.

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

In one embodiment, the cytotoxic agent and linker is selected from any combinations of those described in WO 2016/067035 (the cytotoxic agents and linkers thereof are herein incorporated by reference). In one embodiment the cytotoxic agent is MMAE.

In one embodiment, the linker between said cytotoxic agent and said bicyclic peptide comprises 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 a further embodiment, the cytotoxic agent is MMAE and the linker is selected from: -Val-Cit-, -Trp-Cit-, -Val-Lys- or -D-Trp-Cit-. In a yet further embodiment, the cytotoxic agent is MMAE and the linker is -Val-Cit- or -Val-Lys-. In a still yet further embodiment, the cytotoxic agent is MMAE and the linker is -Val-Cit-.

In an alternative embodiment, the linker between said cytoxic agent comprises a disulfide bond, such as a cleavable disulfide bond. Thus, in a further embodiment, the cytotoxic agent is DM1 and the linker is selected from: -S-S-, -SS(S03H)-, -SS-(Me)-, -(Me)-SS-(Me)-, -SS- (Me2)- or -SS-(Me)-S03H-. In a further embodiment, the cytotoxic agent is DM1 and the linker comprises an -S-S- moiety, such as (N-succinimidyl 3-(2-pyridyldithio)propionate (SPDB), or an -SS(S03H)- moiety, such as SO3H-SPDB. In a yet further embodiment, the cytotoxic agent is DM1 and the linker comprises an -S-S- moiety, such as -S-S- or -S-S- (SO3H)-.

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

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

wherein said bicycle is BCY6099 as defined herein.

In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (A), wherein said bicycle is selected from BCY6099 as defined herein. This BDC is known herein as BCY6027. Data is presented herein which

demonstrates excellent competition binding for BCY6027 in the EphA2 competition binding assay as shown in Tables 4 and 8.

In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (B), wherein said bicycle is selected from BCY6099 as defined herein. This BDC is known herein as BCY6028. Data is presented herein which

demonstrates excellent competition binding for BCY6028 in the EphA2 competition binding assay as shown in Tables 4 and 8. In a further embodiment, the cytotoxic agent is MMAE or DM1 and the drug conjugate is selected from BCY6136 and BCY6173. Data is presented herein which shows that these two Bicycle Drug Conjugates exhibited no significant binding to: closely related human homologs EphA1 , EphA3, EphA4, EphA5, EphA6, EphA7 and EphB4; mouse EphA3 and EphA4; and rat EphA3 and EphB1 as shown in Tables 11 and 12.

In a yet further embodiment, the drug conjugate is selected from any one of: BCY6135, BCY6136, BCY6173, BCY6174 and BCY6175: In a still yet further embodiment, the drug conjugate is BCY6136. Data is presented herein in Studies 7 and 8 which show that BCY6136 showed significant and potent anti-tumor activity in the PC-3 xenograft prostate cancer model (see Figures 5 and 6 and Tables 16 to 19).

Data is also provided herein which show that BCY6136 demonstrated potent antitumor activity in the NCI-H1975 xenograft lung cancer (NSCLC) model (see Figure 8 and Tables 20 to 25). Data is also presented herein in Studies 10 and 11 which show that BCY6136 demonstrated potent anti-tumor effect in both large and small tumour size LU-01-0251 PDX lung cancer (NSCLC) models (see Figures 9 and 10 and Tables 26 to 29) wherein complete tumor regression was observed. Data is also presented herein in Study 12 which show that BCY6136 demonstrated significant anti-tumor effect in the LU-01-0046 PDX lung cancer (NSCLC) model (see Figure 11 and Tables 30 and 31) wherein complete tumor regression was observed for BCY6136. Data is also presented herein in Study 13 which show that BCY6136 demonstrated dose dependent anti-tumor activity in the LU-01-0046 PDX lung cancer (NSCLC) model (see Figure 12 and Tables 32 and 33). Data is also presented herein in Study 14 which show BCY6136 eradicated tumors in the LU-01-0046 PDX lung cancer (NSCLC) model (see Figures 13 to 15 and Tables 34 to 37). Data is also presented herein in Studies 15 and 16 which demonstrate the effects of BCY6136 in two models which make use of cell lines with low/negligible EphA2 expression (namely Lu-01-0412 and Lu-01-0486). This data is shown in Figures 23 and 24 and Tables 38 to 41 and demonstrate that

BCY6136 had no effect upon tumor regression in either cell line but BCYs BCY8245 and BCY8781 , which bind to a target highly expressed in the Lu-01-0412 cell line, completely eradicated the tumour. Data is presented herein in Study 17 which show that BCY6136 demonstrated potent antitumor activity in the MDA-MB-231 xenograft breast cancer model (see Figure 18 and Tables 42 to 45). Data is also presented herein in Study 18 which demonstrates the effects of BCY6136 in a breast cancer model which makes use of a cell line with low/negligible EphA2 expression (namely EMT6). This data is shown in Figure 19 and Tables 46 and 47 and demonstrates that BCY6136 had no effect upon tumor regression in this cell line. Data is also presented herein in Study 19 which show that BCY6136 demonstrated significant antitumor activity in the NCI-N87 xenograft gastric cancer model (see Figure 20 and Tables 48 and 49). Data is also presented herein in Study 20 which show that BCY6136 demonstrated significant antitumor activity in the SK-OV-3 xenograft ovarian cancer model (see Figure 21 and Tables 50 and 51) compared with the ADC MEDI-547 which demonstrated moderate antitumour activity. Data is also presented herein in Study 21 which show that BCY6136 demonstrated significant antitumor activity in the OE-21 xenograft oesophageal cancer model (see Figure 22 and Tables 52 and 53). Data is also presented herein in Study 22 which show that BCY6136 demonstrated dose-dependent antitumor activity in the MOLP-8 xenograft multiple myeloma model (see Figure 23 and Tables 59 and 60). Data is also presented herein in Study 23 which show that BCY6136 demonstrated potent antitumor activity in the HT-1080 xenograft fibrosarcoma model (see Figures 24 to 28 and Tables 56 and 57).

Synthesis

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

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

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

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

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

Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).

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

Similar techniques apply equally to the synthesis/coupling of two bicyclic and bispecific macrocycles, potentially creating a tetraspecific molecule.

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

Pharmaceutical Compositions

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

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

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

The peptide ligands of the present invention may be used as separately administered compositions or in conjunction with other agents. These can include antibodies, antibody fragments and various immunotherapeutic drugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum and immunotoxins. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in conjunction with the protein ligands of the present invention, or even combinations of selected polypeptides according to the present invention having different specificities, such as polypeptides selected using different target ligands, whether or not they are pooled prior to administration.

The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, the peptide ligands of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. Preferably, the pharmaceutical compositions according to the invention will be administered by inhalation. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.

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

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

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

Therapeutic Uses

The bicyclic peptides of the invention have specific utility as EphA2 binding agents.

Eph receptor tyrosine kinases (Ephs) belong to a large group of receptor tyrosine kinases (RTKs), kinases that phosphorylate proteins on tyrosine residues. Ephs and their membrane bound ephrin ligands (ephrins) control cell positioning and tissue organization (Poliakov et al. (2004) Dev Cell 7, 465-80). Functional and biochemical Eph responses occur at higher ligand oligomerization states (Stein et ai (1998) Genes Dev 12, 667-678).

Among other patterning functions, various Ephs and ephrins have been shown to play a role in vascular development. Knockout of EphB4 and ephrin-B2 results in a lack of the ability to remodel capillary beds into blood vessels (Poliakov et ai, supra) and embryonic lethality. Persistent expression of some Eph receptors and ephrins has also been observed in newly- formed, adult micro-vessels (Brantley-Sieders et al. (2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).

The de-regulated re-emergence of some ephrins and their receptors in adults also has been observed to contribute to tumor invasion, metastasis and neo-angiogenesis (Nakamoto et al. (2002) Microsc Res Tech 59, 58-67; Brantley-Sieders et ai, supra). Furthermore, some Eph family members have been found to be over-expressed on tumor cells from a variety of human tumors (Brantley-Sieders et ai, supra) Marme (2002) Ann Hematol 81 Suppl 2, S66; Booth et ai (2002) Nat Med 8, 1360-1).

EPH receptor A2 (ephrin type-A receptor 2) is a protein that in humans is encoded by the EPHA2 gene.

EphA2 is upregulated in multiple cancers in man, often correlating with disease progression, metastasis and poor prognosis e.g.: breast (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 (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 (Nakamura et al (2005) Cancer Sci. 96, 42-47; Yuan et al (2009) Dig Dis Sci 54, 2410-2417), pancreatic (Mudali et al (2006) Clin Exp Metastasis 23, 357-365), prostate (Walker-Daniels et al (1999) Prostate 41 , 275-280), liver (Yang et 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 not defined although there is evidence for interaction at numerous stages of cancer progression including tumour cell growth, survival, invasion and angiogenesis. Downregulation of EphA2 expression suppresses tumour cancer cell propagation (Binda et al (2012) Cancer Cell 22, 765-780), whilst EphA2 blockade inhibits VEGF induced cell migration (Hess et al (2001) Cancer Res. 61 , 3250-3255), sprouting and angiogenesis (Cheng et al (2002) Mol Cancer Res. 1, 2-11; Lin et al (2007) Cancer 109, 332-40) and metastatic progression (Brantley-Sieders et al (2005) FASEB J.

19, 1884-1886).

An antibody drug conjugate to EphA2 has been shown to significantly diminish tumour growth in rat and mouse xenograft models (Jackson et al (2008) Cancer Research 68, 9367- 9374) and a similar approach has been tried in man although treatment had to be

discontinued for treatment related adverse events (Annunziata et al (2013) Invest New drugs 31 , 77-84).

Polypeptide ligands selected according to the method of the present invention may be employed in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, and the like. Ligands having selected levels of specificity are useful in applications which involve testing in non-human animals, where cross-reactivity is desirable, or in diagnostic applications, where cross-reactivity with homologues or paralogues needs to be carefully controlled. In some applications, such as vaccine applications, the ability to elicit an immune response to predetermined ranges of antigens can be exploited to tailor a vaccine to specific diseases and pathogens.

Substantially pure peptide ligands of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human. Once purified, partially or to homogeneity as desired, the selected polypeptides may be used diagnostically or therapeutically (including extracorporeally) or in developing and performing assay procedures, immunofluorescent stainings and the like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY). According to a further aspect of the invention, there is provided a peptide ligand or a drug conjugate as defined herein, for use in preventing, suppressing or treating a disease or disorder characterised by overexpression of EphA2 in diseased tissue (such as a tumour).

According to a further aspect of the invention, there is provided a method of preventing, suppressing or treating a disease or disorder characterised by overexpression of EphA2 in diseased tissue (such as a tumour), which comprises administering to a patient in need thereof an effector group and drug conjugate of the peptide ligand as defined herein.

In one embodiment, the EphA2 is mammalian EphA2. In a further embodiment, the mammalian EphA2 is human EphA2.

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

Examples of cancers (and their benign counterparts) which may be treated (or inhibited) include, but are not limited to tumours of epithelial origin (adenomas and carcinomas of various types including adenocarcinomas, squamous carcinomas, transitional cell carcinomas and other carcinomas) such as carcinomas of the bladder and urinary tract, breast, gastrointestinal tract (including the esophagus, stomach (gastric), small intestine, colon, rectum and anus), liver (hepatocellular carcinoma), gall bladder and biliary system, exocrine pancreas, kidney, lung (for example adenocarcinomas, small cell lung carcinomas, non-small cell lung carcinomas, bronchioalveolar carcinomas and mesotheliomas), head and neck (for example cancers of the tongue, buccal cavity, larynx, pharynx, nasopharynx, tonsil, salivary glands, nasal cavity and paranasal sinuses), ovary, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (for example thyroid follicular carcinoma), adrenal, prostate, skin and adnexae (for example melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic naevus); haematological malignancies (i.e. leukemias, lymphomas) and premalignant haematological disorders and disorders of borderline malignancy including haematological malignancies and related conditions of lymphoid lineage (for example acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma

[DLBCL], follicular lymphoma, Burkitt’s lymphoma, mantle cell lymphoma, T-cell lymphomas and leukaemias, natural killer [NK] cell lymphomas, Hodgkin’s lymphomas, hairy cell leukaemia, monoclonal gammopathy of uncertain significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), and haematological malignancies and related conditions of myeloid lineage (for example acute myelogenousleukemia [AML], chronic myelogenousleukemia [CML], chronic myelomonocyticleukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythaemia vera, essential thrombocythaemia and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocyticleukemia);

tumours of mesenchymal origin, for example sarcomas of soft tissue, bone or cartilage such as osteosarcomas, fibrosarcomas, chondrosarcomas,

rhabdomyosarcomas, leiomyosarcomas, liposarcomas, angiosarcomas, Kaposi’s sarcoma, Ewing’s sarcoma, synovial sarcomas, epithelioid sarcomas, gastrointestinal stromal tumours, benign and malignant histiocytomas, and dermatofibrosarcomaprotuberans; tumours of the central or peripheral nervous system (for example astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumours and schwannomas); endocrine tumours (for example pituitary tumours, adrenal tumours, islet cell tumours, parathyroid tumours, carcinoid tumours and medullary carcinoma of the thyroid); ocular and adnexal tumours (for example retinoblastoma); germ cell and trophoblastic tumours (for example teratomas, seminomas, dysgerminomas, hydatidiform moles and choriocarcinomas); and paediatric and embryonal tumours (for example medulloblastoma, neuroblastoma, Wilms tumour, and primitive neuroectodermal tumours); or syndromes, congenital or otherwise, which leave the patient susceptible to malignancy (for example Xeroderma Pigmentosum).

In a further embodiment, the cancer is selected from: breast cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, liver cancer, glioblastoma and angiogenesis.

In a further embodiment, 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, oesophageal cancer, multiple myeloma and fibrosarcoma.

In a yet further embodiment, the cancer is prostate cancer. Data is presented herein in Studies 7 and 8 which show that BCY6136 showed significant and potent anti-tumor activity in the PC-3 xenograft prostate cancer model (see Figures 5 and 6 and Tables 16 to 19).

In a yet further embodiment, the drug conjugate is useful for preventing, suppressing or treating solid tumours such as fibrosarcomas and breast, and non-small cell lung

carcinomas.

In a yet further embodiment, the cancer is selected from lung cancer, such as non-small cell lung carcinomas (NSCLC). Data is presented herein in Study 9 which show that BCY6136 demonstrated potent antitumor activity in the NCI-H1975 xenograft lung cancer (NSCLC) model (see Figure 8 and Tables 20 to 25). Data is also presented herein in Studies 10 and 11 which show that BCY6136 demonstrated potent anti-tumor effect in both large and small tumour size LU-01-0251 PDX lung cancer (NSCLC) models (see Figures 9 and 10 and Tables 26 to 29) wherein complete tumor regression was observed. Data is also presented herein in Study 12 which show that BCY6136 demonstrated significant anti-tumor effect in the LU-01-0046 PDX lung cancer (NSCLC) model (see Figure 11 and Tables 30 and 31) wherein complete tumor regression was observed for BCY6136. Data is also presented herein in Study 13 which show that BCY6136 demonstrated dose dependent anti-tumor activity in the LU-01-0046 PDX lung cancer (NSCLC) model (see Figure 12 and Tables 32 and 33). Data is also presented herein in Study 14 which show that BCY6173 demonstrated antitumor activity and BCY6136 and BCY6175 eradicated tumors in the LU-01-0046 PDX lung cancer (NSCLC) model (see Figures 13 to 15 and Tables 34 to 37). Data is also presented herein in Studies 15 and 16 which demonstrate the effects of BCY6136 in two models which make use of cell lines with low/negligible EphA2 expression (namely Lu-01- 0412 and Lu-01-0486). This data is shown in Figures 23 and 24 and Tables 38 to 41 and demonstrate that BCY6136 had no effect upon tumor regression in either cell line but BCYs BCY8245 and BCY8781 , which bind to a target highly expressed in the Lu-01-0412 cell line, completely eradicated the tumour. In a further embodiment, the cancer is breast cancer. In a yet further embodiment, the breast cancer is triple negative breast cancer. Data is presented herein in Study 17 which show that BCY6136 demonstrated potent antitumor activity in the MDA-MB-231 xenograft breast cancer model (see Figure 18 and Tables 42 to 45). Data is also presented herein in Study 18 which demonstrates the effects of BCY6136 in a breast cancer model which makes use of a cell line with low/negligible EphA2 expression (namely EMT6). This data is shown in Figure 19 and Tables 46 and 47 and demonstrates that BCY6136 had no effect upon tumor regression in this cell line. In an alternative embodiment, the breast cancer is Herceptin resistant breast cancer. Without being bound by theory, EphA2 is believed to be implicated in the resistance to Herceptin, therefore, an EphA2- targeting entity has potential utility in patients who have failed to respond to Herceptin.

In a further embodiment, the cancer is gastric cancer. Data is presented herein in Study 19 which show that BCY6136 demonstrated significant antitumor activity in the NCI-N87 xenograft gastric cancer model (see Figure 20 and Tables 48 and 49).

In a further embodiment, the cancer is ovarian cancer. Data is presented herein in Study 20 which show that BCY6136 demonstrated significant antitumor activity in the SK-OV-3 xenograft ovarian cancer model (see Figure 21 and Tables 50 and 51) compared with the ADC MEDI-547 which demonstrated moderate antitumour activity.

In a further embodiment, the cancer is oesophageal cancer. Data is presented herein in Study 21 which show that BCY6136 demonstrated significant antitumor activity in the OE-21 xenograft oesophageal cancer model (see Figure 22 and Tables 52 and 53).

In a further embodiment, the cancer is multiple myeloma. Data is presented herein in Study 22 which show that BCY6136 demonstrated dose-dependent antitumor activity in the MOLP- 8 xenograft multiple myeloma model and BCY6082 demonstrated significant antitumor activity (see Figure 23 and Tables 59 and 60).

In a further embodiment, the cancer is fibrosarcoma. Data is presented herein in Study 23 which show that BCY6173, BCY6135, BCY6174 and BCY6175 demonstrated dose dependent antitumor activity and BCY6136 demonstrated potent antitumor activity in the HT- 1080 xenograft fibrosarcoma model (see Figures 24 to 28 and Tables 56 and 57).

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

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

Furthermore, data is presented herein in Study 3 which demonstrates an association between copy number variation (CNV) and gene expression for EphA2 from multiple tumor types. Thus, according to a further aspect of the invention, there is provided a method of preventing, suppressing or treating cancer, which comprises administering to a patient in need thereof an effector group and drug conjugate of the peptide ligand as defined herein, wherein said patient is identified as having an increased copy number variation (CNV) of EphA2.

In one embodiment, the cancer is selected from those identified herein as having increased CNV of EphA2. In a further embodiment, the cancer is breast cancer. The invention is further described below with reference to the following examples.

Examples

Materials and Methods

Peptide Synthesis

Peptides were synthesized by solid phase synthesis. Rink Amide MBHA Resin was used. To a mixture containing Rink Amide MBHA (0.4-0.45 mmol/g) and Fmoc-Cys(Trt)-OH (3.0 eq) was added DMF, then DIC (3 eq) and HOAt (3 eq) were added and mixed for 1 hour. 20% piperidine in DMF was used for deblocking. Each subsequent amino acid was coupled with 3 eq using activator reagents, DIC (3.0 eq) and HOAT (3.0 eq) in DMF. The reaction was monitored by ninhydrin color reaction or tetrachlor color reaction. After synthesis completion, the peptide resin was washed with DMF x 3, MeOH x 3, and then dried under N ₂ bubbling overnight. The peptide resin was then treated with 92.5% TFA/2.5% TIS/2.5% EDT/2.5% H ₂ 0 for 3h. The peptide was precipitated with cold isopropyl ether and centrifuged (3 min at 3000 rpm). The pellet was washed twice with isopropyl ether and the crude peptide was dried under vacuum for2 hours and then lyophilised. The lyophilised powderwas dissolved in of ACN/H ₂ 0 (50:50), and a solution of 100 mM TATA in ACN was added, followed by ammonium bicarbonate in H ₂ 0 (1 M) and the solution mixed for 1 h. Once the cyclisation was complete, the reaction was quenched with 1M aq. Cysteine hydrochloride (10 eq relative to TATA), then mixed and left to stand for an hour. The solution

was lyophilised to afford crude product. The crude peptide was purified by Preparative HPLC and lyophilized to give the product o

All amino acids, unless noted otherwise, were used in the L- configurations.

o o

BCY6099

m

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

73

C

m

ho

m

n

H

O

Cd Os

Preparation of Bicyclic Peptide Drug Conjugates

The general schematic for preparing Bicycle drug conjugates (BDCs) is shown in Figure 1 and Table A describes the component targeting bicycle and linker/toxin within each BDC. Table A

BCY6135

BCY6099 (114.1 mg, 35.84 pmol) was used as the bicycle reagent. 22.4 mg Compound BCY6135 (5.30 pmol, 17.74% yield, 95.14% purity) was obtained as a white solid.

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

c

CD

W

H

H

C

H

m

w

n

m

m

73

C

m

ho

m

n

H

o

C bOd o o

H BCY6099 (200.15 mg, 62.89 pmol) was used as the bicycle reagent. 57.1 g compound BCY6173 (3.40 pmol, 22.79% yield, 95.80% purity) was

C

H

m obtained as a white solid.

w o

n

m

m

73

C

m

ho

m

n

H

o

Cd o o

BCY6174

m

c

CD

W

H

H

C

H

m

w

n

m

m

BCY6099 (389.77 mg, 122.47 pmol, 1.2 eq) was used as the bicycle reagent. Dde-BCY6174 (0.250 g, 55.10 pmol, 53.99% yield) was obtained

73

C as a white solid.

m

ho

m

n

H

Dde-BCY6174 (0.250 g, 55.10 qmol, 1.0 eq) was deprotected using hydrazine according to the general procedure to give BCY6174 (0.1206 g, o

Cd 27.45 pmol, 49.82% yield) as a white solid. o o o

m

c

CD

W

H

H

C

H

m

w M oe

n

m

m

73

C r_

m

ho

O)

BCY6175

General procedure for preparation of Compound 10A

To a solution of BCY6099 (195.15 mg, 61.32 pmol, 1.1 eq) in DMA (3 ml_) were added DIEA (21.61 mg, 167.23 pmol, 29.13 mI_, 3 eq) and compound 9 (0.085 g, 55.74 pmol, 1.0 eq). The mixture was stirred at 25 °C for 16 hr. LC-MS showed compound 9 was consumed completely and one main peak with desired m/z was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to afford a residue (light yellow oil). The reaction was directly purified by prep-HPLC (neutral condition). Compound 10A (0.160 g, 34.84 pmol, 62.50% yield) was obtained as a white solid.

General procedure for preparation of 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 min. LC-MS showed compound 10A was consumed completely and one main peak with desired m/z was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to afford a residue, which was purified by prep-HPLC (TFA condition). Compound BCY6175 (61.40 mg, 13.56 pmol, 31.13% yield) was obtained as a white solid.

BIOLOGICAL DATA

Study 1: Fluorescence polarisation measurements

(a) Direct Binding Assay

Peptides with a fluorescent tag (either fluorescein, SIGMA or Alexa Fluor488™, Fisher Scientific) were diluted to 2.5nM in PBS with 0.01 % tween 20 or 50mM HEPES with 100mM NaCI and 0.01 % tween pH 7.4 (both referred to as assay buffer). This was combined with a titration of protein in the same assay buffer as the peptide to give 1 nM peptide in a total volume of 25pL in a black walled and bottomed low bind low volume 384 well plates, typically 5pL assay buffer, 10pL protein (Table 1) then 10pL fluorescent peptide. One in two serial dilutions were used to give 12 different concentrations with top concentrations ranging from 500nM for known high affinity binders to 10mM for low affinity binders and selectivity assays. Measurements were conducted on a BMG PHERAstar FS equipped with an“FP 485 520 520” optic module which excites at 485nm and detects parallel and perpendicular emission at 520nm. The PHERAstar FS was set at 25°C with 200 flashes per well and a positioning delay of 0.1 second, with each well measured at 5 to 10 minute intervals for 60 minutes. The gain used for analysis was determined for each tracer at the end of the 60 minutes where there was no protein in the well. Data was analysed using Systat Sigmaplot version 12.0. mP values were fit to a user defined quadratic equation to generate a Kd value: f = ymin+(ymax- ymin)/Lig*((x+Lig+Kd)/2-sqrt((((x+Lig+Kd)/2) ^A 2)-(Lig*x))). “Lig” was a defined value of the concentration of tracer used.

(b) Competition Binding Assay

Peptides without a fluorescent tag were tested in competition with a peptide with a fluorescent tag and a known Kd (Table 2). Reference Compound A has the sequence Fl-G-Sars- ACPWGPAWCPVNRPGCA (FI-G-Sar ₅ -(SEQ ID NO: 4)). Reference Compound B has the sequence FI-G-Sar ₅ -ACPWGPFWCPVNRPGCA (FI-G-Sar ₅ -(SEQ ID NO: 5)). Reference Compound C has the sequence FI-G-Sar ₅ -ADVTCPWGPFWCPVNRPGCA (FI-G-Sar ₅ -(SEQ ID NO: 6). Each of Reference Compounds A, B and C contain a TBMB molecular scaffold. Peptides were diluted to an appropriate concentration in assay buffer as described in the direct binding assay with a maximum of 5% DMSO, then serially diluted 1 in 2. Five pl_ of diluted peptide was added to the plate followed by 10mI_ of human or mouse EphA2 (Table 1) at a fixed concentration which was dependent on the fluorescent peptide used (Table 2), then 10mI_ fluorescent peptide added. Measurements were conducted as for the direct binding assay, however the gain was determined prior to the first measurement. Data analysis was in Systat Sigmaplot version 12.0 where the mP values were fit to a user defined cubic equation to generate a Ki value: f=ymin+(ymax-ymin)/Lig*((Lig*((2*((Klig+Kcomp+Lig+Comp-Prot* c) ^A 2-3*(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*(Lig-Prot*c)+K lig*(Comp-

Prot*c)+Klig*Kcomp)-27*(-1*Klig*Kcomp*Prot*c))/(2*((((Kli g+Kcomp+Lig+Comp-Prot*c) ^A 2-

3*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)) ^A 3) ^A 0.5)))/3))-

(Klig+Kcomp+Lig+Comp-Prot*c)))/((3*Klig)+((2*((Klig+Kcomp +Lig+Comp-Prot*c) ^A 2-

3 ^* (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*(Lig-

Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)-27*(-

1*Klig*Kcomp*Prot*c))/(2*((((Klig+Kcomp+Lig+Comp-Prot*c) ^A 2-3*(Kcomp*(Lig- Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)) ^A 3) ^A 0.5)))/3))-(Klig+Kcomp+Lig+Comp-Prot*c)))). “Lig”,“KLig” and“Prot” were all defined values relating to: fluorescent peptide concentration, the Kd of the fluorescent peptide and EphA2 concentration respectively.

Table 1 : Ephrin receptors and source

Table 2: Final concentrations of fluorescent peptide and EphA2 as used with Competition Binding Assays

Certain peptide ligands of the invention were tested in the above mentioned assays and the results are shown in Tables 3 and 4: Table 3: Biological Assay Data for Peptide Ligand of the Invention (TATA peptides, Competition Binding Assay)

Table 4: Biological Assay Data for Peptide Ligands of the Invention (BDC competition binding data with TATA Scaffolds)

Study 2: Fluorescence polarisation measurements ( Alternative Protocol)

(a) Competition binding

Peptides without a fluorescent tag were tested in competition with a peptide with a fluorescent tag and a known Kd (Table 9). Five pL of increasing (2 fold) concentrations of test compound was added to the plate followed by 10pL of EphA2 protein (Table 8) at a fixed concentration which was dependent on the fluorescent peptide used (Table 9), then 10pL fluorescent peptide added. Buffer was assay buffer as above with DMSO <1 %. Measurements were conducted on a BMG PHERAstar FS equipped with an“FP 485 520 520” optic module which excites at 485nm and detects parallel and perpendicular emission at 520nm. The PHERAstar FS was set at 25°C with 200 flashes per well and a positioning delay of 0.1 second, with each well measured at 5 to 10 minute intervals for 60 minutes. Alternatively, measurements were done on at similar time intervals on a Perkin Elmer Envision equipped with 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 a G-Factor of 1.2. Data analysis was in Systat Sigmaplot version 12.0 or 13.0 where the mP values at 60 minutes were fit to a user defined cubic equation to generate a Ki value:

f=ymin+(ymax-ymin)/Lig*((Lig*((2*((Klig+Kcomp+Lig+Comp-Pr ot*c) ^A 2-3*(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*(Lig-Prot*c)+K lig*(Comp- Prot*c)+Klig*Kcomp)-27*(-1*Klig*Kcomp*Prot*c))/(2*((((Klig+K comp+Lig+Comp-Prot*c) ^A 2- 3*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)) ^A 3) ^A 0.5)))/3))- (Klig+Kcomp+Lig+Comp-Prot*c)))/((3*Klig)+((2*((Klig+Kcomp+Li g+Comp-Prot*c) ^A 2- 3 ^* (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*(Lig-

Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)-27*(-

1*Klig*Kcomp*Prot*c))/(2*((((Klig+Kcomp+Lig+Comp-Prot*c) ^A 2-3*(Kcomp*(Lig-

Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)) ^A 3) ^A 0.5)))/3))-(Klig+Kcomp+Lig+Comp-Prot*c)))). “Lig”,“KLig” and“Prot” were all defined values relating to: fluorescent peptide concentration, the Kd of the fluorescent peptide and EphA2 concentration respectively.

Table 5: Eph receptors and source

Table 6: Final concentrations of fluorescent peptide and EphA2 as used with competition binding assays

Certain peptide ligands and bicycle drug conjugates of the invention were tested in the above mentioned competition binding assay and the results are shown in Table 7: Table 7: Competition Binding with Selected Bicyclic Peptides

The results from the competition binding assay in Table 7 show that Bicycle peptides targeting human EphA2 (BCY6099) bind with high affinity to mouse and rat EphA2. These results show that the peptide of the invention can be used in in vivo mouse and rat efficacy and toxicology models.

Table 8: Competition Binding with Selected Bicycle Drug Conjugates (BDCs)

Table 8 shows that certain Bicycle Drug Conjugates of the invention exhibit excellent cross reactivity between human, mouse and rodent EphA2. The peptide of the invention can therefore be used in mouse and rat efficacy and toxicology in vivo models.

(b) SPR Measurements Non-Fc fusion proteins were biotinylated with EZ-Link™ Sulfo-NHS-LC-Biotin for 1 hour in 4mM sodium acetate, 100mM NaCI, pH 5.4 with a 3x molar excess of biotin over protein. The degree of labelling was determined using a Fluorescence Biotin Quantification Kit (Thermo) after dialysis of the reaction mixture into PBS. For analysis of peptide binding, a Biacore T200 instrument was used utilising a XanTec CMD500D chip. Streptavidin was immobilized on the chip using standard amine-coupling chemistry at 25°C with HBS-N (10 mM HEPES, 0.15 M NaCI, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated with a 7 min injection of a 1 : 1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 mI/min. For capture of streptavidin, the protein was diluted to 0.2 mg/ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120mI onto the activated chip surface. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5):HBS-N (1 : 1). Buffer was changed to PBS/0.05% Tween 20 and biotinylated EphA2 was captured to a level of 500-1500 RU using a dilution of protein to 0.2mM in buffer. A dilution series of the peptides was prepared in this buffer with a final DMSO concentration of 0.5% with a top peptide concentration was 50 or 100nM and 6 further 2-fold dilutions. The SPR analysis was run at 25°C at a flow rate of 90pl/min with 60 seconds association and 900-1200 seconds dissociation. Data were corrected for DMSO excluded volume effects. All data were double-referenced for blank injections and reference surface using standard processing procedures and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Data were fitted using simple 1 : 1 binding model allowing for mass transport effects where appropriate.

For binding of Bicycle Drug Conjugates a Biacore 3000 instrument was used. For biotinylated proteins immobilisation levels were 1500 RU and the top concentration was 100nM. Otherwise the method was the same as described above using either the CMD500D or a CM5 chip (GE Healthcare). For the Fc-tagged proteins, a CM5 chip was activated as described above and then goat anti-human IgG antibody (Thermo-Fisher H 10500) was diluted to 20pg/ml in 10mM sodium acetate pH5.0 and captured to approximately 3000 RU. The surface was then blocked as described above. Subsequent capture of the Fc-tagged proteins was carried out to obtain approximately 200-400 RU of the target protein. The proteins used are described below. All proteins were reconstituted as per manufacturer’s suggested buffers and concentrations and captured using 5-10pg/ml protein in PBS/0.05% Tween 20.

Table 9

Certain peptide ligands and bicycle drug conjugates of the invention were tested in the above mentioned competition binding assay and the results are shown in Tables 10 to 12: Table 10: SPR Binding Analysis with Selected Bicyclic Peptides and Bicycle Drug Conjugates of the Invention

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Table 11 : SPR Binding Analysis with Selected Bicycle Drug Conjugates of the Invention with Human Eph Homologs

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Table 11 illustrates binding results with four Bicycle Drug Conjugates (BCY6136 and BCY6173) in the SPR assay with closely related human Ephrin homologs. The results show that compounds of the invention exhibit no significant binding to closely related human homologs: EphA1 , EphA3, EphA4, EphA5, EphA6, EphA7 and EphB4.

Table 12: SPR Binding Analysis with Selected Bicycle Drug Conjugates of the

Invention with Mouse and Rat Eph Orthologs

The results in Table 12 show that certain Bicycle Drug Conjugates of the invention (BCY6136 and BCY6173) are also selective for mouse and rat EphA2 and exhibit no significant binding to closely related homologs: 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 adopted for each study:

(a) Materials

(il Animals and Housing Condition

Animals

Species: Mus Musculus

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 Housing condition

The mice were kept in individual ventilation cages at constant temperature and humidity with 3-5 animals in each cage.

• Temperature: 20-26 °C.

• Humidity 40-70%.

Cages: Made of polycarbonate. The size is 300 mm x 180 mm x 150 mm. The bedding material is corn cob, which is changed twice per week.

Diet: Animals had free access to irradiation sterilized dry granule food during the entire study period.

Water: Animals had free access to sterile drinking water.

Cage identification: The identification labels for each cage contained the following information: number of animals, sex, strain, the date received, treatment, study number, group number and the starting date of the treatment.

Animal identification: Animals were marked by ear coding. (ii) Test and Postitive Control Articles

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

(b) Experimental Methods and Procedures

(i) Observations

All the procedures related to animal handling, care and the treatment in the study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec, following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). At the time of routine monitoring, the animals were daily checked for any effects of tumor growth and treatments on normal behavior such as mobility, food and water consumption (by looking only), body weight gain/loss, eye/hair matting and any other abnormal effect as stated in the protocol. Death and observed clinical signs were recorded on the basis of the numbers of animals within each subset.

(ii) Tumor Measurements and the Endpoints

The major endpoint was to see if the tumor growth could be delayed or mice could be cured. Tumor volume was measured three times weekly in two dimensions using a caliper, and the volume was expressed in mm ³ using the formula: V = 0.5 a x b ² where a and b are the long and short diameters of the tumor, respectively. The tumor size was then used for calculations of T/C value. The T/C value (in percent) is an indication of antitumor effectiveness; T and C are the mean volumes of the treated and control groups, respectively, on a given day. TGI was calculated for each group using the formula: TGI (%) = [1-(T-To)/ (Vi-Vo)] ^c 100; T , is the average tumor volume of a treatment group on a given day, To is the average tumor volume of the treatment group on the day of treatment start, V, is the average tumor volume of the vehicle control group on the same day with T, and V _o is the average tumor volume of the vehicle group on the day of treatment start.

(iii) Sample Collection

At the end of study the tumors of all groups were collected for FFPE.

(iv) Statistical Analysis

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

Statistical analysis of difference in tumor volume among the groups was conducted on the data obtained at the best therapeutic time point after the final dose.

A one-way ANOVA was performed to compare tumor volume among groups, and when a significant F-statistics (a ratio of treatment variance to the error variance) was obtained, comparisons between groups were carried out with Games-Howell test. All data were analyzed using GraphPad Prism 5.0. P < 0.05 was considered to be statistically significant.

Study 3: Investigation of Association between Copy Number Variation ( CNV ) and gene expression for EohA2 from multiple tumour types

Methods

1. Select all studies in cBioPortal (http://www.cbioportal.org/) and search for EPHA2.

(a) Remove provisional studies.

(b) Deselect studies with overlapping samples to prevent sample bias (based on warning in cBioPortal)- always keep PanCancer study if this is an option.

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

Table 13: Studies analysed from cBioPortal and units in study

2. Export CNV and RNA expression data from cBioPortal.

3. Test if CNVs are statistically significantly associated with changes in mRNA expression for EphA2 (log2 not applied).

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

(i) GraphPad Prism: set up column table, run non-parametric test with no matching or pairing and do not assume Gaussian distribution.

(ii) Packages used in R:

1. XLConnect

2. dplyr

3. Kruskal-Wallis Rank Sum Test: Kruskal.test.

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

(a) dunn. test with multiple comparison method _^ “bonferonni”.

Results

The results are shown in Table 14 below. Across 41 publicly available datasets compiled in cBioPortal that report both Copy Number Variation (CNV) and mRNA gene expression for EphA2, there are numerous cancer types where cases have been reported with EphA2 shallow-deletions (<2 copies). Although less common, in these same cancer types a subset of tumors harbored EphA2 deep deletions (>1 copy loss or biallelic loss), EphA2 gains (2-3 copies) or EphA2 amplifications (>3 copies). Indications where >33% of tumors had either shallow-deletions or deep deletions in EphA2 included: kidney chromophobe,

cholangiocarcinoma, pheochromocytoma and paraganglioma, lung squamous cancer, breast, rectum, brain lower grade glioma, liver, adrenocortical carcinoma, mesothelioma, esophageal adenocarcinoma and colon cancer. In contrast, there were no studies where >33% of samples had either gains or amplification in EphA2. Taken together these results demonstrate that deletions in EphA2 DNA are found across a variety of indications.

Approximately one third of all samples analyzed in the 41 studies harbored EphA2 CNVs. Based on this high percentage of CNVs across studies, and the high percentage of shallow deletions within specific tumor types, statistical testing was performed to identify possible associations between copy number changes and RNA expression. Tumors per indication were allocated to 1 of 5 classes:

a) Deep deletion;

b) Shallow deletion;

c) Diploid;

d) Gain; or

e) Amplification.

Kruskall-Wallis testing was then performed to detect if the distributions of mRNA expression values per classes differed between classes (P < 0.01). For those TCGA data sets with P < 0.01 and to identify which classes were different to one another post-hoc testing was performed by calculating Z-statistics with adjusted P-values calculated (Bonferroni). For simplicity of interpretation pair-wise comparisons vs. diploid per indication were reviewed (although all pair-wise P-values were calculated). 19/41 of these studies had a Kruskall- Wallis p-value of <0.01 demonstrating that copy number is statistically significantly associated with RNA expression. Of these 19 studies, 17 of them had a Bonferroni adjusted P < 0.025 for Diploid vs. Shallow Deletion indicating an association of decreased EphA2 mRNA expression with decreased EphA2 copy number. Only 2 of these 19 studies had a Bonferroni adjusted P < 0.025 for Diploid vs. Gain and both were breast cancer studies. Furthermore, one of these breast cancer studies (Breast Invasive Carcinoma (TCGA, PanCancer Atlas)) had a Bonferroni adjusted P<0.025 for both Diploid vs. Shallow Deletion and Diploid vs. Gain suggesting that copy number alterations may have a strong impact on EphA2 RNA expression in breast cancer.

The central dogma of genetics suggests that reduced copy number in EphA2 lead to reduced RNA and protein expression. Therefore, the observed associations between copy number loss of EphA2 and reduced mRNA expression in a variety of tumor types suggest that EphA2 protein expression may also be reduced. Similarly, copy number gains of EphA2 in breast cancer that were associated with increased mRNA expression may also suggest increased EphA2 protein expression. Moreover, higher EphA2 protein expression (measured by FACS) is associated with increased efficacy of certain EphA2 bicyclic drug conjugates of the invention (measured by tumor volume) in preclinical in vivo models. Taken together if copy number alterations that are associated with mRNA expression changes do predict protein expression levels then patients with tumors containing copy number deletions of EphA2 may be less likely to respond to EphA2 bicyclic drug conjugates of the invention. Similarly, if patients with tumor copy number gains in EphA2 (e.g. breast cancer) it is possible that these patients would be more likely to respond to EphA2 bicyclic drug conjugates of the invention. Therefore, if patients were stratified by EphA2 copy number status, then this information could be used to both exclude and select patients for treatment with EphA2 bicyclic drug conjugates of the invention to increase efficacy.

Table 14: Results of Investigation of Association between Copy Number Variation (CNV) and gene expression for EphA2

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PCT/GB 2018/053 675 - Ϊ8 Ϊ.20Ϊ9 ^{: BIC} WO 2019/122860 PCT/GB2018/053675

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in the

LU-01-0251 PDX model in Balb/c nude mice.

(b) Experimental Design

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with LU-01-0251 of tumor

fragment (~30 mm ³ ) for tumor development. The treatment was started when the average

tumor volume reached 174 mm ³ for efficacy study. The test article administration and the

animal number in each group are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

(d) Results

(i) Tumor Growth Curve

Tumor growth curve are shown in Figure 9.

(ii) Tumor Volume Trace

Mean tumor volume on day 28 after the start of treatment in female Balb/c nude mice bearing

LU-01-0251 xenograft is shown in Table 26.

Table 26: Tumor volume trace overtime

Day Group 1 Group 2 Group 3 Group 4 Group 5

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

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

(a) Experimental method

Balb/c mice were inoculated subcutaneously with tumour cells at the right flank and drug treatment started when the average the average tumour volume reached between 150 and 200 mm ³ . Tumour measurements and statistical analysis were performed as described above. Tumour bearing animals were treated once weekly with BCY6136 or vehicle.

(b) Discussion

Figures 4-6 show that BCY6136 is effective in breast, lung and fibrosarcoma xenograft models following once weekly dosing.

The HT1080 fibrosarcoma model:

In the HT1080 model complete regression of tumour growth was achieved by day 14 following once weekly dosing with BCY6136 on days 0 and 7 at 3 and 5 mg/kg (Figure 2). Once weekly dosing with BCY6136 at 2 mg/kg on days 0 and 7 gave rise to tumour stasis (partial regression) (Figure 2). BCY6136 treatment gave rise to no significant body weight loss and there were no adverse clinical observations on drug treated mice throughout the study.

The NCI-H1975 NSCLC model:

Complete regression of tumour growth in the NCI-H 1975 model was observed by around day 28 following 2 and 3 mg/kg once weekly dosing with BCY6136 (Figure 3). Following dosing cessation on day 35 no tumour regrowth was observed in the 3 mg/kg treated animals from day 35 to day 72 when the 3 mg/kg arm measurements ended (Figure 3). Dosing with BCY6136 at 2 mg/kg gave rise to complete regression in this model from around day 28. Following dosing cessation on day 35 there was no tumour regrowth until around day 51 at the 2 mg/kg dose. At this dose level moderate tumour re-growth was observed from around day 51 until study termination on day 77. 1 mg/kg treatment with BCY6136 gave rise to tumour stasis (partial regression) (Figure 3). BCY6136 treatment gave rise to no significant body weight loss and there were no adverse clinical observations on drug treated mice throughout the study.

The MDA-MB-231 breast model:

71 Tumour stasis (partial regression) was observed in the MDA-MB231 model following once weekly dosing at 2 and 3 mg/kg from days 0 to day 45 (Figure 4). Some body weight loss (attributed to tumour burden) was observed in the 2 mg/kg treated animals. These results demonstrate that BCY6136 gives rise to profound tumour growth inhibition in mice implanted with fibrosarcoma, breast and lung CDX xenografts following once daily dosing.

Study 5: Safety studies in the rat

Six (6) female rats were randomly assigned to 3 groups of 2 rats/group to determine the toxicity of BCY6136, following administered by IV bolus injection at 5, 7.5 and 10 mg/kg on days 1 and 8. The study was terminated on day 15.

No significant effects on coagulation parameters (Prothrombin time (sec), Activated partial thromboplastin time (sec) or Fibroginogen levels (g/L) were observed on days 2, 12 and 15 (data not shown). No in-life bleeding events were reported and no evidence of internal bleeding was detected following pathology examination.

Study 6: Safety studies in the cvnomologous monkeys

Twenty eight day toxicology studies with BCY6136 we conducted in cynomologous monkeys. BCY6136 was dosed at 1.0 and 2.0 mg/kg on days 1 , 8, 15 and 22. Animals were euthanised and necropsied on day 29 (7 days after the final dose).

No significant effects on coagulation parameters relative to baseline were observed on days 18, 22 and 25 (data not shown) and day 29 (Table 15). No in-life bleeding events were reported and no evidence of internal bleeding was detected following pathology examination.

72 Table 15: Day 29 coagulation parameters following 1.0 and 2.0 mg/kg BCY6136 dosing to cynomolgus monkeys

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

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in treatment of PC-3 xenograft.

(b) Experimental Design

(c) Experimental Methods and Procedures

(i) Cell Culture

The PC-3 tumor cells will be maintained in F12K medium supplemented with 10% heat inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. The tumor cells will be routinely subcultured twice weekly. The cells growing in an exponential growth phase will be harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

73 Each mouse will be inoculated subcutaneously at the right flank with PC-3 (10*10 ⁶ ) tumor cells for tumor development. The animals will be randomized and treatment will be started when the average tumor volume reaches approximately 150 mm ³ . The test article administration and the animal numbers in each group are shown in the following experimental design table. (iii) Testing Article Formulation Preparation

(d) Results

(il T umor Growth Curve

Tumor growth curve are shown in Figures 5 and 6.

(iil Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing PC-3 xenograft is shown in Table 16.

74 Table 16: Tumor volume trace over time

Gr Treatment 0 2 4 7 9 11 14 16 18 21 o

O

Vehicle,

1 149±9 235±9 377±9 718±30 1126±41 1431±79 1792±69 2070±152

qw

o

BCY6136, o

2 150±11 185±25 228±31 201 ±17 183±23 153±38 137±33 107±32 64±28 45±23

1 mpk, qw

BCY6136,

3 149±18 179±28 158±22 137±16 122±15 114±20 101±16 79±20 57±19 42±17 m 2 mpk, qw

c

CD

w BCY6136

H 4 149±2 155±8 144±16 132±20 107±28 94±23 83±22 70±27 38±16 35±17

H 3 mpk, qw

C

H ADC

m 5 151±27 203±10 210±12 189±11 185±16 190±37 158±36 124±35 103±27 74±14 w 3 mpk, qw

n

m

m

73

C Gr Treatment 23 25 28 30 32 35 37 39 42

m Vehicle,

ho

m 1

qw

BCY6136,

2 35±18 28±14 37±19 34±17 42±21 42±23 43±21 28±14 18±9

1 mpk, qw

n

H

BCY6136,

3 21 ±1 1 22±12 22±12 24±12 33±16 22±11 26±14 22±12 16±9 o w 2 mpk, qw o

BCY6136 o

4 21 ±10 23±12 27±14 22±11 24±12 20±11 27±14 12±6 12±6

3 mpk, qw

ADC

5 53±16 50±22 46±23 70±35 78±39 53±27 60±30 53±27 40±22

3 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles 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 analysis

m

c Tumor P value compare

Gr Treatment T/C ^b (%) TGI (%)

le

h o

m 3 mpk, qw

5 ^ADC ’ 124±35 6.0 101.4 p<0.001

3 mpk, qw

a. Mean ± SEM.

b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the PC-3 xenograft model was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in the Figures 5 and 6 and Tables 16 and 17.

The mean tumor size of vehicle treated mice reached 2070 mm ³ on day 16. BCY6136 at 1 mg/kg, qw (TV=107 mm ³ , TGI=102.2%, p<0.001), BCY6136 at 2 mg/kg, qw (TV=79 mm ³ , TGI=103.6%, p<0.001) and BCY6136 at 3 mg/kg, qw (TV=70 mm ³ , TGI=104.1%, p<0.001) showed potent anti-tumor effect. In this study, animal body weight was monitored regularly. All mice maintained their body weight well.

Study 8. In vivo efficacy study ofBCY6136 in treatment of PC-3 xenograft in Balb/c nude mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in treatment of PC-3 xenograft in Balb/c nude mice.

(b) Experimental Design

a. N, the number of animals in each group.

b. After 4 weeks’ treatment demonstrated in the experimental design table, the mice of group 3, 5 and 6 were treated with BCY6136 1.5 mg/kg qw from day 52 during the monitoring schedule.

c. Due to the severe body weight loss of the Docetaxel treated mice after the first dosing, the treatment was suspended for 2 weeks, then a lower dosage (Docetaxel, 10 mg/kg) was performed on day 28. After that, the mice were treated with BCY6136 1.5 mg/kg qw from day 42 to day 70.

(c) Experimental Methods and Procedures

77 (i) Cell Culture

The tumor cells were maintained in F-12K medium supplemented with 10% heat inactivated fetal bovine serum at 37°C in an atmosphere of 5% CCL in air. The tumor cells were routinely subcultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with PC-3 tumor cells (10 x 10 ⁶ ) in 0.2 ml of PBS for tumor development. 52 animals were randomized when the average tumor volume reached 454 mm ³ . The test article administration and the animal numbers in each group were shown in the experimental design table.

(iii) Testing Article Formulation Preparation

78

(c) Results

(i) Tumor Growth Curve

Tumor growth curve is shown in Figure 7.

(ii) Tumor Volume Trace

Mean tumor volume over time in male Balb/c nude mice bearing PC-3 xenograft is shown in Table 18.

79 Table 18: Tumor volume trace over time (Day 0 to day 20)

O

Days after the start of treatment O

Gr. Treatment -

0 2 4 6 8 10 13 15 17 20

o

456+ 648± 1022 ±2 1178±1 1327±1 1631 ±9 1868±9 2052±1 2364±1 o

1 Vehicle, qw 880 ±23

25 50 9 18 33 3 0 39 02

BCY6136 450+ 631 ± 1089±7 1124±9 1188±1

2 695 ±78 739 ±39 850 ±68 904 ±73 975 ±47

m 0.167 mpk, qw 33 55 4 2 11 c

CD

W BCY6136 451± 622±

H 3 519 ±70 460 ±55 398 ±50 329 ±38 260 ±33 249 ±33 231 ±38 234 ±42

H 0.5 mpk, qw 47 96

C

H

m BCY6136 458+ 587±

w 4 494 ±54 363 ±32 283 ±32 237 ±24 192 ±13 164 ±16 155 ±20 131 ±19

o

n 1.5 mpk, qw 49 63

m

m BCY6136 454+ 643± 531 458 411 382 430 522 560 530

5

73 0.5 mpk, q2w 37 25 ±37 ±33 ±32 ±49 ±88 ±124 ±129 ±147

C

BCY6136

m 452± 590± 457 375 328 242 206 197 182 128 ho

m 6 1.5 mpk, q2w

_ 42 75 ±49 ±44 ±47 ±63 ±61 ±62 ±55 ±36

1.5 mpk, qw

EphA2-ADC 457± 636± 712 792 870 900 1049±6 1242±1 1443±1 1637±1

7

0.33 mpk, qw 43 57 ±70 ±78 ±87 ±58 6 23 29 81 n

H

o

EphA2-ADC 450+ 617± 673 721 782 755 840 913 978 981 w

8 o

1 mpk, qw 49 48 ±50 ±61 ±78 ±67 ±93 ±91 ±100 ±100

o

9 EphA2-ADC 643 593 433 290 268 232 225 184 o -

452± 593± ±141 ±106 ±103 ±81 ±64 ±60 ±66 ±62

3 mpk, qw

60 98 O

Docetaxel 453± 584± 632 636 568 408 374 388 361 419 O

10

15 mpk, qw 62 72 ±56 ±48 ±50 ±31 ±26 ±36 ±25 ±31

o o

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the PC-3 xenograft model was calculated based on tumor volume measurements at day 20 after m the start of the treatment.

c

CD

W

H

H

C

H

m

w

n

m

m

73

C r_

m

ho

m

n

H

O

Cd

O

O

Os

Table 19: Tumor growth inhibition analysis

Tumor P value

Volume T/C ^b compared

Gr Treatment TGI (%)

(%) with

(mm 3 ³ \)a

vehicle

1 Vehicle, qw 2364±102

2 BCY6136, 0.167 mpk, qw 1188±111 50.2 61.4 p<0.001

3 BCY6136, 0.5 mpk, qw 234±42 9.9 111.4 p<0.001

4 BCY6136, 1.5 mpk,qw 131±19 5.5 117.2 p<0.001

5 BCY6136, 0.5 mpk, q2w 530±147 22.4 96.0 p<0.001

6 BCY6136, 1.5 mpk, q2w 128±36 5.4 117.0 p<0.001

7 EphA2-ADC, 0.33 mpk, qw 1637±181 69.2 38.1 p<0.001

8 EphA2-ADC, 1 mpk,qw 981±100 41.5 72.2 p<0.001

9 EphA2-ADC, 3 mpk,qw 184±62 7.8 114.0 p<0.001

10 Docetaxel, 15 mpk,qw 419±31 17.7 101.8 p<0.001 a. Mean ± SEM.

b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(d) Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the PC-3 xenograft model was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in the Figure 7 and Tables 18 and 19.

The mean tumor size of vehicle treated mice reached 2364 mm ³ on day 20. BCY6136 at 0.167 mg/kg, qw (TV=1188 mm ³ , TGI=61.4%, p<0.001), 0.5 mg/kg, q2w (TV=530 mm ³ , TGI=96.0%, p<0.001), 0.5 mg/kg, qw (TV=234 mm ³ , TGI=111.4%, p<0.001) and 1.5 mg/kg, qw (TV=131 mm ³ , TGI=117.2%, pO.001) produced significant anti-tumor activity in dose or dose- frequency dependent manner on day 20. BCY6136 at 1.5 mg/kg, q2w (TV-128 mm ³ , TGI=117.0%, pO.001) produced comparable anti-tumor activity with BCY6136 1.5 mg/kg qw. Among them, the mice treated with BCY6136, 0.5 mg/kg qw or BCY6136, 0.5 mg/kg q2w showed obvious tumor relapse after ceasing the treatment, further treatment with BCY6136,

82 1.5 mg/kg qw from day 52 worked well on the tumor regression. The mice treated with BCY6136, 1.5 mg/kg q2w also showed tumor relapse after ceasing the treatment, but further dosing didn’t work on complete tumor regression. The mice treated with BCY6136, 1.5 mpk qw didn’t show any tumor relapse until day 48.

EphA2-ADC at 0.33 mg/kg, qw (TV=1637 mm ³ , TGI=38.1 %, p<0.001), 1 mg/kg, qw (TV=981 mm ³ , TGI=72.2%, p<0.001) and 3 mg/kg, qw (TV=184 mm ³ , TGI=114.0%, p<0.001) produced significant anti-tumor activity in dose dependent manner on day 20. The mice treated with EphA2-ADC, 3 mg/kg qw didn’t show any tumor relapse until day 59.

Docetaxel at 15 mg/kg, qw (TV=419 mm ³ , TGI=101.8%, p<0.001) produced significant anti tumor activity but caused severe animal body weight loss. After ceasing the treatment, the mice showed obvious tumor relapse. The treatment with BCY6136, 1.5 mg/kg qw from day 42 worked well on tumor regression of these mice.

Study 9. In vivo efficacy test of BCY6136 in treatment of NCI-H1975 xenograft in

Balb/c nude mice

(a) Study Objective

The objective of the research was to evaluate the in vivo anti-tumor efficacy of BCY6136 in treatment of NCI-H1975 xenograft model in Balb/c nude mice.

(b) Experimental Design

(c) Experimental Methods and Procedures

(i) Cell Culture

The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with NCI-H1975 tumor cells (10* 10 ^L 6) in 0.2ml of PBS for tumor development. 36 animals were randomized when the average tumor volume reached 149 mm ³ . The test article administration and the animal numbers in each group were shown in the experimental design table.

(iii) Testing Article Formulation Preparation

83 PCT/GB 2018/053 675 - Ϊ8ΌΪ.20Ϊ9=

BIC

WO 2019/122860 PCT/GB2018/053675

(iyl Sample Collection

On PG-D44, we fixed the tumors of Group 2 for FFPE.

At the end of study, we the tumors of Group 3 for FFPE.

(d) Results

(i) Tumor Growth Curve

Tumor growth are shown in Figure 8.

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing NCI-H1975 xenograft is

shown in Table 20 to 24.

Table 20: Tumor volume trace (PG-D0~PG-D17)

Gr Days after the start of treatment

Treatment . . ... —

0 2 4 7 9 11 14 17

Vehicle, 148 195±1 297±3 466±6 732±1 1028±1 1278±2 1543±2

BCY6136, 150 178±2 232±4 336±4 400±2 299±1 1 261±12

2 407±42

1 mpk, qw ±6 0 9 3 4 3 7

BCY6136, 150 181 ±2 237±2 277±3 297±3

3 306±55 256±53 218±49

2 mpk, qw ±14 6 7 6 7

BCY6136, 148 168±1 365±1 390±1

4 231 ±6 423±42 319±26 228±16

3 mpk, qw ±9 0 6 3

Table 21 : Tumor volume trace (PG-D18~PG-D35)

Days after the start of treatment

Gr. Treatment

18 21 23 25 28 30 33 35

84 2371±

1 Vehicle, qw 4±3

470

95

215

BCY6136, 205±1 197±1 200±1 202±1 202±1 230±1 241 ±1

2 ±1 1

1 mpk, qw 17 13 05 12 17 42 27

BCY6136, 149

3 99±30 69±22 42±13 30±10 16±8 20±9 4±2

2 mpk, qw ±31

BCY6136, 149

4 94±30 50±15 41 ±21 21 ±8 6±6 10±6 3±1

3 mpk, qw ±17

Table 22: Tumor volume trace (PG-D37-PG-D53)

Days after the start of treatment

Gr. Treatment -

37 39 42 44 46 49 51 53

BCY6136,

2 277±149 294±159 351±188

1 mpk, qw

BCY6136,

3 7±4 2±1 1 ±0 3±1 2±1 3±2 6±3 14±10

2 mpk, qw

BCY6136,

4 3±3 2±1 1 ±0 0±0 0±0 0±0 1 ±0 1 ±0

3 mpk, qw

Table 23: Tumor volume trace (PG-D56-PG-D74)

Treatmen Days after the start of treatment

Gr. - t 56 58 60 63 65 67 70 72 74

BCY6136,

16± 1 11 ±7 122±7

3 2 mpk, 27±18 34±23 45±31 63±40 71±47 95±70

1 1 3 5 qw

BCY6136,

4 3 mpk, 1±0 1±0 1 ±0 0±0 0±0 0±0 0±0 0±0

qw

Table 24: Tumor volume trace (PG-D77-PG-D98)

Days after the start of treatment

Gr. Treatment -

77 81 84 88 91 95 98

85 PCT/GB 2018/053 675 - Ϊ8 Ϊ.20Ϊ9 ^:

BIC WO 2019/122860 PCT/GB2018/053675

BCY6136,

3 208±112 337±123 501 ±172 626±182 856±245 1035±169 1266±39

2 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the NCI-H1975 xenograft model was calculated

based on tumor volume measurements at day 21 after the start of treatment.

Table 25: Tumor growth inhibition analysis

Tumor

Gr Treatment , T/C ^b (%) TGI (%) P value

Volume (mm ³ ) ^a

Ϊ Vehicle, qw 2371 ±470 - ^" ~

BCY6136,

2 205±117 8.6 97.5 p<0.001

1 mpk, qw

BCY6136,

3 99±30 4.2 102.3 p<0.001

2 mpk, qw

BCY6136,

4 94±30 4.0 102.4 p< 0.001

3 mpk, qw

a. Mean ± SEM.

b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the

treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in the NCI-H1975 xenograft model was

evaluated. The measured tumor volumes of all treatment groups at various time points are

shown in the Figure 8 and Tables 20 to 25.

The mean tumor size of vehicle treated mice reached 2371 mm ³ on day 21. BCY6136 at 1

mg/kg (TV=205 mm ³ , TGI=97.5%, p<0.001), 2 mg/kg (TV=99 mm ³ , TGI=102.3%, p<0.001)

and 3 mg/kg (TV=94 mm ³ , TGI=102.4%, p<0.001) produced potent antitumor activity.

BCY6136 at 2 mg/kg and 3 mg/kg eradicated the tumors or regressed the tumor to small

size. The treatments was suspended from day 35, and the tumors in 3 mg/kg group didn’t

show obvious re-growth in following 5-6 weeks monitoring, however tumors in 2 mg/kg group

showed obvious regrowth and didn’t show significant tumor inhibition when resuming the

dosing. In this study, mice maintained the bodyweight well.

Study 10. in vivo efficacy study of BCY6136 in the LU-01-0251 PDX model in

Balb/c nude mice

(a) Study Objective

86 The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in the LU-01-0251 PDX model in Balb/c nude mice.

(b) Experimental Design

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with LU-01-0251 of tumor fragment (~30 mm ³ ) for tumor development. The treatment was started when the average tumor volume reached 174 mm ³ for efficacy study. The test article administration and the animal number in each group are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

(d) Results

(i) Body Weight change and T umor Growth Curve

Body weight and tumor growth curve are shown in Figure 9.

(ii) Tumor Volume Trace

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

Table 26: Tumor volume trace over time

Day Group 1 Group 2 Group 3 Group 4 Group 5

87 BCY6136, BCY6136, BCY6136, ADC,

Vehicle

1 mpk, qw 2 mpk, qw 3 mpk, qw 3 mpk, qw

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) T umor Growth Inhibition Analysis

Tumor growth inhibition rate 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 the treatment. Table 27: Tumor growth inhibition analysis

Tumor

Group Treatment T/C ^b (%) TGI (%) P value

Volume (tnmj

1 Vehicle, qw 2208±512

2 BCY6136, 1 mpk, qw 499+128 2Z6 840 p< 0.001

3 BCY6136, 2 mpk, qw 32±30 1.4 107.0 p<0.001

4 BCY6136, 3 mpk, qw 0±0 0.0 108.6 p<0.001

5 ADC, 3 mpk, qw 39+14 18 106.6 p< 0.001 a. Mean ± SEM; b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C). (e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 and ADC in LU-01-0251 PDX model was evaluated. The measured tumor volume of all treatment groups at various time points are shown in the Figure 9 and Tables 26 and 27.

In this study, the mean tumor volume of vehicle treated mice reached 2208 mm ³ on day 28 after the start of treatment. BCY6136 at 1 mg/kg, qw (TV=499 mm ³ , TGI=84.0%, p<0.001), 2 mg/kg, qw (TV=32 mm ³ , TGI=107.0%, p<0.001) and 3 mg/kg, qw (TV=0 mm ³ , TGI=108.6%,

88 p<0.001) produced dose-dependent anti-tumor activity. ADC at 3 mg/kg, qw (TV=39 mm ³ , TGI=106.6%, p<0.001) showed significant anti-tumor activity.

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

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in the LU-01-0251 PDX model in Balb/c nude mice.

(b) Experimental Design

a. The dosing schedule was kept from day 0 to day 70 for all the mice of this group, then the mouse 3-2 and mouse 3-4 were further dosed with BCY6136 3 mg/kg qw from day 77 while the treatment of the other 3 mice was suspended. The dosing schedule was kept from day 0 to day 56 for all the mice of this group

b. The dosing schedule was kept from day 0 to day 70 for all the mice of this group.

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with LU-01-0251 of tumor fragment (~30 mm ³ ) for tumor development. The treatment was started when the average tumor volume reached 960 mm ³ for efficacy study. The test article administration and the animal number in each group are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

89

fiii) Sample Collection

Tumor of mouse #3-2 was collected for FFPE on Day 94. Tumors of mice #5-2 and 5-3 were collected and embed into 1 FFPE block on Day 140.

(d) Results

(i) Tumor Growth Curve

Tumor growth curve are shown in Figure 10.

(ii) Tumor Volume Trace

Mean tumor volume on day 0 to day 28 after the start of treatment in female Balb/c nude mice bearing LU-01-0251 xenograft is shown in Table 28.

Table 28: Tumor volume trace over time

Group 1 Group 2 Group 3 Group 4 Group 5

Day BCY6136, BCY6136, BCY6136, ADC,

Vehicle

1 mpk, qw 2 mpk, qw 3 mpk, qw 3 mpk, qw

0 962±102 963±97 962±137 960±103 959±124

3 1176±108 1003±121 973±105 989±128 1043±158

7 1351±142 1056±151 873±125 890±98 1100±156

10 1591±179 1122±139 722±157 674±96 1172±188

14 1951 ±225 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) T umor Growth Inhibition Analysis

Tumor growth inhibition rate 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 the treatment.

Table 29: Tumor growth inhibition analysis

Tumor

Group Treatment T/C ^b (%) TGI (%) P value

Volume (mnr) ^a

1 Vehicle, qw 2301 ±344

2 BCY6136, 1 mpk, qw 1672±262 72.7 47.0 p> 0.05

90 3 BCY6136, 2 mpk, qw 398±160 17.3 142.1 p<0.001

4 BCY6136, 3 mpk, qw 216±43 9.4 155.6 p <0.001

5 ADC, 3 mpk, qw 1143±186 49.7 86.3 p<0.01 a. Mean ± SEM;

b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 and ADC in LU-01-0251 PDX model was evaluated. The measured tumor volume of all treatment groups at various time points are shown in the Figure 10 and Tables 28 and 29.

In this study, the treatment was started when the average tumor volume reached 960 mm ³ . On day 17 after the start of treatment, the mean tumor volume of vehicle treated mice reached 2301 mm ³ . BCY6136 at 1 mg/kg qw (TV=1672 mm ³ , TGI=47.0%, p>0.05) didn’t show obvious antitumor activity; BCY6136 at 2 mg/kg qw (TV=398 mm ³ , TGI=142.1%, p<0.001) and 3 mg/kg qw (TV=216 mm ³ , TGI=155.6%, p<0.001) produced dose-dependent anti-tumor activity on day 17.

After 70 days’ treatment with BCY6136 at 2 mg/kg qw, 3 in 5 of these mice showed complete tumor regression, the other 2 mice showed obvious tumor relapse from day 42 to day 77. Then further treatment with BCY6136 3 mg/kg qw was performed to the two relapse tumors from day 7, one of tumor showed obvious tumor regress while another one showed resistance to the treatment.

After 56 days’ treatment with BCY6136 at 3 mg/kg qw, all the mice of this group showed complete tumor regression.

ADC at 3 mg/kg qw (TV=1143 mm ³ , TGI=86.3%, p<0.01) showed obvious anti-tumor activity on day 17, after another 53 day’ treatment, these mice showed further but not complete tumor regression.

In this study, there were some mice showed sudden bodyweight loss, this may have the relationship with the long term feeding of the immune-deficiency mice.

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

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in large LU-01-0046 PDX tumors in Balb/c nude mice.

(b) Experimental Design

91

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with LU-01-0046 of tumor fragment (~30 mm ³ ) for tumor development. The treatment was started when the average tumor volume reaches 1039 mm ³ . The test article administration and the animal numbers in each group are shown in the experimental design table.

(iil Testing Article Formulation Preparation

(d) " Results

(i) Tumor Growth Curve

T umor growth curve are shown in Figure 11.

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing LU-01-0046 is shown in Table 30.

92 Table 30: Tumor volume trace over time (BCYs Section)

Days after the start of treatment O

Group Treatment O

0 4 8 11 15 18 22

Vehicle, K

o

1 qw 1044±115 1762±178 2404±262 o

3 mpk, qw

BCY6136,

2 1037±130 1163±146 1927±283 2483±530

m

c 1 mpk, qw

CD

W BCY6136,

H 3 1036±100 784±146 548±107 362±110 325±122 275±152 233±187

H 3 mpk, qw

C

H

m ADC,

w

n 4 3 mpk, qw 1033±114 1155±230 2200±505

m

m 3 mpk, qw

73 Note: the tumor volume trace didn’t show after the day 22 for the group 2 and 4.

C r_

m

ho

m

n

H

o

Cd o o

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the LU-01-0046 PDX model was calculated based on tumor volume measurements at day 22 and day 28 respectively for the two section studies after the start of the treatment.

Table 31 : Tumor growth inhibition analysis (BCYs section on day 22)

BCY6136,

3 233±187 3.8 115.6 p<0.001

3 mpk, qw

ADC,

4 5446±1250 ^* 88.0 14.2 p> 0.05

3 mpk, qw

a. Mean ± SEM;

b. Tumor Growth Inhibition is calculated by dividing the average tumor volume of the treated group by the average tumor volume of the control group (T/C).

*Some groups was terminated before day 22, and the tumor size was calculated by exponential growth equation acquisition as below:

Vehicle group: Y = 995.4 * exp (0.1134 * X).

BCY6136, 1mpk group: Y = 855.0 * exp (0.0974 * X).

ADC, 3mpk group: Y = 757.4* exp (0.1312 * X).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in large LU-01-0046 tumors was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in the Figure 11 and Tables 30 and 31.

In this study, the mean tumor size Of vehicle treated mice was calculated as 6186 mm ³ on day 22. BCY6136 at 1 mg/kg and ADC at 3mg/kg didn’t show obvious anti-tumor activity when starting treatment from tumor size of 1000mm ³ .

BCY6136 (TV=233 mm ³ , TGI=115.6%, p<0.001) at 3 mg/kg produced significant anti-tumor antitumor activity. In particular, BCY6136 eradicated 2/5 and 4/5 tumors completely.

Study 13: In vivo efficacy of BCY6136 in Balb/c nude mice bearing LU-01-0046 NSCLC PDX model

(a) Study Objective

94 The objective of the research was to evaluate the in vivo therapeutic efficacy of BCY6136 in Balb/c nude mice bearing LU-01-0046 NSCLC PDX model.

(b) Experimental Design

Note: Groups were terminated when average tumor volume reached over 2000 mm ³ and tumors were harvested for FFPE: Group 1 on PG-D14, group 5 on PG-D18, group 2 & 6 on PG-D21 and group 3 & 4 on PG-D31.

(c) Experimental Methods and Procedures

iil Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with certain kind of tumor fragment (~30 mm ³ ) for tumor development. The treatments were started when the average tumor volume reached approximately 198 mm ³ . The test article administration and the animal numbers in each group are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

95

(iii) Sample Collection

Groups were terminated when average tumor volume reached over 2000 mm ³ and tumors were harvested for FFPE after the last measurement: Group 1 on PG-D14, group 5 on PG- D18, group 2 &6on PG-D21 and group 3 &4on PG-D31.

(d) Results

(i) T umor Growth Curve

Tumor growth curve are shown in Figure 12.

(iil Tumor Volume T race

Mean tumor volume over time in female Balb/c nude mice bearing LU-01-0046 NSCLC PDX model is shown in Table 32.

Table 32: Tumor volume trace over time (mm ³ )

Gr 1 2 3 4 5 6

ADC

BCY6136 BCY6136 BCY6136

Vehicle ADC 5

Treatment 1 mpk, 2 mpk, 3 mpk,

qw 3 mpk, qw mpk,

qw qw qw

qw

201 ± 195 ±

0 37 198 ±39 201 ± 40 200 ± 46 195 ±28 40

441 ± 389 ±

3 82 310 ±59 283 ±77 155 ±40 418 ±99 68

927 ± 423 ± 596 ±

7 171 547 ±88 132 74 ±19 643 ±159 116

1546 ± 747 ± 321 ± 882 ±

10 377 121 108 31 ±8 938 ±230 134

2307 ± 1058 ± 1215

14 594 140 264 ±95 26 ±11 1475 ± 466 ±193

1390 ± 1576

17 205 127 ±41 26 ±13 2281 ± 556 ± 228

2138 ± 2049

21 301 118 ±34 64 ±42 ±242

24 101 ±40 99 ±63

96 255 1 276 +

28 - - 140 176

582 + 477 +

31 346 283

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in Balb/c nude mice bearing LU-01-0046 PDX model was calculated based on tumor volume measured on PG-D14.

Table 33: Tumor growth inhibition analysis

P value

Tumor

compared

Gr Treatment Volume T/C (%) ^b TGI (%) ^c

with

(mm ¹ ) ³

vehicle

Vehicle

1 2307 ± 594

qw

BCY6136

2 1058 1 140 45.9 59.1 p<0.05

1 mpk, qw

BCY6136

3 264 1 95 11.4 97.0 p<0.001

2 mpk, qw

BCY6136

4 26 + 11 1.1 108.3 p<0.001

3 mpk, qw

ADC

5 1475 + 466 63.9 39.2 p> 0.05

3 mpk, qw

ADC

6 1215 + 193 52.7 51.6 p>0.05

5 mpk, qw

a. Mean + SEM.

b. Tumor Growth Inhibition was calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

c. TGI was calculated for each group using the formula: TGI (%) = [1-(T-To)/ (V-Vo)] *100 (e) Results Summary and Discussion

In the present study, the therapeutic efficacy of test articles in the LU-01-0046 PDX model was evaluated. The measured tumor volumes of all treatment groups at various time points were shown in the Figure 12 and Tables 32 and 33.

The mean tumor size of vehicle treated mice reached 2307 mm ³ on PG-D14. BCY6136 at 1 mg/kg (TV=1058 mm ³ , TGI=59.1%, p<0.05), at 2 mg/kg (TV=264 mm ³ , TGI=97.0%, p<0.001) and at 3 mg/kg (TV=26 mm ³ , TGI=108.3%, p<0.001) produced dose-dependent antitumor activity. ADC at 3 mg/kg and 5 mg/kg did not show obvious antitumor activity (p>0.05).

97 In this study, all of the group’s animals maintained the body weight well.

Study 14: In vivo efficacy study of BCY6136. BCY6173 and BCY6175 in the LU-01-

0046 NSCLC PDX model in Balb/c nude mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of test articles in the LU-01-0046 NSCLC PDX model in Balb/c nude mice.

(b) Experimental Design

(C) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with LU-01-0046 of tumor fragment (~30 mm ³ ) for tumor development. The treatment was started when the average tumor volume reaches 200 mm ³ for part 1 study and 192 mm ³ for part 2 study. The test article administration and the animal numbers in each group are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

98

(d) Results

(0 Tumor Growth Curve

Tumor growth curve are shown in Figures 13 to 15.

(ip Tumor Volume Trace

Mean tumor volume on day 21 after the start of treatment in female Balb/c nude mice bearing LU-01-0046 is shown in Tables 34 and 35.

Table 34: Tumor volume trace over time (Part 1)

Treatmen Days after the start of treatment

Gr .

t 0 3 6 10 14 17 21

Vehicle, 202±2 328±4 536±6 953±10 1833±13 2551±24

1 1386±97

qw 6 8 8 7 2 2

BCY6136,

200±3 293±5 426±9 682±15 1285±23

2 1 mpk, 964±194 976±258

3 6 1 1 4 qw

BCY6136,

201 ±3 194±3 135±2

3 3 mpk, 52±18 13±9 4±4 0±0

3 1 7

qw

Table 35: Tumor volume trace over time (Part 2)

G Days after the start of treatment

Treatment

r 0 3 7 10 14 17 21

Vehicle, 192±3 31 1±8 562±14 830±23 1320±44 1652±52 2342±65

4

qw 0 3 6 0 4 8 1

BCY6173, 191±3 318±5 817±16 1314±27 1546±27 2151±26

5 553±88

1 mpk, qw 3 8 5 6 6 2

BCY6173, 192±3 259±5

6 400±53 455±28 636±92 646±138 890±260

3 mpk, qw 7 1

99 BCY6175, 192±4 186±5

7 92±38 19±11 0±0 0±0 0±0

3 mpk, qw 2 7

(in) Tumor Growth Inhibition Analysis

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

Table 36: Tumor growth inhibition analysis (Part 1)

Group Treatment T/C ^b (%) TGI (%) P value

Volume

Vehicle,

1 2551 ±242

qw

BCY6136,

2 1285±234 50.4 53.9 p<0.001

1 mpk, qw

BCY6136,

3 0±0 0.0 108.5 p<0.001

3 mpk, qw

a. Mean ± SEM; b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C). Table 37: Tumor growth inhibition analysis (Part 2)

Group Treatment T/C ^b (%) TGI (%) P value

Volume

Vehicle,

4 2342±651

qw

BCY6173

5 ’ 2151 ±262 91.8 8.9 p>0.05

1 mpk, qw

BCY6173,

6 890±260 38.0 67.5 p<0.05

3 mpk, qw

BCY6175,

7 0±0 0.0 108.9 p<0.001

3 mpk, qw

a. Mean ± SEM; b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C). (e) Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the LU-01-0046 PDX model was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in the Figures 13 to 15 and Tables 34 to 37.

In part 1 study, the mean tumor size of vehicle treated mice reached 2551 mm ³ on day 21 after the start of treatment.

100 BCY6136 at 1/2 mg/kg, qw (TV=1285 mm ³ , TGI=53.9%, p<0.001) produced significant anti tumor activity, but didn’t exhibit any tumor regression. BCY6136 at 3 mg/kg, qw (TV=0 mm ³ , TGI=108.5%, p<0.001) completely eradicated the tumors, 1 of 5 tumors respectively in BCY6136 3 mg/kg groups showed regrowth after the dosing suspension and the tumors were resistant to BICY6136 treatment when resuming the dosing. The remaining tumors in the BCY6136 groups (4/5 for each group) showed no regrowth after 80 days of dosing suspension. In part 2 study, the mean tumor size of vehicle treated mice reached 2342 mm ³ on day 21 after the start of treatment. BCY6173 at 1 mg/kg, qw (TV=2151 mm ³ , TGI=8.9%,p>0.05) did not show anti-tumor antitumor activity. BCY6173 at 3 mg/kg, qw (TV=890 mm ³ , TGI=67.5%, p< 0.05) produced obvious anti-tumor activity.

BCY6175 at 3 mg/kg, qw (TV=0 mm ³ , TGI=108.9%, p<0.001) completely eradicated 4/5 tumors on day 14.

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

(a) Study Objective

The objective of the project 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

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with LU-01-0412 tumorfragment (~30 mm ³ ) for tumor development. Animals were randomized when the average tumor volume reached 159 mm ³ . The test article administration and the animal numbers in each group were shown in the experimental design table.

(ii) Testing Article Formulation Preparation

101

(iiil Sample Collection

Plasma from vehicle and 3 extra mice treated with BCY6136, BCY8245 and BCY8781 were collected at 30 min and 24 h post dosing. Tumor from vehicle and 3 extra mice treated with BCY6136, BCY8245 and BCY8781 were collected at 24 h post dosing.

(d) Results

(il T umor Growth Curve

Tumor growth curves are shown in Figure 16.

(iil T umor Volume T race

Mean tumor volume over time in female BALB/c nude mice bearing LU-01-0412 xenograft is shown in Table 38.

Table 38: Tumor volume trace over time

Group 1 Group 2 Group 3 Group 4 Group 5

Days Vehicle BCY6136 BCY6136 BCY8245 BCY8781

Qw*4 1 mpk, Qw*4 3 mpk, Qw*4 3 mpk, Qw*4 3 mpk, Qw*4

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

11 395±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

102 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 Analysis

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

Table 39: Tumor growth inhibition analysis

Group Treatment Tumor T/C TGI (%) P value

3 ^a

, q

BCY6136, 1mpk,

2 p<0.05 qw*4 758±70 68.6 36.7

BCY6136, 3mpk,

3 p<0.001 qw*4 416±52 37.6 72.9

BCY8245, 3 mpk,

4 p<0.001 qw*4 1±1 0.1 116.8

BCY8781 , 3 mpk,

5 p<0.001 qw*4 12±5 1.0 115.6 a. Mean ± SEM;

b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136, BCY8245 and BCY8781 in the LU-01-0412 xenograft model was evaluated. The measured tumor volume of all treatment groups at various time points are shown in Figure 16 and Tables 38 and 39.

The mean tumor volume of vehicle treated mice reached 1104 mm ³ on day 32 after the start of treatment. BCY6136 at 1 mg/kg, qw *4 (TV=758 mm ³ , TGI=36.7%, p<0.05) and 3 mg/kg, qw*4 (TV=416 mm ³ , TGI=72.9%, p< 0.001) produced dose-dependent antitumor activity, but didn’t show any tumor regression. BCY8245 at 3 mg/kg, qw ^* 4 (TV=1 mm ³ , TGI=116.8%, p<0.001) and BCY8781 at 3 mg/kg, qw*4 (TV=12 mm ³ , TGI=115.6%, p<0.001) regressed the tumors obviously. Among them, 5 of 6 tumor treated with BCY8245 3 mg/kg and 2 of 6 tumor treated with d BCY8781 3 mg/kg were completely eradicated on day 32.

In this study, animals in all groups maintained the body weight well.

103 Study 16: In vivo efficacy study of BCY6136 in treatment of LU-01-0486 PDX model in Balb/c nude mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in the LU-01-0486 PDX model in Balb/c nude mice.

(b) Experimental Design

(C) Experimental Methods and Procedures

Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with LU-01-0486 of tumor fragment (~30 mm ³ ) for tumor development. The treatment was started when the average tumor volume reached 180 mm ³ for efficacy study. The test article administration and the animal number in each group are shown in the experimental design table.

(iil Testing Article Formulation Preparation

(d) Results

(i) Tumor Growth Curve

Tumor growth curve are shown in Figure 17.

(ii) Tumor Volume Trace

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

Table 40: Tumor volume trace overtime

Group Treatment Days after the start of treatment

104 0 3 7 10 14

Vehicle,

1 179±20 232±30 358±45 450±47 651 ±112 qw

BCY6136,

2 180±23 221 ±20 326±34 420±34 638±71

1 mpk, qw

BCY6136,

3 179±27 222±26 365±44 459±82 645±105

2 mpk, qw

BCY6136,

4 180±25 209±37 304±51 348±77 449±115

3 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the LU-01-0486 PDX model was calculated based on tumor volume measurement at day 14 after the start of the treatment.

Table 41 : Tumor growth inhibition analysis

Tumor

Group Treatment T/C ^b (%) TGI (%) P value

Volume (mm ³ ) ^a

1 Vehicle, qw 651 ±112

2 BCY6136, 1 mpk, qw 638±71 98.0 3.0 p>0.05

3 BCY6136, 2 mpk, qw 645+105 99Ί 12 p>0.05

4 BCY6136, 3 mpk, qw 449±115 68.9 43.1 p> 0.05 a. Mean ± SEM; b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C). (e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in LU-01-0486 PDX model was evaluated. The measured tumor volume of all treatment groups at various time points are shown in the Figure 17 and Tables 40 and 41.

In this study, the mean tumor volume of vehicle treated mice reached 651 mm ³ on day 14 after the start of treatment. BCY6136 at 1 mg/kg, qw (TV=638 mm ³ , TGI=3.0%, p>0.05) and 2 mg/kg, qw (TV=645 mm ³ , TGI=1.2%, p>0.05) didn’t show any anti-tumor activity. BCY6136 at 3 mg/kg, qw (TV=449 mm ³ , TGI=43.1 %, p>0.05) produced slight anti-tumor activity without statistical significance.

Study 17: In vivo efficacy test of BCY6136 in treatment of MDA-MB-231-luc xenograft in Balb/c nude mice

105 (a) Study Objective

The objective of the research was to evaluate the in vivo anti-tumor efficacy of BCY6136 in treatment of MDA-MB-231-luc xenograft model in Balb/c nude mice.

(b) Experimental Design

(c) Experimental Methods and Procedures

(P Cell Culture

The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

fii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with MDA-MB-231-luc tumor cells (10* 10 ^L 6) in 0.1ml of PBS with 0.1 ml matrigel for tumor development. 36 animals were randomized when the average tumor volume reached 159 mm ³ . The test article administration and the animal numbers in each group were shown in the experimental design table.

(iii) Testing Article Formulation Preparation

(iv) Sample Collection

On PG-D33, we collected and fixed the tumors of Group 2 for FFPE.

At the end of study, we collected and fixed the tumors of Group 3 and 4 for FFPE.

(d) Results

(i) Tumor Growth Curve

Tumor growth are shown in Figure 18.

106 (ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing MDA-MB-231-luc xenograft is shown in Tables 42 to 44.

Table 42: Tumor volume trace (PG-D0-PG-D17)

Gr Treatme Days after the start of treatment

nt 0 2 4 7 9 11 14 17

Vehicle, 159±1 306±1 425±5 688±5 908±5 1064±9 1315±9

1 269±8

qw 4 9 2 4 4 8 5

BCY6136

, 159±1 226±3 221±5 310±7 416±8 526±7 809±13

2 636±92

1 mpk, 0 6 4 2 9 7 5 qw

BCY6136

159±1 218±1 182±2 182±2 101 ±2

3 77±24 36±4 41±10

2 mpk, 6 7 2 6 0

qw

BCY6136

241±1 325±1 258±1 246±1

4 158±5 259±6 162±19 178±10

3 mpk, 2 4 2 5

qw

Table 43: Tumor volume trace (PG-D19-PG-D33)

Gr Treatme Days after the start of treatment

nt 19 21 24 26 28 31 33

Vehicle, 1453±12 1661 ±17

1

qw 8 3

BCY6136

, 1253±31 1431 ±35 1507±25 2181±60

2 879±190 994±213

1 mpk, 3 3 3 9

qw

BCY6136

87±7

3 ’ 35±9 33±9 31 ±17 41 ±32 59±45 82±59

2 mpk, 1 qw

107 BCY6136

92±1

4 ’ 171±21 132±19 108±19 85±15 81±8 87±14

3 mpk, 8 qw

Table 44: Tumor volume trace (PG-D35~PG-D47)

Days after the start of treatment

Gr. Treatment

35 38 40 42 45 47

BCY6136

3 ’ 124±106 156±120 179±142 239±197 285±239 350±298

2 mpk, qw

BCY6136,

4 129±38 173±65 181±65 269±113 293±114 371 ±128

3 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the MDA-MB-231-luc xenograft model was calculated based on tumor volume measurements at day 21 after the start of treatment. Table 45: Tumor growth inhibition analysis

Tumor

Gr Treatment , T/C ^b (%) TGI (%) P value

Volume (mm ¹ ) ⁸

1 Vehicle, qw 1661 ±173

BCY6136,

2 994±213 59.8 44.4 p<0.01

1 mpk, qw

BCY6136,

3 33±9 2.0 108.4 p<0.001

2 mpk, qw

BCY6136,

4 132±19 8.0 101.7 p<0.001

3 mpk, qw

a. Mean ± SEM.

b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in the MDA-MB-231-luc xenograft model was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in the Figure 18 and Tables 42 to 45.

The mean tumor size of vehicle treated mice reached 1661 mm ³ on day 21. BCY6136 at 1 mg/kg (TV=994 mm ³ , TGI=44.4%, p<0.01) showed moderate antitumor activity, BCY6136 at 2 mg/kg (TV=33 mm ³ , TGI=108.4%, p<0.001) and 3 mg/kg (TV=132 mm ³ , TGI=101.1%,

108 p<0.001) produced potent antitumor activity, but the tumors showed obvious re-growth from day 28. In this study, one mouse treated with BCY6136 2 mg/kg lost over 15% bodyweight during the treatment schedule, other mice maintained the bodyweight well. Study 18: in vivo efficacy test of BCY6136 in treatment of EMT-6 syngeneic model in B ALB/c mice

(a) Study Objective

The objective of the research was to evaluate the in vivo anti-tumor efficacy of BCY6136 in treatment of EMT-6 syngeneic model in BALB/c mice.

(b) Experimental Design

a. The injection volume of each mouse is 10 ml/kg.

b. The dosage of group 3 and group 4 was changed to 5 mpk and 3 mpk from Day 14.

(c) Experimental Methods and Procedures

(i) Cell Culture

The 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. The tumor cells were routinely subcultured twice weekly by trypsin-EDTA treatment. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(iil Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with EMT-6 tumor cells (5 x 10 ⁶ ) in 0.1 ml of PBS for tumor development. 44 animals were randomized when the average tumor volume reached 75 mm ³ . The test article administration and the animal numbers in each group were shown in the experimental design table.

(iii) Testing Article Formulation Preparation

109

(iv) Sample Collection

3 tumors from spare mice were collected for FACS on day 1 1. The data was supplied by biology team.

(d) Results

(il Tumor Growth Curve

Tumor growth curve are shown in Figure 19.

(ii) T umor Volume T race

Mean tumor volume over time in female BALB/c mice bearing EMT-6 syngeneic is shown in Table 46.

Table 46: Tumor volume trace over time

Days after the start of treatment

Gr

Treatment

3 mpk, qw 4 1 2 18 23 23 22 37 50 61

1 10 BCY6136

’ 82± 108± 204± 350± 426± 588± 691± 850± 1018 1272

3 1/5 ^a mpk,

4 18 27 57 49 72 65 98 ±115 ±140 qw

BCY6136, 1082

82± 130± 255± 358± 450± 607± 731± 872± 1394 4 0.3/3 ^a mpk, +

4 16 35 34 67 94 112 119 ±161 qw 133

The dosage of group 3 and group 4 was changed to 5 mpk and 3 mpk from Day 14.

(iii) T umor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in EMT-6 syngeneic model was calculated based on tumor volume measurements on day 21 after the start of treatment.

Table 47: Tumor growth inhibition analysis

Tumor _ P value compare

Gr Treatment T/C ^b (%) TGI (%)

Volume (mm ³ ) ^a with vehicle

1 Vehicle, qw 1499±340

2 BCY6136,3 mpk, qw 561±61 37.4 66.2 p<0.05

BCY6136,1/5 ^C mpk,

3 1272±140 84.8 16.1 ns

qw

BCY6136,0.3/3 ^C

4 1394±161 93.0 7.4 ns

mpk, qw

a. Mean ± SEM.

b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

c. The dosage of group 3 and group 4 was changed to 5 mpk and 3 mpk from Day 14.

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in EMT-6 syngeneic model was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in the Figure 19 and Tables 46 and 47.

The mean tumor size of vehicle treated mice reached 1499 mm ³ on day 21. BCY6136 at 3 mg/kg, qw (TV=561 mm ³ , TGI=66.2%, p< 0.05) showed obvious antitumor activity. BCY6136 at 1/5 mg/kg, qw (TV=1272 mm ³ , TGI=16.1 %, p>0.05) and BCY6136 at 0.3/3 mg/kg, qw (TV=1394 mm ³ , TGI=7.4%, p> 0.05) didn’t show any antitumor activity.

The dosage of group 3 and group 4 was changed to 5 mpk and 3 mpk from day 14. Tumor ulceration was found in mouse 3-5 on Day 14, and the mice was deal with antibiotic cream. In this study, all mice maintained the bodyweight well.

111 Study 19: In vivo efficacy study of BCY6136 in treatment of NCI-N87 xenograft in

Balb/c nude mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in treatment of NCI-N87 xenograft in Balb/c nude mice.

(b) Experimental Design

(c) Experimental Methods and Procedures

(i) Cell Culture

The 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. The tumor cells were routinely subcultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(ih Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with NCI-N87 tumor cells (10 x 10 ⁶ ) with matrigel (1:1) in 0.2 ml of PBS for tumor development. The animals were randomized and treatment was started when the average tumor volume reached approximately 176 mm ³ . The test article administration and the animal number in each group are shown in the experimental design table.

(iiil Testing Article Formulation Preparation

(d) Results

(i) Tumor Growth Curve

Tumor growth curve is shown in Figure 20.

(ii) Tumor Volume Trace

112 Mean tumor volume over time in female Balb/c nude mice bearing NCI-N87 xenograft is shown in Table 48.

113 Table 48: Tumor volume trace over time

Treatme Days after the start of treatment

Gr.

nt 0 2 4 7 9 11 14 16 18 21 23 25 28 30

17 21 26 42 53 59 73 82 91

Vehicle, 1024 1 151 1305 1407 1465

1 4± 3± 6± 1 ± 7± 8± 4± 1 ± 8±

qw ±83 ±68 ±57 ±64 ±90

7 5 6 10 17 30 46 55 91

BCY613

17 20 21 22 23 18 24 27 30

m 6, 343 390 406 422 425

c 2 6± 0± 0± 4± 8± 4± 4± 6± 8±

CD

W 1 mpk, ±37 ±43 ±48 ±42 ±47

H 7 8 14 27 21 18 23 35 44

H qw

C

H BCY613

m 17 19 16 17 16 96 13 15 16

w 6, 190 203 218 201 210

n 3 6± 7± 8± 0± 5± ±2 3± 0± 0±

m 2 mpk, ±63 ±65 ±66 ±53 ±60

m 18 25 25 26 34 7 35 52 49

qw

73

C r_ BCY613

m 17 19 16 15 14 95 14 14 16

6, 202 205 201 196 201

ho

m 4 7± 7± 9± 8± 8± ±1 1 ± 5± 4±

3 mpk, ±28 ±30 ±16 ±21 ±22

8 9 7 3 8 6 12 24 28

qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the NCI-N87 xenograft was calculated based on tumor volume measurements at day 30 after the start of treatment.

Table 49: Tumor growth inhibition analysis

Tumor

Group Treatment Volume T/C ^b (%) TGI (%) P value

(mm ³ ) ^a

1 Vehicle, qw 1465±90

2 BCY6136, 1 mpk, qw 425+47 29Ό 807 p<0.001

3 BCY6136, 2 mpk, qw 210+60 Ϊ 3 974 p<0.001

4 BCY6136, 3 mpk, qw 201 ±22 137 9&Ϊ p<0.001 a. Mean ± SEM.

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

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in the NCI-N87 model was evaluated. The measured tumor volume of all treatment groups at various time points are shown in the Figure 20 and Tables 48 and 49.

The mean tumor size of vehicle treated mice reached 1465 mm ³ on day 30. BCY6136 at 1 mg/kg, qw (TV=425 mm ³ , TGI=80.7%, p<0.001) and 2 mg/kg, qw (TV=210 mm ³ , TGI=97.4%, p<0.001) produced significant antitumor activity in a dose-dependent manner, BCY6136 at 3 mg/kg, qw (TV=201 mm ³ , TGI=98.1%, p<0.001) showed comparable antitumor activity with BCY6136 at 2 mpk.

In this study, no obvious body weight loss was found in all the groups during the treatment schedule.

Study 20: In vivo efficacy study of BCY6136 in treatment of SK-OV-3 xenograft in Balb/c nude mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in treatment of SK-OV-3 xenograft in Balb/c nude mice.

(b) Experimental Design

115 (c) Experimental Methods and Procedures

(i) Cell Culture

The SK-OV-3 tumor cells were maintained in McCoy ^’ s 5a medium supplemented with 10% heat inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. The tumor cells were routinely subcultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with SK-OV-3 tumor cells (10 x 10 ⁶ ) with matrigel (1 :1) in 0.2 ml of PBS for tumor development. The animals were randomized and treatment was started when the average tumor volume reached approximately 186 mm ³ .

The test article administration and the animal number in each group are shown in the experimental design table.

(iii) Testing Article Formulation Preparation

(d) Results

(i) Tumor Growth Curve

Tumor growth curve is shown in Figure 21.

(ii) T umor Volume T race

Mean tumor volume over time in female Balb/c nude mice bearing SK-OV-3 xenograft is shown in Table 50.

116 Table 50: Tumor volume trace over time

Days after the start of treatment

Gr. Treatment

0 2 5 7 9 12 14 16 19 21 23 26 28

18 24 31 39 47 60 742

Vehicle, 891 1076 1173 1340 1490 1560

1 7± 3± 3± 9± 0± 6± ±10

qw ±133 ±185 ±214 ±236 ±273 ±305

16 24 28 37 23 61 3

18 18 21 26 26 33

ADC, 353 392 449 481 573 647 684

m 2 7± 1± 2± 3± 8± 5±

c 3 mpk, qw ±18 ±63 ±4 ±27 ±33 ±26 ±111

CD

W 16 15 16 35 14 23

H

H 18 22 29 33 35 44

C BCY6136, 503 587 702 752 893 1002 1035

H 3 6± 2± 3± 1± 6± 0±

m 2 mpk, qw ±28 ±33 ±43 ±26 ±34 ±68 ±67

w 23 19 34 21 23 8

n

m

m 18 17 16 18 18 20

BCY6136, 200 230 229 231 236 240 277

4 6± 0± 4± 8± 0± 2±

73 2 mpk, qw ±29 ±46 ±48 ±58 ±49 ±48 ±58

C 23 18 28 33 34 29

m

ho 18 16 15 16 15 18

m BCY6136, 187 212 208 204 205 227 254

5 4± 8± 0± 4± 8± 0±

3 mpk, qw ±4 ±17 ±29 ±12 ±17 ±31 ±48

24 18 12 12 8 8

(iii) Tumor Growth Inhibition Analysis

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

Table 51 : Tumor growth inhibition analysis

Tumor o

Group Treatment T/C ^b (%) TGI (%) P value

Volume (mnr) ^a O

1 Vehicle, qw 1560±305

2 ADC, 3 mpk, qw 684+ΐϊϊ

3 BCY6136, 1 mpk, qw 1035+67 6&4 3 p>0.05

7>

m

n 4 BCY6136, 2 mpk, qw 277+58 l i 93^3 p<0.001

H

31 5 BCY6136, 3 mpk, qw 254+48 16^3 95Ό p<0.001

m

a a. Mean ± SEM.

in

T b. Tumor growth inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for m

m the control group (T/G).

o 5 (e) Results Summary and Discussion

c=

r~

m In this study, the therapeutic efficacy of BCY6136 in the SK-OV-3 model was evaluated. The measured tumor volume of all treatment groups at to various time points are shown in the Figure 21 and Tables 50 and 51.

The mean tumor size of vehicle treated mice reached 1560 mm ³ on day 28. ADC at 3 mg/kg, qw (TV=684 mm ³ , TGI=63.8%, p<0.01) showed in

> moderate anti-tumor efficacy. BCY6136 at 1 mg/kg, qw (TV=1035 mm ³ , TGI=38.1%, p> 0.05) didn ^’ t show obvious anti-tumor activity. BCY6136

10 at 2 mg/kg, qw (TV=277 mm ³ , TGI=93.3%, p<0.001) and 3 mg/kg, qw (TV=254 mm ³ , TGI=95.0%, p<0.001) produced significant anti-tumor

activity.

n

In this study, no obvious body weight loss was found in all the groups during the treatment schedule. H

O

w o b o o

C -/I

118

Study 21: In vivo efficacy study of BCY6136 in treatment of OE21 xenograft in

Balb/c nude mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in treatment of OE21 xenograft in Balb/c nude mice.

(b) Experimental Design

(c) Experimental Methods and Procedures

(il Cell Culture

The 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% C0 ₂ in air. The tumor cells were routinely subcultured twice weekly. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(iil Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with OE21 tumor cells (5 x 10 ⁶ ) with matrigel (1 :1) in 0.2 ml of PBS for tumor development. The animals were randomized and treatment was started when the average tumor volume reached approximately 157 mm ³ . The test article administration and the animal number in each group are shown in the experimental design table.

(iiil Testing Article Formulation Preparation

119 (d) Results

(i) Tumor Growth Curve

Tumor growth curve is shown in Figure 22.

(ii) T umor Volume T race

Mean tumor volume over time in female Balb/c nude mice bearing OE21 xenograft is shown in Table 52.

120 Table 52: Tumor volume trace over time

Days after the start of treatment

Gr. Treatment _

0 2 4 7 9 11 14 16 18 21 23

1 Vehicle, qw 155±9 211±16 291±16 379±14 456±32 539±13 828±42 955±40 1035±58 1250±46 1586±57

BCY6136,

2 159±14 202±28 251 ±29 282±6 331 ±19 392±35 609±56 694±44 777±68 1083±85 1155±98

1 mpk, qw

BCY6136,

m 3 157±19 197±13 219±6 235±27 268±35 243±37 346±78 371 ±98 396±109 515±94 537±122 c 2 mpk, qw

CD

W BCY6136,

H 4 155±19 200±16 197±7 209±11 229±26 211 ±14 289±38 318±53 330±40 474±42 489±51

H 3 mpk, qw

C

H

m

w

n (\\\) Tumor Growth Inhibition Analysis

m

m Tumor growth inhibition rate for BCY6136 in the OE21 xenograft was calculated based on tumor volume measurements at day 23 after the start of treatment.

73

C r_

m

ho Table 53: Tumor growth inhibition analysis

m

Tumor

Group Treatment T/C ^b (%) TGI (%) P value

Volume

1 Vehicle, qw 1586±57

2 BCY6136, 1 mpk, qw 1 155±98 72.8 30.4 p< 0.05

3 BCY6136, 2 mpk, qw 537±122 33.9 73.4 p<0.001

4 BCY6136, 3 mpk, qw 489±51 308 707 p<0.001

a. Mean ± SEM.

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

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in the OE21 model was evaluated. The measured tumor volume of all treatment groups at various time points are shown in the Figure 22 and Tables 52 and 53.

The mean tumor size of vehicle treated mice reached 1586 mm ³ on day 23. BCY6136 at 1 mg/kg, qw (TV= 1155 mm ³ , TGI = 30.4% p<0.05) showed slight anti-tumor activity. BCY6136 at 2 mg/kg, qw (TV=537 mm ³ , TGI=73.4%, p<0.001) and 3 mg/kg, qw (TV=489 mm ³ , TGI=76.7%, p<0.001) produced significant anti-tumor activity.

In this study, no obvious body weight loss was found in all the groups during the treatment schedule.

Study 22: In vivo efficacy test of BCY6136 in treatment of MOLP-8 xenograft in CB17- SCID mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumor efficacy of BCY6136 in treatment of MOLP-8 xenograft in CB17-SCID mice.

(b) Experimental Design

(c) Experimental Methods and Procedures

(i) Cell Culture

The MOLP-8 tumor cells were maintained in vitro as a monolayer culture in RMPI-1640 supplemented with 20% heat inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. The tumor cells were routinely subcultured by trypsin-EDTA treatment. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with MOLP-8 tumor cells (10 x 10 ⁶ ) in 0.2 ml PBS with 50% matrigel for tumor development. 36 animals were randomized

122 when the average tumor volume reached 141 mm ³ . The test article administration and the animal numbers in each group were shown in the experimental design table.

(iii) Testing Article Formulation Preparation

(d) Results

(i) Tumor Growth Curve

Tumor growth curve are shown in Figure 23.

(ii) T umor Volume T race

Mean tumor volume over time in female CB17-SCID mice bearing MOLP-8 xenograft is shown in Table 54.

Table 54: Tumor volume trace over time

Gr Days after the start of treatment

Treatment

BCY6136,2 mpk, 140±1 271 ±4 509+2 1218±1

3 250±2 662±78 873±49

qw 5 3 3 44

BCY6136,3 mpk, 142±1 239±6 197±2 342±7 693±13 938±15

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the MOLP-8 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment.

Table 55: Tumor growth inhibition analysis

123 Tumor P value

T/C ^b TGI

Gr Treatment Volume compared

(%) (%)

(mm ³ ) ^a with vehicle

1 Vehicle, qw 2528±295

BCY6136, 1

2 1446±234 57.2 45.5 p>0.05

mpk, qw

BCY6136, 2

3 1218±144 48.2 54.9 p<0.05

mpk, qw

BCY6136, 3

4 938±155 37.1 66.7 p<0.01

mpk, qw

a. Mean ± SEM.

b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in the MOLP-8 xenograft model was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in the Figure 23 and Tables 54 and 55.

The mean tumor size of vehicle treated mice reached 2528 mm ³ on day 14. BCY6136 at 1 mg/kg (TV=1146 mm ³ , TGI=45.5%, p> 0.05), 2 mg/kg (TV=1218 mm ³ , TGI=54.9%, p<0.05) and 3 mg/kg (TV=938 mm ³ , TGI=66.7%, p<0.01) produced dose-dependent antitumor activity, but all of dosage didn’t regress the tumors in MOLP-8 xenografts. In this study, all of mice maintained the bodyweight well.

Study 23: In vivo efficacy test of BCYs in treatment of HT1080 xenograft in BALB/c nude mice

(a) Study Objective

The objective of the research was to evaluate the in vivo anti-tumor efficacy of BCYs in treatment of HT1080 xenograft model in BALB/c nude mice.

(b) Experimental Design

124

Note: n: animal number; Dosing volume: adjust dosing volume based on body weight 10 mI/g. (c) Experimental Methods and Procedures

(il Cell Culture

The HT1080 tumor cells will be maintained in medium supplemented with 10% heat inactivated fetal bovine serum at 37°C in an atmosphere of 5% C0 ₂ in air. The tumor cells will be routinely subcultured twice weekly. The cells growing in an exponential growth phase will be harvested and counted for tumor inoculation.

(iil Tumor Inoculation

Each mouse will be inoculated subcutaneously at the right flank with HT1080 tumor cells (5*10 ⁶ ) for tumor development. The animals will be randomized and treatment will be started when the average tumor volume reaches approximately 150-200 mm ³ . The test article administration and the animal numbers in each group are shown in the following experimental design table.

(iii) Testing Article Formulation Preparation

125

(d) Results

(il T umor Growth Curve

Tumor growth curve are shown in Figures 24 to 29.

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing HT 1080 xenograft is shown in Table 56.

Table 56: Tumor volume trace over time

Gr Treatmen Days after the start of treatment

t 5 2 4 7 9 Tϊ 14

Vehicle, 179±2 312±8 529+13 886+20 1185±17 1467+22 1737±25

1

qw 2 4 5 7 2 4 8

BCY6173

2 1 mpk, 178±2 1074±15 qw 6 276±8 328±73 594±62 745±22 960±53 0

BCY6173

3

2 mpk, 178±2 277±6 262±12 309±23

qw 8 1 5 8 425±334 436±323 480±347

126 BCY6173

3 mpk, 179±4 182±7

qw 3 1 133±88 87±68 77±65 60±54 47±42

BCY6135

1 mpk, 178±2 267±6

qw 2 6 262±58 436±67 599±89 703±36 871 ±28

BCY6135

2 mpk, 178±2 176±4

qw 3 8 117±43 70±23 67±23 52±21 62±7

BCY6135

3 mpk, 177±3 178±7

qw 9 9 92 ±67 62±46 62±51 57±51 44±40

BCY6136 178±1 249±2

mpk, qw 9 2 115±8 126±53 158±71 140±89 245±116

BCY6136

3 mpk, 178±3 168±2

qw 6 1 72±18 22±7 21±15 8±6 3±2

BCY6136

5 mpk, 178±2 165±3

qw 6 3 52±10 18±7 9±4 5±2 2±1

BCY6174

1 mpk, 180±3 231±1 1066±13 qw 5 9 226±29 432±37 602±63 742±62 0

BCY6174 178±3 203±5

m pk, qw 1 0 123±29 216±47 291 ±40 326±68 532±91

BCY6174

3 mpk, 178±3 195±1

qw 3 3 110±39 58±23 34±17 21±11 1 1 ±7

BCY6175

1 mpk, 178±2 248±6

qw 7 2 244±74 347±18 435±18 558±38 769±26

BCY6175

2 mpk, 178±2 223±4

qw 2 2 158±59 1 16±35 156±52 166±51 295±88

127 BCY6175

16 3 mpk, 179±3 189±4

qw 9 8 116±50 43±18 33±18 25±13 1 1 ±9

ADC

17 3 mpk, 180±2 158±3

qw 6 0 58±8 18±2 7±1 2 ±2 0±0

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCYs in the HT1080 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment.

Table 57: Tumor growth inhibition analysis

Tumor P value

Gr Treatment T/C ^b (%) TGI (%)

Volume (mm 3 ³ \)a compare

1 Vehicle, qw 1737±258

2 BCY6173, 1 mpk, 1074±150 61.8 42.5 p> 0.05

~~ 3 BCY6173, 2 mpk, 480+347 2T6 806 p<0.05

4 BCY6173, 3 mpk, 47±42 2.7 108.4 p<0.01

5 BCY6135, 1 mpk, 871±28 50.1 55.5 p<0.01

6 BCY6135, 2 mpk, 62±7 3.5 107.5 p<0.001

7 BCY6135, 3 mpk, 44±40 2.5 108.6 p<0.001

8 BCY6136, 2mpk, qw 245±1 16 14.1 95.7 p<0.001

9 BCY6136, 3 mpk, 3±2 0.2 1 1 1.2 p<0.001

~0 BCY6136, 5 mpk, 2±i oi 1 1 1.3 p<0.001

11 BCY6174, 1 mpk, 1066±130 61.4 43.1 p<0.05

~ 12 BCY6174, 2m pk, qw 532+91 306 7T3 p<0.01

~ 13 BCY6174, 3 mpk, ΐϊ+7 06 1 10.7 p<0.001

~ 14 BCY6175, 1 mpk, 769+26 443 6Z 1 p<0.01

~ 15 BCY6175, 2 mpk, 295+88 17Ό 9Z5 p<0.001

~ 16 BCY6175, 3 mpk, TT±9 06 1 10.8 p<0.001

~ 17 ADC, 3 mpk, qw 0+0 OO 1 1 1.5

a. Mean ± SEM.

b. Tumor Growth Inhibition is calculated by dividing the group average tumor volume for the treated group by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

128 In this study, the therapeutic efficacy of BCYs in the HT1080 xenograft model was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in the Figures 24 to 29 and Tables 56 and 57.

The mean tumor size of vehicle treated mice reached 1737 mm ³ on day 14.

BCY6173 at 1 mg/kg, qw (TV=1074 mm ³ , TGI=42.5%, p>0.05), 2 mg/kg, qw (TV=480 mm ³ ,

TGI=80.6%, p< 0.05) and 3 mg/kg, qw (TV=7 mm ³ , TGI=108.4%, p<0.01) produced dose- dependent antitumor activity.

BCY6135 at 1 mg/kg, qw (TV=871 mm ³ , TGI=55.5%, p<0.01), 2 mg/kg, qw (TV=62 mm ³ , TGI=107.5%, p<0.001) and 3 mg/kg, qw (TV=44 mm ³ , TGI=108.6%, p<0.001) produced dose- dependent antitumor activity.

w (TV=3 mm ³ , showed potent anti-tumor activity.

BCY6174 at 1 mg/kg, qw (TV=1066 mm ³ , TGI=43.1 %, p< 0.05), 2 mg/kg, qw (TV=532 mm ³ , TGI=77.3%, p<0.01) and 3 mg/kg, qw (TV=11 mm ³ , TGI=1 10.7%, p<0.001) produced dose- dependent antitumor activity.

BCY6175 at 1 mg/kg, qw (TV=769 mm ³ , TGI=62.1 %, p<0.01), 2 mg/kg, qw (TV=295 mm ³ , TGI=92.5%, p<0.001) and 3 mg/kg, qw (TV=11 mm ³ , TGI=110.8%, p<0.001) produced dose- dependent antitumor activity.

ADC at 3 mg/kg, qw (TV=0 mm ³ , TGI=1 11.5%) completely eradicated the tumors by day 14.

129