Patent Grant
11696956
This application is a U.S. national stage application under 35 U.S.C. § 371 of International Application No. PCT/GB2018/053678, filed Dec. 19, 2018, which claims priority under 35 U.S.C. § 119 to United Kingdom Application No. GB1721259.8, filed Dec. 19, 2017, United Kingdom Application No. GB1804102.0, filed Mar. 14, 2018, and United Kingdom Application No. GB1818603.1, filed Nov. 14, 2018, each of which is incorporated herein by reference in its entirety.
A Sequence Listing was submitted electronically via EFS in the form of a txt file on Jun. 9, 2020 and named “174752_SL.txt” (28,061 bytes), the contents of which are incorporated herein by reference in their entirety.
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).
Cyclic peptides are able to bind with high affinity and target specificity to protein targets and hence are an attractive molecule class for the development of therapeutics. In fact, several cyclic peptides are already successfully used in the clinic, as for example the antibacterial peptide vancomycin, the immunosuppressant drug cyclosporine or the anti-cancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). Good binding properties result from a relatively large interaction surface formed between the peptide and the target as well as the reduced conformational flexibility of the cyclic structures. Typically, macrocycles bind to surfaces of several hundred square angstrom, as for example the cyclic peptide CXCR4 antagonist CVX15 (400 Å2; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with the Arg-Gly-Asp motif binding to integrin αVb3 (355 Å2) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1 binding to urokinase-type plasminogen activator (603 Å2; Zhao et al. (2007), J Struct Biol 160 (1), 1-10).
Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, leading to a smaller loss of entropy upon binding to targets and resulting in a higher binding affinity. The reduced flexibility also leads to locking target-specific conformations, increasing binding specificity compared to linear peptides. This effect has been exemplified by a potent and selective inhibitor of matrix metalloproteinase 8, (MMP-8) which lost its selectivity over other MMPs when its ring was opened (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties achieved through macrocyclization are even more pronounced in multicyclic peptides having more than one peptide ring as for example in vancomycin, nisin and actinomycin.
Different research teams have previously tethered polypeptides with cysteine residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and co-workers had used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclisation of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for the generation of candidate drug compounds wherein said compounds are generated by linking cysteine containing polypeptides to a molecular scaffold as for example TATA (1,1′,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.
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.
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).
Where error bars are present in the above Figures, these represent standard error of the mean (SEM).
In one embodiment, said loop sequences comprise 2, 3, 5, 6 or 7 amino acid acids.
In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences one of which consists of 2 amino acids and the other of which consists of 7 amino acids (such as those listed in Table 4).
In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences both of which consist of 5 amino acids (such as those listed in Tables 3 and 4).
In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences both of which consist of 6 amino acids (such as those listed in Tables 3 to 5).
In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences both of which consist of 6 amino acids (such as those listed in Table 10).
In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 3 amino acids (such as those listed in Table 4).
In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 7 amino acids (such as those listed in Table 5).
In one embodiment, the peptide ligand comprises an amino acid sequence selected from:
Ci-X1-Cii-X2-Ciii
wherein X1 and X2 represent the amino acid residues between the cysteine residues listed in Tables 3 to 5 and Ci, Cii and Ciii represent first, second and third cysteine residues, respectively or a pharmaceutically acceptable salt thereof.
In a further embodiment, the peptide ligand comprises an amino acid sequence selected from one or more of the peptide ligands listed in one or more Tables 3 to 5.
In a further embodiment, the peptide ligand comprises an amino acid sequence selected from:
Ci-X1-Cii-X2-Ciii
wherein X1 and X2 represent the amino acid residues between the cysteine residues listed in Table 10 and Ci, Cii and Ciii represent first, second and third cysteine residues, respectively or a pharmaceutically acceptable salt thereof.
In a further embodiment, the peptide ligand comprises an amino acid sequence selected from one or more of the peptide ligands listed in Table 10.
In one embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences both of which consist of 6 amino acids and the peptide ligand has an amino acid sequence selected from:
wherein HyP is hydroxyproline, HArg is homoarginine and Ci, Cii and Ciii represent first, second and third cysteine residues, respectively or a pharmaceutically acceptable salt thereof.
In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences both of which consist of 6 amino acids and the peptide ligand has the following amino acid sequence:
wherein HyP is hydroxyproline, HArg is homoarginine and Ci, Cii and Ciii represent first, second and third cysteine residues, respectively or a pharmaceutically acceptable salt thereof.
In one embodiment, the peptide ligand of the invention is a peptide ligand which is other than the amino acid sequence:
In one embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences both of which consist of 6 amino acids and the peptide ligand has an amino acid sequence selected from:
wherein Sar is sarcosine, HArg is homoarginine and HyP is hydroxyproline.
In a further embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences both of which consist of 6 amino acids and the peptide ligand has the following amino acid sequence:
wherein Sar is sarcosine, HArg is homoarginine and HyP is hydroxyproline.
In one embodiment, the peptide ligand of the invention is a peptide ligand which is other than the amino acid sequence:
In one embodiment, the molecular scaffold is selected from 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) and the peptide ligand is selected from any one of the peptide ligands listed in Tables 3 to 5.
In an alternative embodiment, the molecular scaffold is selected from 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) and the peptide ligand is selected from any one of the peptide ligands listed in Table 10.
In one embodiment, the molecular scaffold is selected from 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) and the peptide ligand is selected from:
wherein Sar is sarcosine, HArg is homoarginine and HyP is hydroxyproline.
In a further embodiment, the molecular scaffold is selected from 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) and the peptide ligand is:
wherein Sar is sarcosine, HArg is homoarginine and HyP is hydroxyproline.
In one embodiment, the peptide ligand is selected from any one of Compounds 1-113 or a pharmaceutically acceptable salt thereof.
In a further embodiment, the peptide ligand is Compound 66 (BCY6099) or Compound 67 (BCY6014) or a pharmaceutically acceptable salt thereof.
In a yet further embodiment, the peptide ligand is Compound 66 (BCY6099) or a pharmaceutically acceptable salt thereof.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as in the arts of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al., Short Protocols in Molecular Biology (1999) 4th 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 (Ci, Cii and Ciii) 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:
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 Ci, Cii, 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 (β-Ala)-Sar10-Ala tail would be denoted as:
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 become C-terminus and vice versa) and their stereochemistry is likewise also reversed (i.e. D-amino acids become L-amino acids and vice versa).
Peptide Ligands
A peptide ligand, as referred to herein, refers to a peptide, peptidic or peptidomimetic covalently bound to a molecular scaffold. Typically, such peptides, peptidics or peptidomimetics comprise a peptide having natural or non-natural amino acids, two or more reactive groups (i.e. cysteine residues) which are capable of forming covalent bonds to the scaffold, and a sequence subtended between said reactive groups which is referred to as the loop sequence, since it forms a loop when the peptide, peptidic or peptidomimetic is bound to the scaffold. In the present case, the peptides, peptidics or peptidomimetics comprise at least three cysteine residues (referred to herein as Ci, Cii and Ciii), and form at least two loops on the scaffold.
Advantages of the Peptide Ligands
Certain bicyclic peptides of the present invention have a number of advantageous properties which enable them to be considered as suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include:
Pharmaceutically Acceptable Salts
It will be appreciated that salt forms are within the scope of this invention, and references to peptide ligands include the salt forms of said ligands.
The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.
Acid addition salts (mono- or di-salts) may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include mono- or di-salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic), isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1,5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.
One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.
If the compound is anionic, or has a functional group which may be anionic (e.g., —COOH may be —COO−), then a salt may be formed with an organic or inorganic base, generating a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li+, Na+ and K+, alkaline earth metal cations such as Ca2+ and Mg2+, and other cations such as Al3+ or Zn+. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4+) and substituted ammonium ions (e.g., NH3R+, NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.
Where the 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.
Modified Derivatives
It will be appreciated that modified derivatives of the peptide ligands as defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from: N-terminal and/or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues (such as replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of a spacer group; replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues; replacement of one or more amino acid residues with one or more replacement amino acids, such as an alanine, replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds within the bicyclic peptide ligand; replacement of one or more peptide bonds with a surrogate bond; peptide backbone length modification; substitution of the hydrogen on the alpha-carbon of one or more amino acid residues with another chemical group, modification of amino acids such as cysteine, lysine, glutamate/aspartate and tyrosine with suitable amine, thiol, carboxylic acid and phenol-reactive reagents so as to functionalise said amino acids, and introduction or replacement of amino acids that introduce orthogonal reactivities that are suitable for functionalisation, for example azide or alkyne-group bearing amino acids that allow functionalisation with alkyne or azide-bearing moieties, respectively.
In one embodiment, the modified derivative comprises an N-terminal and/or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N-terminal modification using suitable amino-reactive chemistry, and/or C-terminal modification using suitable carboxy-reactive chemistry. In a further embodiment, said N-terminal or C-terminal modification comprises addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator or a chromophore.
In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, the N-terminal residue is capped with acetic anhydride or other appropriate reagents during peptide synthesis leading to a molecule which is N-terminally acetylated. This embodiment provides the advantage of removing a potential recognition point for aminopeptidases and avoids the potential for degradation of the bicyclic peptide.
In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group which facilitates the conjugation of effector groups and retention of potency of the bicyclic peptide to its target.
In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal residue is synthesized as an amide during peptide synthesis leading to a molecule which is C-terminally amidated. This embodiment provides the advantage of removing a potential recognition point for carboxypeptidase and reduces the potential for proteolytic degradation of the bicyclic peptide.
In one embodiment, the modified derivative comprises replacement of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids may be selected having isosteric/isoelectronic side chains which are neither recognised by degradative proteases nor have any adverse effect upon target potency.
Alternatively, non-natural amino acids may be used having constrained amino acid side chains, such that proteolytic hydrolysis of the nearby peptide bond is conformationally and sterically impeded. In particular, these concern proline analogues, bulky sidechains, C□-disubstituted derivatives (for example, aminoisobutyric acid, Aib), and cyclo amino acids, a simple derivative being amino-cyclopropylcarboxylic acid.
In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine (Ci) and/or the C-terminal cysteine (Ciii).
In one embodiment, the modified derivative comprises replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues. In a further embodiment, the modified derivative comprises replacement of a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the advantage of improving the pharmaceutical stability profile of the resultant bicyclic peptide ligand.
In one embodiment, the modified derivative comprises replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged versus hydrophobic amino acid residues is an important characteristic of the bicyclic peptide ligands. For example, hydrophobic amino acid residues influence the degree of plasma protein binding and thus the concentration of the free available fraction in plasma, while charged amino acid residues (in particular arginine) may influence the interaction of the peptide with the phospholipid membranes on cell surfaces. The two in combination may influence half-life, volume of distribution and exposure of the peptide drug, and can be tailored according to the clinical endpoint. In addition, the correct combination and number of charged versus hydrophobic amino acid residues may reduce irritation at the injection site (if the peptide drug has been administered subcutaneously).
In one embodiment, the modified derivative comprises replacement of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and by a propensity of D-amino acids to stabilise □-turn conformations (Tugyi et al (2005) PNAS, 102(2), 413-418).
In one embodiment, the modified derivative comprises removal of any amino acid residues and substitution with alanines, such as D-alanines. This embodiment provides the advantage of identifying key binding residues and removing potential proteolytic attack site(s).
It should be noted that each of the above mentioned modifications serve to deliberately improve the potency or stability of the peptide. Further potency improvements based on modifications may be achieved through the following mechanisms:
(for reviews see Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem (2009), 16, 4399-418).
Isotopic Variations
The present invention includes all pharmaceutically acceptable (radio)isotope-labeled peptide ligands of the invention, wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature, and peptide ligands of the invention, wherein metal chelating groups are attached (termed “effector”) that are capable of holding relevant (radio)isotopes, and peptide ligands of the invention, wherein certain functional groups are covalently replaced with relevant (radio)isotopes or isotopically labelled functional groups.
Examples of isotopes suitable for inclusion in the peptide ligands of the invention comprise isotopes of hydrogen, such as 2H (D) and 3H (T), carbon, such as 11C, 13C and 14C, chlorine, such as 36Cl, fluorine, such as 18F, iodine, such as 123I, 125I and 131I, nitrogen, such as 13N and 15N, oxygen, such as 15O, 17O and 18O, phosphorus, such as 32P, sulfur, such as 35S, copper, such as 64Cu, gallium, such as 67Ga or 68Ga, yttrium, such as 90Y and lutetium, such as 177Lu, and Bismuth, such as 213Bi.
Certain isotopically-labelled peptide ligands of the invention, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies, and to clinically assess the presence and/or absence of the EphA2 target on diseased tissues. The peptide ligands of the invention can further have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes or receptors. The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The radioactive isotopes tritium, i.e. 3H (T), and carbon-14, i.e. 14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
Substitution with heavier isotopes such as deuterium, i.e. 2H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.
Substitution with positron emitting isotopes, such as 11C, 18F, 15O and 13N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.
Isotopically-labeled compounds of peptide ligands of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.
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 α unsaturated carbonyl containing compound is 1,1′,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 tβ 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 tβ 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, p 821; 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 p 10444), the 18 amino acid ‘model amphipathic peptide’ (Oehlke et al (1998) Biochim Biophys Acts Volume 1414 p 127) 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 p 153). Other chemical strategies to add guanidinium groups to molecules also enhance cell penetration (Elson-Scwab et al (2007) J Biol Chem Volume 282 p 13585). 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 tβ 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 tβ range 12 to 60 hours. In a further embodiment, it will have a tβ 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 carboxypeptidase 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 yet further particular embodiment of the invention, the cytotoxic agent is selected from maytansinoids (such as DM1). Data is presented herein in Table 6 which demonstrates the effects of peptide ligands conjugated to toxins containing DM1.
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 selected from DM1 or 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: 98). 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(SO3H)—, —SS-(Me)-, -(Me)-SS-(Me)-, —SS-(Me2)- or —SS-(Me)-SO3H—. 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(SO3H)— moiety, such as SO3H-SPDB.
In an alternative embodiment, the cytotoxic agent comprises a non-cleavable cytotoxic agent. Thus, in one embodiment the cytotoxic agent is non-cleavable MMAE (such as the cytotoxic agent within BCY6063) or non-cleavable DM1 (such as the cytotoxic agent within BCY6064).
In one embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (A):
wherein said bicycle is selected from any one of BCY6099 and BCY6014 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 selected from any one of BCY6099 and BCY6014 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 Table 6.
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 Table 6.
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 BCY6014 as defined herein. This BDC is known herein as BCY6031. Data is presented herein which demonstrates excellent competition binding for BCY6031 in the EphA2 competition binding assay as shown in Table 6. Data is also presented herein in Table 11 and
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 BCY6014 as defined herein. This BDC is known herein as BCY6032. Data is presented herein which demonstrates excellent competition binding for BCY6032 in the EphA2 competition binding assay as shown in Table 6.
In an alternative embodiment, the cytotoxic agent is MMAE or DM1 and the drug conjugate is selected from any of the BDCs listed in Table 11. Data is presented herein which shows that these BDCs exhibited excellent cross reactivity between human, mouse and rodent EphA2 as shown in Table 11.
In a further embodiment, the cytotoxic agent is MMAE or DM1 and the drug conjugate is selected from any of the BDCs listed in Table 13.
In a further embodiment, the cytotoxic agent is MMAE or DM1 and the drug conjugate is selected from BCY6033, BCY6082, BCY6136 and BCY6173. Data is presented herein which shows that these four 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 14 and 15.
In a yet further embodiment, the drug conjugate is selected from any one of: BCY6031, BCY6033, BCY6082, BCY6135, BCY6136, BCY6173, BCY6174 and BCY6175:
In one embodiment, the drug conjugate is other than BCY6027, BCY6028, 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
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 USA. 1994 Dec. 20; 91(26):12544-8 or in Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 Nov. 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 cyclosporine, 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 al. (1998) Genes Dev 12, 667-678).
Among other patterning functions, various Ephs and ephrins have been shown to play a role in vascular development. Knockout of EphB4 and ephrin-B2 results in a lack of the ability to remodel capillary beds into blood vessels (Poliakov et al., supra) and embryonic lethality. Persistent expression of some Eph receptors and ephrins has also been observed in newly-formed, adult micro-vessels (Brantley-Sieders et al. (2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).
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 al., 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 al., supra); Marme (2002) Ann Hematol 81 Suppl 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).
EPH receptor A2 (ephrin type-A receptor 2) is a protein that in humans is encoded by the 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 BCY6033 and BCY6136 showed significant and potent anti-tumor activity in the PC-3 xenograft prostate cancer model (see
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 which demonstrates that a BDC of the invention (BCY6031) completely eradicated non-small cell lung carcinomas from day 32 and no tumour regrowth occurred following dosing suspension on day 28. This data clearly demonstrates the clinical utility of the BDCs of the present invention in cancers such as lung cancers, in particular non-small cell lung carcinomas. Data is also presented herein in Study 9 which show that BCY6033 demonstrated dose dependent anti-tumor activity, BCY6082 demonstrated significant antitumor activity and BCY6136 demonstrated potent antitumor activity in the NCI-H1975 xenograft lung cancer (NSCLC) model (see
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
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
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
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
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 BCY6082, BCY6031, BCY6033 and BCY6136 demonstrated potent antitumor activity in the HT-1080 xenograft fibrosarcoma model (see
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 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.
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×3, MeOH×3, and then dried under N2 bubbling overnight. The peptide resin was then treated with 92.5% TFA/2.5% TIS/2.5% EDT/2.5% H2O for 3 h. 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 for 2 hours and then lyophilised. The lyophilised powder was dissolved in of ACN/H2O (50:50), and a solution of 100 mM TATA in ACN was added, followed by ammonium bicarbonate in H2O (1 M) and the solution mixed for 1 h. Once the cyclisation was complete, the reaction was quenched with 1 M 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
All amino acids, unless noted otherwise, were used in the L-configurations.
8.0 g of resin was used to generate 2.1 g BCY6099 (99.2% purity; 16.3% yield) as a white solid.
4.79 g of resin was used to generate 1.07 g BCY6014 (Q1: 131.9 mg, 97.99% purity; Q2: 141.7 mg, 99.04% purity; Q3: 800.7 mg, 92.35% purity; 16.9% yield) as white a solid.
4.44 g of resin was used to generate 700 mg BCY6104 (95.87% purity, 10.5% yield) as white a solid.
4.44 g of resin was used to generate 700 mg BCY6103 (98.9% purity, 11.1% yield) as white a solid.
4.44 g of resin was used to generate 700 mg BCY6101 (95.9% purity, 10.9% yield) as white a solid.
4.44 g of resin was used to generate 900 mg BCY6102 (95.9% purity, 14.1% yield) as white a solid.
4.44 g of resin was used to generate 900 mg BCY6139 (97.4% purity, 11.2% yield) as white a solid.
1.11 g of resin was used to generate 200 mg BCY6138 (95.2% purity, 12.2% yield) as white a solid.
4.44 g of resin was used to generate 600 mg BCY6137 (98.9% purity, 9.06% yield) as white a solid.
1.11 g of resin was used to generate 99.2 ma BCY6042 (99.2% purity 7.0% yield) as white a solid
4.79 g of resin was used to generate 732.0 mg BCY6019 (92.82% purity, 12.2% yield) as white a solid.
To a solution of BCY6019 (0.05 g, 17.82 μmol, 1.00 eq) in H2O (3 mL) was adjusted PH=11 by Na2CO3 (aq) and added acetyl acetate (5.46 mg, 53.46 μmol, 5.01 μL, 3.00 eq). The mixture was stirred at 15° C. for 1 hr. LC-MS showed BCY6019 was consumed completely and one main peak with desired MS was detected. The reaction was adjusted PH=7 by 1N HCl and directly purified by prep-HPLC (TFA condition). Compound BCY6059 (18.1 mg, 6.36 μmol, 35.67% yield) was obtained as a white solid.
1.11 g of resin was used to generate 45.2 mg BCY6160 (95.5% purity, 2.5% yield) as white a solid.
4.79 g of resin was used to generate 2.42 g BCY6009 (>88.92% purity, 36.0% yield) as white a solid.
1.19 g of resin was used to generate 189.9 mg BCY6017 (95.05% purity, 16.8% yield) as white a solid.
1.19 g of resin was used to generate 289.1 mg BCY6018 (97.92% purity, 21.0% yield) as white a solid.
1.11 g of resin was used to generate 150.0 mg BCY6152 (98.75% purity; 9.5% yield) as white a solid.
1.11 g of resin was used to generate 120.0 mg BCY6141 (97.91% purity; 7.3% yield) as white a solid.
1.11 g of resin was used to generate 285.0 mg BCY6026 (97.7% purity; 24.2% yield) as white a solid.
1.11 g of resin was used to generate 140.0 mg BCY6153 (98.59% purity; 9.9% yield) as white a solid.
The general schematic for preparing Bicycle drug conjugates (BDCs) is shown in
The synthesis of Bicyclic Peptide Drug Conjugates BCY6027, BCY6028, BCY6031 and BCY6032 listed in Table 6 were performed using the protocol disclosed in WO 2016/067035.
Activated bicycle peptides with formula (C) and (D):
were synthesised by reacting the free amino group of the bicycle precursors with, respectively, SPP (N-succinimidyl 4-(2-pyridyldithio)pentanoate, Annova Chem) and SPDB (N-succinimidyl 3-(2-pyridyldithio)propionate, Annova Chem) in DMSO. Concentrations of bicycle precursors were 10 mM or higher, with a 1.3-fold excess of SPP or SPDB, and a 20-fold excess of diisopropylethylamine, at room temperature. The reaction was judged complete after 1 hour, as judged by LCMS. Purification was performed by reverse phase as described above. Appropriate fractions were lyophilised.
Activated bicycle peptides with formula (C) and (D) were disulphide exchanged with 1.15 equivalents of DM1 (as the free thiol), in semi aqueous conditions (50% dimethylacetamide and 50% 100 mM sodium acetate pH 5.0 supplemented with 2 mM EDTA) for 21 hours at room temperature under a nitrogen gas blanket. Concentrations of activated bicycle peptides with structure C and D in the reaction were at 10 mM or higher.
This was followed by standard reverse phase purification using a C18 column. Fractions at purity greater than 95% were isolated and lyophilised. The materials did not contain measurable quantities of free toxin.
The peptide was synthesized by solid phase synthesis. 50 g CTC Resin (sub: 1.0 mmol/g) was used. To a mixture containing CTC Resin (50 mmol, 50 g, 1.0 mmol/g) and Fmoc-Cit-OH (19.8 g, 50 mmol, 1.0 eq) was added DCM (400 mL), then DIEA (6.00 eq) was added and mixed for 3 hours. And then MeOH (50 mL) was added and mixed for 30 min for capping. 20% piperidine in DMF was used for deblocking. Boc-Val-OH (32.5 g, 150 mmol, 3 eq) was coupled with 3 eq using HBTU (2.85 eq) and DIPEA (6.0 eq) in DMF (400 mL). The reaction was monitored by ninhydrin colour reaction test. After synthesis completion, the peptide resin was washed with DMF×3, MeOH×3, and then dried under N2 bubbling overnight. After that the peptide resin was treated with 20% HFIP/DCM for 30 min for 2 times. The solution was removed on a rotary evaporator to give the crude. The crude peptide was dissolved in ACN/H2O, then lyophilized twice to give the peptide product (17.3 g crude).
A solution of Compound 2 (4.00 g, 10.68 mmol, 1.00 eq) in DCM (40.00 mL) and MeOH (20.00 mL) was stirred at room temperature, then (4-aminophenyl)methanol (1.58 g, 12.82 mmol, 1.20 eq) and EEDQ (5.28 g, 21.37 mmol, 2.00 eq) were added and the mixture stirred in the dark for 9 hrs. TLC (dichloromethane/methanol=5/1, Rf=0.56) indicated one new spot had formed. The reaction mixture was concentrated under reduced pressure to remove solvent. The resulting residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0˜20% MeOH/DCM @ 80 mL/min). Compound 3 (3.00 g, 6.26 mmol, 58.57% yield) was obtained as a white solid.
To a solution of Compound 3 (3.00 g, 6.26 mmol, 1.00 eq) in anhydrous THF (35.00 mL) and anhydrous DCM (15.00 mL) was added (4-nitrophenyl) chloroformate (6.31 g, 31.30 mmol, 5.00 eq) and pyridine (2.48 g, 31.30 mmol, 2.53 mL, 5.00 eq), and the mixture was stirred at 25° C. for 5 hrs. TLC (dichloromethane/methanol=10/1, Rf=0.55) indicated a new spot had formed. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0˜10% DCM/MeOH© 80 mL/min). Compound 4 (2.00 g, 3.10 mmol, 49.56% yield) was obtained as a white solid.
A mixture of Compound 4 (278.43 mg, 387.80 μmol, 1.00 eq) and DIEA (501.19 mg, 3.88 mmol, 677.29 μL, 10.00 eq) in DMF (5.00 mL) was stirred under nitrogen for 10 min. MMAE (250.00 mg, 387.80 μmol, 1.00 eq) and HOBt (52.40 mg, 387.80 μmol, 1.00 eq) were added and the mixture was stirred at 0° C. under nitrogen for 20 min and stirred at 30° C. for additional 18 hrs. LC-MS showed one main peak with desired mass was detected. The resulting mixture was purified by flash C18 gel chromatography (ISCO®; 130 g SepaFlash® C18 Flash Column, Eluent of 0˜50% MeCN/H2O @ 75 mL/min). Compound 5 (190.00 mg, 155.29 μmol, 40.04% yield) was obtained as a white solid.
To a solution of Compound 5 (170.00 mg, 138.94 μmol, 1.00 eq) in DCM (2.70 mL) was added 2,2,2-trifluoroacetic acid (413.32 mg, 3.62 mmol, 268.39 μL, 26.09 eq), and the mixture was stirred at 25° C. for 1 hr. LC-MS showed Compound 5 was consumed completely. The mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in THF (10.00 mL) and was added K2CO3 (192.03 mg, 1.39 mmol, 10.00 eq), the mixture was stirred at room temperature for additional 3 hrs. LC-MS showed one main peak with desired mass was detected. The resulting reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by flash C18 gel chromatography (ISCO®; 130 g SepaFlash® C18 Flash Column, Eluent of 0-50% MeCN/H2O @ 75 mL/min). Compound 6 (110.00 mg, 97.92 μmol, 70.48% yield) was obtained as a white solid.
To a solution of Compound 6 (110.00 mg, 97.92 μmol, 1.00 eq) in DMA (5 mL), DIEA (25.31 mg, 195.83 μmol. 34.20 μL, 2.00 eq) and tetrahydropyran-2,6-dione (22.34 mg, 195.83 μmol, 2.00 eq). The mixture was stirred at room temperature for 18 hrs. LC-MS showed Compound 6 was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by flash C18 gel chromatography (ISCO®; 130 g SepaFlash® C18 Flash Column, Eluent of 0˜50% MeCN/H2O @ 75 mL/min). Compound 7 (100.00 mg, 80.81 μmol, 82.53% yield) was obtained as a white solid.
Compound 8 (MMAE-PABC-Cit-Val-Glutarate-NHS)
To a solution of Compound 7 (100.00 mg, 80.81 μmol, 1.00 eq) in DMA (4.5 mL) and DCM (1.5 mL) was added 1-hydroxypyrrolidine-2,5-dione (27.90 mg, 242.42 μmol, 3.00 eq) under N2, the mixture was stirred at 0° C. for 30 min. EDCI (46.47 mg, 242.43 μmol, 3.00 eq) was added in the mixture, and the mixture was stirred at 25° C. for additional 16 hrs. LC-MS showed Compound 7 was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by flash C18 gel chromatography (ISCO®; 130 g SepaFlash® C18 Flash Column, Eluent of 0˜50% MeCN/H2O @ 75 mL/min). Compound 8 (90.00 mg, 60.69 μmol, 75.11% yield) was obtained as a white solid.
General Procedure for Coupling MMAE-PABC-Cit-Val-Glutarate-NHS with Targeting Bicycles
To a solution of Bicycle (1.0-1.3 eq) in DMA was added DIEA (3 eq) and MMAE-PABC-Cit-Val-Glutarate-NHS (1 eq). The mixture was stirred at 25° C. for 18 hr. The reaction was monitored by LC-MS and once complete, was directly purified by preparative HPLC.
BCY6099 (71.5 mg, 22.48 μmol) was used as the bicycle reagent. Compound BCY6136 (40.9 mg, 9.05 μmol, 40.27% yield, 97.42% purity) was obtained as a white solid.
BCY6014 (70.00 mg, 22.47 μmol, 1.00 eq) was used as the bicycle reagent. Compound BCY6033 (33.90 mg, 7.96 μmol, 34.57% yield) was obtained as a white solid.
BCY6009 (70.0 mg, 22.47 μmol, 1 eq) was used as the bicycle reagent. Compound BCY6029 (32.9 mg, 7.75 μmol, 33.49% yield) was obtained as a white solid.
BCY6104 (71.59 mg, 22.48 μmol, 1.00 eq) was used as the bicycle reagent. Compound BCY6122 (38.30 mg, 8.57 μmol, 38.14% yield, 98.58% purity) was obtained as a white solid.
BCY6018 (72.40 mg, 26.97 μmol, 1.2 eq) was used as the bicycle reagent. Compound BCY6053 (38.3 mg, 9.81 μmol, 43.65% yield) was obtained as a white solid.
BCY6017 (50.75 mg, 22.48 μmol, 1.2 eq) was used as the bicycle reagent. Compound BCY6049 (22.5 mg, 6.47 μmol, 34.54% yield) was obtained as a white solid.
BCY6019 (65.00 mg, 22.47 μmol, 1.00 eq) was used as the bicycle reagent. Compound BCY6037 (26.80 mg, 6.66 μmol, 28.74% yield) was obtained as a white solid.
General Procedure for Preparation of 3
To a solution of compound 2 (4.00 g, 8.67 mmol, 1.00 eq), DIC (1.61 g, 12.78 mmol, 1.97 mL, 9.00 eq) and HOBt (10.54 g, 78.00 mmol, 9.00 eq) in DMF (30.00 mL) was added (4-aminophenyl)methanol (9.61 g, 78.00 mmol, 9.00 eq). The mixture was stirred at 15° C. for 1 hour. LC-MS showed compound 2 was consumed completely and one main peak with desired MS was detected. The mixture was purified by prep-HPLC. Compound 3 (4.20 g, 7.41 mmol, 85.49% yield) was obtained as a white solid.
General Procedure for Preparation of 4
To a solution of compound 3 (4.20 g, 6.30 mmol, 1.00 eq), DIPEA (1.09 g, 8.40 mmol, 1.47 mL, 7.00 eq) in DMF (30.00 mL) was added bis(4-nitrophenyl) carbonate (11.50 g, 37.79 mmol, 6.00 eq) in one part. The mixture was stirred at 0-15° C. for 1.5 hour. LC-MS showed compound 3 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (TFA condition). Compound 4 (2.00 g, 2.40 mmol, 38.16% yield) was obtained as a white solid.
General Procedure for Preparation of 5
To a solution of compound 4 (300.00 mg, 360.63 μmol, 1.00 eq), DIEA (93.22 mg, 721.27 μmol, 125.97 μL, 3.00 eq) in DMF (10.00 mL) was added MMAE (233.03 mg, 324.57 μmol, 0.90 eq) and HOBt (48.73 mg, 360.63 μmol, 1.00 eq) at 0° C. The mixture was stirred at 30° C. for 18 hour. LC-MS showed compound 4 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition). Compound 5 (250.00 mg, 190.75 μmol, 52.89% yield) was obtained as a yellow solid.
General Procedure for Preparation of 6
To a solution of compound 5 (240.00 mg, 183.12 μmol, 1.00 eq) in DCM (10.00 mL) was added TFA (1.54 g, 13.51 mmol, 1.00 mL, 73.76 eq). The mixture was stirred at 15° C. for 2 hour. And the mixture was concentrated under reduced pressure to remove solvent to give a residue, the residue was dissolved in THF and added K2CO3 and stirred at 15° C. for 2 h. LC-MS showed compound 5 was consumed completely and one main peak with desired MS was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (neutral condition). The crude product 6 (125.00 mg, 94.37 μmol, 51.53% yield, TFA) was used into the next step without further purification.
General Procedure for Preparation of 7
To a solution of compound 6 (125.00 mg, 94.37 μmol, 1.00 eq, TFA) in DMA (5.00 mL) was added DIEA (24.39 mg, 188.75 μmol, 32.96 μL, 2.00 eq), tetrahydropyran-2,6-dione (21.54 mg, 188.75 μmol, 2.00 eq). The mixture was stirred at 15° C. for 2 hour. LC-MS showed compound 6 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition). Compound 7 (100.00 mg, 75.49 μmol, 80.00% yield) was obtained as a white solid.
General Procedure for Preparation of 8 (MMAE-PABC-Cit-Trp-Glutarate-NHS)
To a solution of compound 7 (100.00 mg, 75.49 μmol, 1.00 eq), 1-hydroxypyrrolidine-2, 5-dione (26.07 mg, 226.48 μmol, 3.00 eq) in DMA (3.00 mL) and DCM (1.00 mL) was added EDCI (43.42 mg, 226.48 μmol, 3.00 eq). The mixture was stirred at 15° C. for 4 hour. LC-MS showed compound 7 was consumed completely and one main peak with desired MS was detected. The DCM was removed. Directly was purified by prep-HPLC (neutral condition). Compound 8 (60.00 mg, 42.20 μmol, 55.91% yield) was obtained as a white solid.
General Procedure for Coupling MMAE-PABC-Cit-Trp-Glutarate-NHS with Targeting Bicycles
To a solution of Bicycle (1.0-1.3 eq) in DMA was added DIEA (3 eq) and MMAE-PABC-Cit-Trp-Glutarate-NHS (1 eq). The mixture was stirred at 25° C. for 18 hr. The reaction was monitored by LC-MS and once complete, was directly purified by preparative HPLC.
BCY6009 (47.29 mg, 14.07 μmol, 1.00 eq) was used as the bicycle reagent. Compound BCY6030 (0.0156 g, 3.51 μmol, 24.93% yield, 97.41% purity) was obtained as a white solid.
BCY6014 (88.21 mg, 23.21 μmol, 1.10 eq) was used as the bicycle reagent. Compound BCY6034 (27.70 mg, 6.05 μmol, 28.70% yield, 95.02% purity) was obtained as a white solid.
BCY6017 (57.17 mg, 25.32 μmol, 1.2 eq) was used as the bicycle reagent. Compound BCY6050 (0.0519 g, 14.56 μmol, 69.01% yield) was obtained as a white solid.
BCY6018 (67.97 mg, 25.32 μmol, 1.2 eq) was used as the bicycle reagent. Compound BCY6054 (40.10 mg, 10.05 μmol, 47.62% yield) was obtained as a white solid.
BCY6019 (81.39 mg, 23.21 μmol, 1.10 eq) was used as the bicycle reagent. Compound BCY6038 (34.10 mg, 8.02 μmol, 38.00% yield, 96.68% purity) was obtained as a white solid.
General Procedure for Preparation of Compound 2
To a mixture of Compound 1 (3.00 g, 5.89 mmol, 1 eq) and (4-aminophenyl)methanol (869.93 mg, 7.06 mmol, 1.2 eq) in DCM (35 mL) and MeOH (18 mL) was added EEDQ (2.91 g, 11.77 mmol, 2 eq) in the dark under nitrogen, the mixture was stirred at 25° C. for 5 hr. LC-MS showed Compound 1 was consumed completely and one main peak with desired MS was detected. The resulting reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0˜20% MeOH/DCM @ 80 mL/min). Compound 2 (2.2 g, 3.58 mmol, 60.79% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 3
To a solution Compound 2 (500 mg, 813.31 μmol, 1 eq) in THF (10 mL) was added DIEA (630.69 mg, 4.88 mmol, 849.98 μL, 6 eq) at 0° C. under nitrogen with stirring for 30 mins. Then bis(4-nitrophenyl) carbonate (1.48 g, 4.88 mmol, 6 eq) was added thereto, the mixture was stirred at 25° C. under nitrogen for additional 21 hr. LC-MS showed one main peak with desired MS was detected. The resulting reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0˜20% MeOH/DCM @ 40 mL/min). Compound 3 (500 mg, 641.13 μmol, 78.83% yield) was obtained as a yellow solid.
General Procedure for Preparation of Compound 4
To a mixture of Compound 3 (500 mg, 512.90 μmol, 1.23 eq) in DMF (8 mL) was added DIEA (135.01 mg, 1.04 mmol, 181.95 μL, 2.5 eq) with stirring at 0° C. for 30 mins. Then MMAE (300 mg, 417.84 μmol, 1 eq) and HOBt (84.69 mg, 626.76 μmol, 1.5 eq) was added thereto, and the mixture was stirred at 40° C. for 15 hr. LC-MS showed compound 3 was consumed completely and one main peak with desired MS was detected. The residue was purified by flash C18 gel chromatography (ISCO®; 130 g SepaFlash® C18 Flash Column, Eluent of 0˜60% MeCN/H2O @ 75 mL/min). Compound 4 (330 mg, 242.87 μmol, 58.13% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 5
To a solution of Compound 4 (325 mg, 239.19 μmol, 1 eq) in DCM (18 mL) was added TFA (3.03 g, 26.60 mmol, 1.97 mL, 111.22 eq) at 0° C., the mixture was stirred at 25° C. for 2 hr. LC-MS showed compound 4 was consumed completely. Then the reaction mixture was concentrated under reduced pressure to give a residue, the residue was dissolved in THF (10 mL) and K2CO3 (661.16 mg, 4.78 mmol, 20 eq) was added thereto. The mixture was stirred at 25° C. for 15 hrs. LC-MS showed one main peak with desired MS was detected. The resulting reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash C18 gel chromatography (ISCO®; 130 g SepaFlash® C18 Flash Column, Eluent of 0˜60% MeCN/H2O @ 75 mL/min). Compound 5 (170 mg, 135.07 μmol, 56.47% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 6
A round bottle containing a solution of compound 5 (140 mg, 111.23 μmol, 1 eq) in DMA (5 mL) was purged using a nitrogen balloon and added DIEA (28.75 mg, 222.46 μmol, 38.75 μL, 2 eq) at 0° C. with stirring for 10 mins, tetrahydropyran-2,6-dione (25.38 mg, 222.46 μmol, 2 eq) was added as a solution in DMA. The mixture was stirred at 25° C. for 12 hr. LC-MS showed compound 5 was consumed completely and one main peak with desired MS was detected. The resulting reaction mixture was purified by flash C18 gel chromatography (ISCO®; 43 g SepaFlash® C18 Flash Column, Eluent of 0˜60% MeCN/H2O @ 40 mL/min). Compound 6 (120 mg, 87.42 μmol, 78.59% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 7 (MMAE-PABC-Lys(Dde)-Val-Glutarate-NHS)
To a solution of compound 6 (120 mg, 87.42 μmol, 1 eq) in DMA (9 mL) and DCM (3 mL) was added 1-hydroxypyrrolidine-2,5-dione (30.18 mg, 262.25 μmol, 3 eq) with stirring, and EDCI (50.27 mg, 262.25 μmol, 3 eq) was added thereto, the mixture was stirred at 0° C. for 30 mins and at 25° C. for additional 19 hr. LC-MS showed compound 6 was consumed completely and one main peak with desired MS was detected. The resulting reaction mixture was concentrated under reduced pressure to remove DCM. The mixture was purified by flash C18 gel chromatography (ISCO®; 43 g SepaFlash® C18 Flash Column, Eluent of 0˜60% MeCN/H2O @ 40 mL/min). Compound 7 (60 mg, 40.82 μmol, 46.70% yield) was obtained as a white solid.
General Procedure for Coupling MMAE-PABC-Lys(Dde)-Val-Glutarate-NHS with Targeting Bicycles
To a solution of Bicycle (1.0-1.3 eq) in DMA was added DIEA (3 eq) and MMAE-PABC-Lys(Dde)-Val-Glutarate-NHS (1 eq). The mixture was stirred at 25° C. for 18 hr. The reaction was monitored by LC-MS and once complete, was directly purified by preparative HPLC.
General Procedure for Dde Deprotection
To a solution of Dde protected peptide (1 eq) in DMF was added hydrazine hydrate (6500 eq), and the mixture was stirred at 25° C. for 1 hr. LC-MS was used to monitor the reaction, and once complete, the mixture was purified by preparative HPLC and the clean fractions lyophilised.
BCY6014 (124.12 mg, 40.82 μmol, 1.2 eq) was used as the bicycle reagent. Dde-BCY6038 (80 mg, 18.20 μmol, 53.51% yield) was obtained as a white solid.
Dde-BCY6061 (78 mg, 17.75 μmol) was deprotected using hydrazine according to the general procedure to give BCY6061 (47.1 mg, 11.13 μmol, 62.73% yield) as a white solid.
BCY6099 (389.77 mg, 122.47 μmol, 1.2 eq) was used as the bicycle reagent. Dde-BCY6174 (0.250 g, 55.10 μmol, 53.99% yield) was obtained as a white solid.
Dde-BCY6174 (0.250 g, 55.10 μmol, 1.0 eq) was deprotected using hydrazine according to the general procedure to give BCY6174 (0.1206 g, 27.45 μmol, 49.82% yield) as a white solid.
General Procedure for Preparation of Compound 3
To a solution of compound 1 (2 g, 4.33 mmol, 1.00 eq), DIC (4.92 g, 39.00 mmol, 6.00 mL, 9.00 eq), HOBt (5.27 g, 39.00 mmol, 9.00 eq) in DMF (30.00 mL) was added (4-aminophenyl)methanol (4.80 g, 39.00 mmol, 9.00 eq). The mixture was stirred at 15° C. for 1 hour. LC-MS showed compound 1 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition). Compound 2 (2 g, 3.53 mmol, 81.45% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 4
To a solution of compound 2 (2 g, 3.00 mmol, 1 eq), DIEA (2.71 g, 21.00 mmol, 3.66 mL, 7 eq) in DMF (20 mL) was added bis(4-nitrophenyl) carbonate (5.48 g, 18.00 mmol, 6 eq) in one part at 0° C. The mixture was stirred at 0-15° C. for 2 hr. LC-MS showed compound 2 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition). Compound 3 (0.9 g, 1.08 mmol, 36.07% yield) was obtained as a yellow solid.
General Procedure for Preparation of Compound 5
To a solution of compound 3 (350 mg, 420.74 μmol, 1.00 eq), HOBt (56.85 mg, 420.74 μmol, 1 eq) and DIEA (163.13 mg, 1.26 mmol, 219.86 μL, 3 eq) in DMF (10 mL) was added MMAE (302.08 mg, 420.74 μmol, 1 eq) at 0° C. The mixture was stirred at 40° C. for 18 hour. LC-MS showed compound 4 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition). Compound 4 (0.22 g, 155.95 μmol, 37.06% yield) was obtained as a yellow solid.
General Procedure for Preparation of Compound 6
To a solution of compound 4 (0.21 g, 148.86 μmol, 1 eq) in DCM (9 mL) was added TFA (1.54 g, 13.51 mmol, 1 mL, 90.73 eq). The mixture was stirred at 15° C. for 4 h, and concentrated under reduced pressure to give a residue, dissolved in THF, then added K2CO3(s) and stirred at 15° C. for 16 h. LC-MS showed compound 4 was consumed completely and one main peak with desired MS was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (neutral condition). Compound 5 (0.13 g, 102.02 μmol, 68.54% yield, 95% purity) was obtained as a white solid.
General Procedure for Preparation of Compound 7
To a solution of compound 5 (0.12 g, 99.13 μmol, 1 eq) in DMA (5 mL) was added DIEA (38.44 mg, 297.40 μmol, 51.80 μL, 3 eq) and tetrahydropyran-2,6-dione (22.62 mg, 198.26 μmol, 2 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed compound 5 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition). Compound 6 (0.09 g, 67.94 μmol, 68.54% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 8
To a solution of compound 6 (0.09 g, 67.95 μmol, 1 eq), HOSu (23.46 mg, 203.84 μmol, 3 eq) in DMA (6 mL) and DCM (2 mL) was added EDCI (39.08 mg, 203.84 μmol, 3 eq). The mixture was stirred at 15° C. for 16 h. LC-MS showed compound 6 was consumed completely and one main peak with desired MS was detected. DCM was removed and directly purified by prep-HPLC (neutral condition). Compound 7 (0.06 g, 40.09 μmol, 59.01% yield, 95% purity) was obtained as a white solid.
BCY6062
To a solution of BCY6014 (76.99 mg, 25.32 μmol, 1.2 eq) in DMA (5 mL) was added DIEA (8.18 mg, 63.31 μmol, 11.03 μL, 3 eq), compound 7 (0.03 g, 21.10 μmol, 1 eq). The mixture was stirred at 15° C. for 16 hr. The reaction was monitored by LC-MS and once complete, the mixture was purified by preparative HPLC. BCY6062 (0.0255 g, 5.70 μmol, 27.01% yield, 97.15% purity) was obtained as a white solid.
To a solution of 2-(2-pyridyldisulfanyl)pyridine (12.37 g, 56.18 mmol, 1.50 eq) in EtOH (100.00 mL) was added 4-sulfanylbutanoic acid (4.50 g, 37.45 mmol, 1.00 eq). The mixture was stirred at 15° C. for 18 hours under N2. LC-MS showed compound 1 was consumed completely and one main peak with desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by preparative HPLC (C18 360 g, neutral condition). Compound SPDB (1.9 g, 8.29 mmol, 22.12% yield) was obtained as a yellow solid.
1H NMR: ES6446-8-P1A 400 MHz CDCl3
δ ppm 1.98 (q, J=7.09 Hz, 2H), 2.45 (t, J=7.15 Hz, 2H), 2.79 (t, J=7.03 Hz, 2H), 7.03 (dd, J=7.15, 4.89 Hz, 1H), 7.19 (s, 1H), 7.56-7.65 (m, 2H), 8.41 (d, J=4.52 Hz, 1H).
A mixture of DM1 (250.00 mg, 338.62 μmol, 1.00 eq) and 4-(2-pyridyldisulfanyl)butanoic acid (100.95 mg, 440.21 μmol, 1.30 eq) was added under nitrogen in a 50 mL of flask with DMF (10.00 mL) purged with N2 for 30 mins. The mixture was stirred at room temperature for 1 hr. LC-MS showed that the DM1 was consumed completely and one main peak with desired mass was detected. The residue was purified by flash C18 gel chromatography (ISCO®; 120 g SepaFlash® C18 Flash Column, Eluent of 0-60% MeCN/H2O @ 85 mL/min). DM1-SPDB (120.00 mg, 140.11 μmol, 41.38% yield) was obtained as a white solid.
To a solution of DM1-SPDB (120.00 mg, 140.11 μmol, 1.00 eq) and 2,3,5,6-tetrafluorophenol (69.81 mg, 420.34 μmol, 3.00 eq) in DCM (1.00 mL) and DMA (3.00 mL) was added EDCI (80.58 mg, 420.34 μmol, 3.00 eq). The mixture was stirred at 15° C. for 4 hours. LC-MS showed DM1-SPDB was consumed completely and one main peak with desired mass was detected. The DCM was removed and the residue The mixture was directly purified by preparative HPLC (neutral condition). Compound DM1-SPDB-TFP (60.00 mg, 59.73 μmol, 42.63% yield) was obtained as a white solid.
General Procedure for Coupling DM1-SPDB-TFP with Targeting Bicycles
To a solution of targeting Bicycle (1.1-1.3 eq) in DMA was added DIEA (3 eq) and DM1-SPDB-TFP (1 eq). The mixture was stirred at 25° C. for 18 hr. The reaction was monitored by LC-MS and once complete, the mixture was directly purified by preparative HPLC.
BCY6099 (114.1 mg, 35.84 μmol) was used as the bicycle reagent. 22.4 mg Compound BCY6135 (5.30 μmol, 17.74% yield, 95.14% purity) was obtained as a white solid.
BCY6014 (121.07 mg, 39.82 μmol) was used as the bicycle reagent. 59.90 mg compound BCY6031 (14.67 μmol, 36.85% yield, 95.02% purity) was obtained as a white solid.
BCY6104 (95.11 mg, 29.87 μmol, 1 eq) was used as the bicycle reagent. BCY6134 (0.0232 g, 5.64 μmol, 18.89% yield, 97.82% purity) was obtained as a white solid.
BCY6009 (60.24 mg, 19.91 μmol, 1.00 eq) was used as the bicycle reagent. BCY6027 (20.40 mg, 5.11 μmol, 25.69% yield, 96.88% purity) was obtained as a white solid.
BCY6017 (61.81 mg, 27.38 μmol, 1.1 eq) was used as the bicycle reagent. BCY6047 (0.032 g, 10.34 μmol, 41.53% yield) was obtained as a white solid.
BCY6019 (115.22 mg, 32.86 μmol, 1.10 eq) was used as the bicycle reagent. BCY6035 (37.80 mg, 10.37 μmol, 34.73% yield) was obtained as a white solid.
BCY6018 (73.48 mg, 27.38 μmol, 1.1 eq) was used as the bicycle reagent. BCY6051 (0.0582 g, 16.52 μmol, 66.39% yield) was obtained as a white solid.
BCY6152 (93.17 mg, 29.87 μmol, 1 eq) was used as the bicycle reagent. BCY6154 (40.10 mg, 9.93 μmol, 33.27% yield, 98.06% purity) was obtained as a white solid.
BCY6153 (82.55 mg, 29.87 μmol, 1 eq) was used as the bicycle reagent. BCY6155 (0.0312 g, 8.55 μmol, 28.62% yield, 98.69% purity) was obtained as a white solid.
To a solution of 4-sulfanylbutanoic acid (2.0 g, 16.64 mmol, 1 eq) and 2-(2-pyridyldisulfanyl)pyridine (11.0 g, 49.93 mmol, 3 eq) in EtOH (50 mL) was added AcOH (1.05 g, 17.48 mmol, 1 mL, 1.05 eq). The mixture was stirred at 40° C. for 16 hr under N2. LC-MS showed one main peak with desired mass was detected and TLC indicated 4-sulfanylbutanoic acid was consumed completely. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by preparative HPLC (neutral condition). Compound 2 (2.0 g, 8.72 mmol, 52.4% yield) was obtained as yellow solid.
1H NMR: 400 MHz CDCl3
δ ppm 2.03-2.11 (m, 2H), 2.54 (t, J=7.20 Hz, 2H), 2.88 (t, J=7.20 Hz, 2H), 7.11-7.14 (m, 1H), 7.67-7.74 (m, 2H), 8.50 (d, J=4.80 Hz, 1H).
To a solution of compound 2 (0.5 g, 2.18 mmol, 1 eq) in DCE (5 mL) was added chlorosulfonic acid (1.5 g, 13.08 mmol, 0.89 mL, 6 eq) in three portions and DIEA (1.13 g, 8.72 mmol, 1.52 mL, 4 eq) in two portions. The mixture was stirred at 75° C. for 2 hr. LC-MS showed compound 2 was consumed completely and one main peak with desired mass was detected. The reaction mixture was quenched by addition 3 mL of H2O and the DCE was removed. The residue was The mixture was directly purified by preparative HPLC (neutral conditions). Compound 3 (0.68 g, 1.76 mmol, 80.6% yield, 80% purity) was obtained as light yellow oil.
1H NMR: 400 MHz CDCl3
δ ppm 2.49-2.54 (m, 2H), 3.63-3.67 (m, 2H), 3.90 (t, J=6.60 Hz, 2H), 7.09-7.12 (m, 1H), 7.66-7.76 (m, 2H), 8.47 (dd, J=4.80 Hz, 0.80 Hz, 1H), 8.56 (s, 1H).
To a solution of DM1 (1.0 g, 1.35 mmol, 1 eq) and compound 3 (502.9 mg, 1.63 mmol, 1.2 eq) in DMF (10 mL) was added NaHCO3(aq) until the pH reached 8. The mixture was stirred at 25° C. for 1 hr. LC-MS showed DM1 was consumed completely and one main peak with desired mass was detected. The residue was The mixture was directly purified by preparative HPLC (neutral condition). Compound DM1-SO3H-SPDB (0.28 g, 299.0 μmol, 22.1% yield) was obtained as a white solid.
To a solution of DM1-SO3H-SPDB (103.2 mg, 896.95 μmol, 3 eq), 1-Hydroxypyrrolidine-2,5-dione (103.2 mg, 896.95 μmol, 3 eq) in DMA (6 mL) and DCM (2 mL) was added EDCI (171.9 mg, 896.95 μmol, 3 eq). The mixture was stirred at 25° C. for 16 hr. LC-MS showed DM1-SO3H-SPDB was consumed completely and one main peak with desired mass was detected. DCM was removed. The residue was The mixture was directly purified by preparative HPLC (neutral condition). Compound DM1-SO3H-SPDB-NHS (0.22 g, 212.85 μmol, 71.2% yield) was obtained as a white solid.
General Procedure for Coupling DM1-SO3H-SPDB-NHS with Targeting Bicycles
To a solution of targeting Bicycle (1.1-1.3 eq) in DMA was added DIEA (3 eq) and DM1-SO3H-SPDB-NHS (1 eq). The mixture was stirred at 25° C. for 16 hr. The reaction was monitored by LC-MS and once complete, the mixture was directly purified by preparative HPLC.
BCY6099 (200.15 mg, 62.89 μmol) was used as the bicycle reagent. 57.1 mg compound BCY6173 (3.40 μmol, 22.79% yield, 95.80% purity) was obtained as a white solid.
BCY6014 (711.9 mg, 234.14 μmol) was used as the bicycle reagent. 308 mg compound BCY6082 (74.97 μmol, 35.2% yield, 96.36% purity) was obtained as a white solid.
BCY6018 (77.91 mg, 29.03 μmol, 1 eq) was used as the bicycle reagent. BCY6150 (0.0249 g, 6.61 μmol, 22.78% yield) was obtained as a white solid.
BCY6104 (120.17 mg, 37.73 μmol, 1.3 eq) was used as the bicycle reagent. BCY6151 (0.0256 g, 6.16 μmol, 21.22% yield) was obtained as a white solid.
BCY6138 (82.80 mg, 26.61 μmol, 1.1 eq) was used as the bicycle reagent. BCY6162 (0.0362 g, 8.98 μmol, 37.13% yield) was obtained as a white solid.
BCY6137 (79.67 mg, 24.48 μmol, 1.1 eq) was used as the bicycle reagent. BCY6161 (0.0232 g, 5.26 μmol, 21.76% yield) was obtained as a white solid.
To a solution of 2-(2-pyridyldisulfanyl)pyridine (2.46 g, 11.18 mmol, 1.50 eq) and AcOH (1.05 g, 17.49 mmol, 1.00 mL, 2.35 eq) in EtOH (50.00 mL) was added 4-sulfanylpentanoic acid (1.00 g, 7.45 mmol, 1.00 eq). The mixture was stirred at 40° C. for 18 hours under N2. LC-MS showed compound 1 was consumed completely and one main peak with desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by preparative HPLC (neutral condition). Compound SPP (1.61 g, 6.62 mmol, 88.81% yield) was obtained as a yellow solid.
1H NMR: 400 MHz DMSO-d6
δ ppm 1.36 (d, J=6.78 Hz, 3H), 1.88-2.07 (m, 2H), 2.56 (td, J=7.53, 1.76 Hz, 2H), 3.00-3.09 (m, 1H), 7.11 (ddd, J=7.34, 4.96, 1.00 Hz, 1H), 7.66 (td, J=7.78, 1.76 Hz, 1H), 7.73-7.77 (m, 1H), 8.48 (dt, J=4.02, 0.88 Hz, 1H).
A solution of DM1 (200 mg, 270.90 μmol, 1.00 eq), 4-(2-pyridyldisulfanyl)pentanoic acid (98.89 mg, 406.35 μmol, 1.50 eq) in H2O (5.00 mL) was adjusted PH=8 using NaHCO3 (aq). The mixture was stirred at 15° C. for 1 hour. LC-MS showed DM1 was consumed completely and one main peak with desired mass was detected (main MS was M+1-18). The mixture was directly purified by preparative HPLC (neutral condition). Compound DM1-SPP (120 mg, 137.86 μmol, 50.89% yield) was obtained as a white solid.
To a solution of DM1-SPP (0.175 g, 201.04 μmol, 1.0 eq), 2,3,5,6-tetrafluorophenol (100.16 mg, 603.13 μmol, 3.0 eq) in DCM (1.0 mL) and DMA (3.0 mL) was added EDCI (115.62 mg, 603.13 μmol, 3.0 eq). The mixture was stirred at 15° C. for 12 hour. LC-MS showed DM1-SPP was consumed completely and one main peak with desired MS was detected. The DCM was removed and the residue was purified by prep-HPLC (neutral condition). Compound DM1-SPP-TFP (0.123 g, 120.76 μmol, 60.07% yield) was obtained as a white solid.
General Procedure for Coupling DM1-SPP-TFP with Targeting Bicycles
To a solution of targeting Bicycle (1.1-1.3 eq) in DMA was added DIEA (3 eq) and DM1-SPP-TFP (1 eq). The mixture was stirred at 25° C. for 16 hr. The reaction was monitored by LC-MS and once complete, the mixture was directly purified by preparative HPLC.
To a solution of DM1-SPP (30.00 mg, 34.46 μmol, 1.00 eq) in DMF (5.00 mL) was added DIEA (13.36 mg, 103.38 μmol, 18.05 μL, 3.00 eq) and HATU (13.10 mg, 34.46 μmol, 1.00 eq). After 1 h, BCY6014 (104.79 mg, 34.46 μmol, 1.00 eq) was added and the mixture was stirred at 15° C. for 2 hours. LC-MS showed 40% of DM1-SPP was remained. Several new peaks were observed on LC-MS and 20% of desired compound was detected. The mixture was directly purified by preparative HPLC (TFA condition). Compound BCY6032 (10.00 mg, 2.57 μmol, 7.45% yield) was obtained as a white solid.
BCY6018 (86.96 mg, 32.40 μmol, 1.1 eq) was used as the bicycle reagent. BCY6052 (32.30 mg, 9.13 μmol, 31.01% yield) was obtained as a white solid.
BCY6017 (66.50 mg, 29.45 μmol, 1.2 eq) was used as the bicycle reagent. BCY6048 (40.80 mg, 13.12 μmol, 53.45% yield) was obtained as a white solid.
BCY6019 (113.60 mg, 32.40 μmol, 1.10 eq) was used as the bicycle reagent. BCY6036 (53.20 mg, 14.00 μmol, 47.54% yield, 96.26% purity) was obtained as a white solid.
BCY6009 (99.00 mg, 29.45 μmol, 1.00 eq) was used as the bicycle reagent. BCY6028 (24.30 mg, 6.05 μmol, 20.56% yield, 96.61% purity) was obtained as a white solid.
To a solution of 2-(2-pyridyldisulfanyl)pyridine (2.46 g, 11.18 mmol, 1.50 eq) and AcOH (1.05 g, 17.49 mmol, 1.00 mL, 2.35 eq) in EtOH (50.00 mL) was added 4-sulfanylpentanoic acid (1A) (1.00 g, 7.45 mmol, 1.00 eq). The mixture was stirred at 40° C. for 18 hours under N2. LC-MS showed 1A was consumed completely and one main peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by preparative HPLC (neutral condition). Compound 2A (1.61 g, 6.62 mmol, 88.81% yield) was obtained as a light yellow solid.
To a solution of 2A (0.01 g, 41.09 μmol, 1.00 eq), 1-hydroxypyrrolidine-2,5-dione (14.19 mg, 123.28 μmol, 3.00 eq) in DMA (1 mL) was added EDCI (23.63 mg, 123.28 μmol, 3.00 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed 2A was consumed completely and one main peak with desired mass was detected. The residue was purified by preparative HPLC (neutral condition). Compound 3A (0.011 g, 32.31 μmol, 78.63% yield) was obtained as a white solid.
To a solution of BCY6014 (98.25 mg, 32.31 μmol, 1.00 eq) in DMA (3 mL) was added DIEA (8.26 mg, 64.62 μmol, 11.26 μL, 2.00 eq) and 3A (0.011 g, 32.31 μmol, 1.00 eq). The mixture was stirred at 15° C. for 18 hr. LC-MS showed 3A was consumed completely and one main peak with desired mass was detected. The mixture was directly purified by preparative HPLC (neutral condition). Compound 4A (0.04 g, 12.25 μmol, 37.90% yield) was obtained as a white solid.
To a solution of 4A (0.04 g, 12.25 μmol, 1.00 eq) in MeCN (4 mL) and H2O (2 mL) was added TCEP (4.21 mg, 14.70 μmol, 4.05 μL, 1.20 eq). The mixture was stirred at 15° C. for 1 hr. LC-MS showed 4A was consumed completely and one main peak with the desired mass was detected. The residue was purified by preparative HPLC (neutral condition). Compound 5A (0.035 g, 11.09 μmol, 90.53% yield) was obtained as a white solid.
To a solution of 4-(2-pyridyldisulfanyl)pentanoic acid (2A) (22.46 mg, 92.29 μmol, 1.20 eq), HATU (35.09 mg, 92.29 μmol, 1.20 eq), DIEA (29.82 mg, 230.71 μmol, 40.19 μL, 3.00 eq) in DMF (5 mL) was added 1B (0.05 g, 76.90 μmol, 1.00 eq). The mixture was stirred at 15° C. for 1 hr. LC-MS showed 1B was consumed completely and one main peak with the desired mass was detected. The residue was purified by preparative HPLC (neutral condition). Compound DM3-SPy (0.025 q, 28.56 μmol, 37.13% yield) was obtained as a white solid.
A solution of DM3-SPy (0.015 g, 17.13 μmol, 1.00 eq) and 5A (54.08 mg, 17.13 μmol, 1.00 eq) in DMF (3 mL) was adjusted to pH=8 using NaHCO3(aq). The mixture was stirred at 15° C. for 1 hr. LC-MS showed DM3-SPy was consumed completely and one main peak with desired mass was detected. The mixture was directly purified by preparative HPLC (TFA condition). Compound BCY6039 (0.0263 g, 6.58 μmol, 38.39% yield) was obtained as a white solid.
To a solution of compound 1 (0.045 g, 126.96 μmol, 1 eq) in H2O (1 mL) was adjusted pH=13 using 1N NaOH solution. The mixture was stirred at 15° C. for 16 hr. LC-MS showed compound 1 was consumed completely and one main peak with the desired mass was detected. The residue was purified by preparative HPLC (neutral condition). Compound 2 (0.03 g, 116.56 μmol, 91.81% yield) was obtained as a yellow solid.
A solution of compound 2 (0.03, 116.56 μmol, 1.0 eq) and DM1 (111.87 mg, 151.53 μmol, 1.3 eq) in DMF (5 mL) was stirred at 15° C. for 2 hours. LC-MS showed DM1 was consumed completely and one main peak with desired mass was detected. The mixture was directly purified by preparative HPLC (NH4HCO3 condition). Compound 3 (0.05 g, 56.53 μmol, 48.50% yield) was obtained as a white solid.
To a solution of compound 3 (0.05 g, 56.53 μmol, 1.0 eq) and 2,3,5,6-tetrafluorophenol (28.16 mg, 169.59 μmol, 3.0 eq) in DMA (3 mL) and DCM (1 mL) was added EDCI (32.51 mg, 169.59 μmol, 3 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed compound 3 was consumed completely and one main peak with desired mass was detected. DCM was removed and the mixture was directly purified by preparative HPLC (neutral condition). Compound 4 (0.03 g, 29.05 μmol, 51.40% yield) was obtained as a white solid.
To a solution of BCY6014 (106.01 mg, 34.87 μmol, 1.2 eq) in DMA (3 mL) was added DIEA (11.27 mg, 87.16 μmol, 15.18 μL, 3.0 eq) and compound 4 (0.03 g, 29.05 μmol, 1.0 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed compound 4 was consumed completely and one main peak with desired mass was detected. The mixture was directly purified by preparative HPLC (TFA condition). Compound BCY6055 (0.0352 g, 9.01 μmol, 31.01% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 2
To a solution of compound 1 (0.1 g, 410.94 μmol, 1 eq) in 1,2-dichloroethane (3 mL) was added sulfurochloridic acid (0.86 g, 7.38 mmol, 491.43 μL, 17.96 eq) on three parts and DIEA (318.67 mg, 2.47 mmol, 429.47 μL, 6 eq) was added on two parts. The mixture was stirred at 75° C. for 16 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired MS was detected MS324, one main peak of byproduct MS 221 was PySSPy. The solvent was removed and dissolved in H2O/MeCN=15/1. Directly purified by prep-HPLC (neutral condition: MeCN/H2O). Compound 2 (0.055 g, 170.06 μmol, 41.38% yield) was obtained as a yellow oil.
General Procedure for Preparation of DM1-SO3H-SPP
To a solution of DM1 (113.00 mg, 153.06 μmol, 1.1 eq), compound 2 (0.045 g, 139.14 μmol, 1 eq) in DMF (2 mL) was adjusted PH=8 used for NaHCO3(aq). The mixture was stirred at 15° C. for 1 hr. LC-MS showed DM1 was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition). Compound DM1-SO3H-SPP (0.075 g, 78.90 μmol, 56.71% yield) was obtained as a white solid.
General Procedure for Preparation of DM1-SO3H-SPP—NHS
To a solution of DM1-SO3H-SPP (0.06 g, 63.12 μmol, 1 eq), 1-hydroxypyrrolidine-2, 5-dione (7.99 mg, 69.43 μmol, 1.1 eq) in DMA (1.5 mL) and DCM (0.5 mL) was added EDCI (13.31 mg, 69.43 μmol, 1.1 eq). The mixture was stirred at 15° C. for 18 hr. LC-MS showed DM1-SO3H-SPP was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (neutral condition: MeCN/H2O). Compound DM1-SO3H-SPP—NHS (0.045 g, 42.96 μmol, 68.05% yield) was obtained as a white solid.
General Procedure for Preparation of BCY6077
To a solution of BCY6014 (101.58 mg, 33.41 μmol, 1 eq) in DMA (1 mL) was added DIEA (12.95 mg, 100.23 μmol, 17.46 μL, 3 eq) and DM1-SO3H-SPP-TFP (0.035 g, 33.41 μmol, 1 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed DM1-SO3H-SPP-TFP was consumed completely and one main peak with desired MS was detected. Directly purified by prep-HPLC (TFA condition). Compound BCY6077 (41.30 mg, 10.03 μmol, 30.01% yield, 96.44% purity) was obtained as a white solid.
To a solution of MMAE (0.2 g, 278.56 μmol, 1.0 eq) in DMA (3 mL) was added DIEA (108.01 mg, 835.68 μmol, 145.56 μL, 3.0 eq) and tetrahydropyran-2,6-dione (63.57 mg, 557.12 μmol, 2.0 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed MMAE was consumed completely and one main peak with desired mass was detected. The mixture was The mixture was directly purified by preparative HPLC (neutral condition). Compound Glutarate-MMAE (0.12 g, 144.22 μmol, 51.77% yield) was obtained as a white solid.
To a solution of Glutarate-MMAE (0.12 g, 144.22 μmol, 1.0 eq), 1-hydroxypyrrolidine-2, 5-dione (49.79 mg, 432.65 μmol, 3.0 eq) in DMA (3 mL) and DCM (1 mL) was added EDCI (82.94 mg, 432.65 μmol, 3.0 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed Glutarate-MMAE was consumed completely and one main peak with desired mass was detected. The mixture was The mixture was directly purified by preparative HPLC (TFA condition). Compound Glutarate-MMAE-NHS (0.055 g, 59.19 μmol, 41.04% yield) was obtained as a white solid.
To a solution of BCY6014 (98.17 mg, 32.29 μmol, 1.2 eq) in DMA (2 mL) were added DIEA (10.43 mg, 80.72 μmol, 14.06 μL, 3 eq) and Glutarate-MMAE-NHS (0.025 g, 26.91 μmol, 1 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed Glutarate-MMAE-NHS was consumed completely and one main peak with desired mass was detected. The mixture was directly purified by preparative HPLC (TFA condition). Compound BCY6063 (32.10 mg, 8.33 μmol, 30.95% yield) was obtained as a white solid.
To a solution of DM1 (0.1 g, 135.45 μmol, 1 eq), 3-[(2-bromoacetyl)amino]propanoic acid (34.14 mg, 162.54 μmol, 1.2 eq) in DMF (5 mL) was added TEA (41.12 mg, 406.35 μmol, 56.56 μL, 3 eq). The mixture was stirred at 15° C. for 1 hr. LC-MS showed DM1 was consumed completely and one main peak with the desired mass was detected. The mixture was directly purified by preparative HPLC (neutral conditions). Compound 1 (0.08 g, 92.23 μmol, 68.09% yield) was obtained as a white solid.
To a solution of compound 1 (0.08 g, 92.23 μmol, 1 eq), 2,3,5,6-tetrafluorophenol (45.95 mg, 276.69 μmol, 3 eq) in DMA (3 mL) and DCM (1 mL) was added EDCI (53.04 mg, 276.69 μmol, 3 eq). The mixture was stirred at 15° C. for 4 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired mass was detected. The mixture was directly purified by preparative HPLC (neutral condition). Compound 2 (0.06 g, 59.09 μmol, 64.06% yield) was obtained as a white solid.
To a solution of BCY6014 (107.79 mg, 35.45 μmol, 1.2 eq) in DMA (3 mL) was added DIEA (11.45 mg, 88.63 μmol, 15.44 μL, 3.0 eq) and compound 2 (0.030 g, 29.54 μmol, 1 eq). The mixture was stirred at 15° C. for 16 hr. LC-MS showed compound 2 was consumed completely and one main peak with desired mass was detected. The mixture was directly purified by preparative HPLC (TFA condition). Compound BCY6064 (28.40 mg, 7.30 μmol, 24.71% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 2
To a solution of compound 1 (3.5 g, 5.68 mmol, 1.0 eq) in DCM (20 mL) and MeOH (10 mL), (4-aminophenyl)methanol (978.5 mg, 7.95 mmol, 1.4 eq) and EEDQ (2.81 g, 11.35 mmol, 2.0 eq) were added in the dark, and the mixture was stirred at 25° C. for 18 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired MS was detected ([M+H+]=722.0). The resulting reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 220 g SepaFlash® Silica Flash Column, Eluent of 0˜10% Methanol/Dichloromethane @ 80 mL/min). Compound 2 (3.0 g, 4.16 mmol, 73.2% yield) was obtained as a yellow solid.
General Procedure for Preparation of Compound 3
To a solution of compound 2 (2.5 g, 3.46 mmol, 1.0 eq) in THF (30 mL) was added DIEA (2.69 g, 20.78 mmol, 3.62 mL, 6.0 eq) and bis(4-nitrophenyl) carbonate (6.32 g, 20.78 mmol, 6.0 eq), and the mixture was stirred at 25° C. for 16 hr. TLC indicated compound 2 was consumed completely and one new spot formed. The reaction was clean according to TLC. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 220 g SepaFlash® Silica Flash Column, Eluent of 0˜5% Methanol/Dichloromethane @ 100 mL/min). Compound 3 (2.2 g, 2.48 mmol, 71.6% yield) was obtained as a yellow solid.
General Procedure for Preparation of Compound 4
To a solution of compound 3 (0.3 g, 338.24 umol, 1.0 eq) in DMF (5 mL), HOBt (50.3 mg, 372.06 umol, 1.1 eq), DIEA (131.1 mg, 1.01 mmol, 176.7 μL, 3.0 eq), and MMAE (218.6 mg, 304.42 umol, 0.9 eq) were added. The mixture was stirred at 40° C. for 16 hr. LC-MS showed one peak with desired MS ([M+H+]=1466.4, [M+2H+]/2=733.2). The reaction mixture was then directly purified by prep-HPLC (neutral condition), and compound 4 (0.2 g, 136.44 umol, 40.3% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 5
Compound 4 (0.175 g, 119.39 umol, 1.0 eq) was first dissolved in TFA (1.8 mL), and then triisopropylsilane (13.5 g, 85.20 mmol, 17.5 mL, 713.7 eq) was added. The mixture was stirred at 0° C. for 30 min. LC-MS showed one peak with desired MS ([M+H+]=1123.4, [M+2H+]/2=562.2). The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (neutral condition). Compound 5 (0.1 g, 89.02 umol, 74.6% yield) was obtained as a yellow solid.
General Procedure for Preparation of Compound 6
To a solution of compound 5 (0.07 g, 62.31 umol, 1.0 eq) in DMA (1.0 mL), DIEA (24.2 mg, 186.94 umol, 32.6 μL, 3.0 eq) and tetrahydropyran-2,6-dione (14.2 mg, 124.62 umol, 2.0 eq) were added. The mixture was stirred at 25° C. for 2 hr. LC-MS showed compound 5 was consumed completely and one main peak with desired MS was detected ([M+H+]=1237.4, [M+2H+]/2=619.3). The reaction mixture was then directly purified by prep-HPLC (neutral condition), and compound 6 (0.04 g, 32.32 umol, 51.8% yield) was obtained as a light yellow solid.
General Procedure for Preparation of Compound 7
To a solution of compound 6 (0.04 g, 32.32 umol, 1.0 eq), 1-hydroxypyrrolidine-2,5-dione (11.2 mg, 96.97 umol, 3.0 eq) in DMA (3.0 mL) and DCM (1.0 mL), EDCI (18.6 mg, 96.97 umol, 3.0 eq) was added. The mixture was stirred at 25° C. for 18 hr. LC-MS showed compound 6 was consumed completely and one main peak with desired MS was detected ([M+H+]=1334.5, [M+2H+]/2=667.7). The reaction mixture was then directly purified by prep-HPLC (TFA condition), and compound 7 (0.025 g, 18.73 umol, 57.9% yield) was obtained as a white solid.
General Procedure for Preparation of BCY6105
To a solution of BCY6014 (82.0 mg, 26.98 umol, 1.2 eq) in DMA (4 mL), DIEA (8.7 mg, 67.44 umol, 11.7 μL, 3.0 eq) and compound 7 (0.03 g, 22.48 umol, 1.0 eq) were added. The mixture was stirred at 25° C. for 18 hr. LC-MS showed compound 7 was consumed completely and one main peak with desired MS was detected ([M+4H+]/4=1065.2). The reaction mixture was then directly purified by prep-HPLC (TFA condition). Compound BCY6105 (0.024 g, 5.41 umol, 24.1% yield, 96.06% purity) was obtained as a white solid.
General Procedure for Preparation of Compound 2
To a solution of compound 1 (5.0 g, 10.67 mmol, 1.0 eq) in DCM (30 mL) and MeOH (10 mL), EEDQ (5.28 g, 21.34 mmol, 2.0 eq) and (4-aminophenyl)methanol (2.63 g, 21.34 mmol, 2.0 eq) were added. The mixture was stirred at 20° C. for 18 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired MS was detected (desired m/z=574, while Boc group falling off and partially falling off corresponded to m/z=474 and 518, respectively). The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by prep-HPLC (neutral condition). Compound 2 (3.7 g, 6.45 mmol, 60.4% yield) was obtained as a yellow solid.
General Procedure for Preparation of Compound 3
To a solution of compound 2 (3.4 g, 5.93 mmol, 1.0 eq) in DMF (20 mL) was added DIEA (5.36 g, 41.49 mmol, 7.23 mL, 7.0 eq) and bis(4-nitrophenyl) carbonate (10.82 g, 35.56 mmol, 6.0 eq) in one part. The mixture was stirred at 25° C. for 2 hr. LC-MS showed one peak with desired MS (m/z=639 corresponded to the mass with Boc group falling off during ESI). The reaction mixture was directly purified by prep-HPLC (neutral condition). Compound 3 (3.0 g, 4.06 mmol, 68.5% yield) was obtained as a yellow solid.
General Procedure for Preparation of Compound 4
To a solution of compound 3 (707.4 mg, 957.55 umol, 1.0 eq) in DMF (15 mL), HOBt (155.3 mg, 1.15 mmol, 1.2 eq), DIEA (371.3 mg, 2.87 mmol, 500.4 μL, 3.0 eq), and MMAE (0.55 g, 766.04 umol, 0.8 eq) were added. The mixture was stirred at 30° C. for 16 hr. LC-MS showed one peak with desired MS (desired m/z=1317, and m/z=609 corresponded to the mass with two protons and Boc group falling off during ESI). The reaction mixture was then directly purified by prep-HPLC (neutral condition). Compound 4 (0.53 g, 402.23 umol, 42.0% yield) was obtained as a yellow solid.
General Procedure for Preparation of Compound 5
To a solution of compound 4 (0.526 g, 399.20 umol, 1.0 eq) in DMF (4 mL), piperidine (862.2 mg, 10.13 mmol, 1.0 mL, 25.4 eq) was added. The mixture was stirred at 25° C. for 30 min. LC-MS showed compound 4 was consumed completely and one main peak with desired MS was detected (desired m/z=1095, and m/z=265 corresponded to Fmoc-piperidine adduct). The reaction mixture was then directly purified by prep-HPLC (neutral condition). Compound 5 (0.230 g, 209.97 umol, 52.6% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 6
To a solution of Fmoc-(D-Ala)-Phe-OH (125.6 mg, 273.87 umol, 1.2 eq) in DMF (10 mL), EDCI (52.5 mg, 273.87 umol, 1.2 eq), HOBt (37.0 mg, 273.87 umol, 1.2 eq), and compound 5 (0.25 g, 228.23 umol, 1 eq) were added. The mixture was stirred at 25° C. for 3 hr. LC-MS showed compound 5 was consumed completely and one peak with desired MS was detected (m/z=718 corresponded to the mass with two protons and Boc group falling off during ESI). The reaction mixture was then directly purified by prep-HPLC (neutral condition). Compound 6 (0.18 g, 117.20 umol, 51.3% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 7
To a solution of compound 6 (0.18 g, 117.20 umol, 1.0 eq) in DMF (8 mL), piperidine (1.72 g, 20.25 mmol, 2.0 mL, 172.8 eq) was added. The mixture was stirred at 25° C. for 1 hr. LC-MS showed compound 7 was consumed completely and one main peak with desired MS was detected (m/z=1314 and 657 corresponded to the desired mass, and m/z=265 corresponded to Fmoc-piperidine adduct). The reaction mixture was then directly purified by prep-HPLC (neutral condition). Compound 7 (0.13 g, 98.96 umol, 84.4% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 8
To a solution of compound 7 (0.105 g, 79.93 umol, 1.0 eq) in DMA (4 mL), DIEA (31.0 mg, 239.79 umol, 41.8 μL, 3.0 eq) and tetrahydropyran-2,6-dione (27.4 mg, 239.79 umol, 3.0 eq) were added. The mixture was stirred at 25° C. for 2 hr. LC-MS showed compound 7 was consumed completely and one main peak with desired MS was detected (m/z 664.5 corresponded to the mass with two protons and Boc group falling off during ESI). The reaction mixture was then directly purified by prep-HPLC (neutral condition), and compound 8 (0.09 g, 63.04 umol, 78.8% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 9
To a solution of compound 8 (0.09 g, 63.04 umol, 1.0 eq), 1-hydroxypyrrolidine-2,5-dione (21.7 mg, 189.11 umol, 3.0 eq) in DMA (3 mL) and DCM (1 mL), EDCI (36.2 mg, 189.11 umol, 3.0 eq) dissolved in 1 mL DCM was added. The mixture was stirred at 25° C. for 18 hr. LC-MS showed compound 8 was consumed completely and one main peak with desired MS was detected (desired m/z=1524 (one proton) and 763 (two protons), while m/z=713 corresponded to the mass with Boc group falling off during ESI). The reaction mixture was directly purified by prep-HPLC (neutral condition). Compound 9 (0.07 g, 45.91 umol, 72.8% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 10
To a solution of BCY6014 (167.5 mg, 55.09 umol, 1.2 eq) in DMF (3 mL), DIEA (11.8 mg, 91.81 umol, 16.0 μL, 2.0 eq) and compound 9 (0.07 g, 45.91 umol, 1.0 eq) were added. The mixture was stirred at 25° C. for 16 hr. LC-MS showed compound 9 was consumed completely and one main peak with desired MS was detected ([M+4H+]/4=1112.9, [M+5H+]/5=890.5). The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The crude product 10 (0.220 g, crude) was used in the next step without further purification.
General Procedure for Preparation of BCY6106
To a solution of compound 10 (0.200 g, 44.95 umol, 1.0 eq) in DCM (4 mL), 1 mL TFA was added. The mixture was stirred at 25° C. for 1 hr. LC-MS showed one main peak with desired MS ([M+4H+]/4=1088.0, [M+5H+]/5=870.8). The reaction mixture was concentrated under reduced pressure to give a residue, which was then directly purified by prep-HPLC (TFA condition). Compound BCY6106 (0.0297 g, 20.06 umol, 14.5% yield, 95.46% purity) was obtained as a white solid.
General Procedure for Preparation of Compound 9
The synthesis of Compound 9 was performed in an analogous to manner to that described in BCY6106.
General Procedure for Preparation of Compound 10A
To a solution of BCY6099 (195.15 mg, 61.32 μmol, 1.1 eq) in DMA (3 mL) were added DIEA (21.61 mg, 167.23 μmol, 29.13 μL, 3 eq) and compound 9 (0.085 g, 55.74 μmol, 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 μmol, 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 μmol, 31.13% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 2
To a solution of compound 1 (3.0 g, 8.49 mmol, 1.0 eq) in DCM (30 mL) and MeOH (10 mL), EEDQ (2.52 g, 10.19 mmol, 1.2 eq) and (4-aminophenyl)methanol (1.25 g, 10.19 mmol, 1.2 eq) were added. The mixture was stirred at 25° C. for 16 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired MS was detected ([M+H]+ 459.5). In addition, TLC indicated compound 1 was consumed completely and new spots formed. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0-60% Ethylacetate/Petroleum ether gradient @ 80 mL/min). Compound 2 (3.5 g, 7.63 mmol, 89.9% yield) was obtained as a yellow solid.
General Procedure for Preparation of Compound 3
To a solution of compound 2 (3.3 g, 7.20 mmol, 1.0 eq) in THF (100 mL), DIEA (4.65 g, 35.98 mmol, 6.27 mL, 5.0 eq) and bis(4-nitrophenyl) carbonate (8.76 g, 28.79 mmol, 4.0 eq) were added. The mixture was stirred at 25° C. for 16 hr. LC-MS showed compound 2 was consumed completely and one main peak with desired MS was detected ([M+H]+ 624.0). In addition, TLC indicated compound 2 was consumed completely and new spots formed. The reaction mixture was concentrated under reduced pressure to remove solvent to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 0-15% Ethylacetate/Petroleum ether gradient @ 80 mL/min). Compound 3 (3.0 g, 4.81 mmol, 66.8% yield) was obtained as a yellow solid.
General Procedure for Preparation of Compound 4
To a solution of compound 3 (124.09 mg, 198.97 umol, 1.0 eq) in DMF (5 mL), HOBt (32.3 mg, 238.77 umol, 1.2 eq), DIEA (77.1 mg, 596.92 μmol, 103.9 μL, 3.0 eq), and MMAE (0.1 g, 139.28 umol, 0.7 eq) were added. The mixture was stirred at 25° C. for 1 hr. LC-MS showed compound 3 was consumed completely and one main peak with desired MS was detected ([M+H]+ 1202.5, [M+Na]+1224.5). The reaction mixture was then directly purified by prep-HPLC (neutral condition). After lyophilization, compound 4 (0.08 g, 66.53 umol, 33.4% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 5
To a solution of compound 4 (0.08 g, 66.53 umol, 1.0 eq) in DMF (4 mL), piperidine (862.2 mg, 10.13 mmol, 1 mL, 152.2 eq) was added. The mixture was stirred at 25° C. for 1 hr. LC-MS showed compound 4 was consumed completely and one main peak with desired MS was detected ([M+H]+ 981.5, [M+Na]+1003.5, while m/z=264.0 corresponded to Fmoc-piperidine adduct). The reaction mixture was directly purified by prep-HPLC (neutral condition). Compound 5 (0.055 g, 56.11 umol, 84.3% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 6
To a solution of Fmoc-Glu(t-Bu)-Pro-Cit-Gly-HPhe-Tyr(t-Bu)-OH (74.1 mg, 66.31 umol, 1.3 eq) in DMF (4 mL), EDCI (12.7 mg, 66.31 umol, 1.3 eq), HOBt (8.9 mg, 66.31 umol, 1.3 eq), and compound 5 (0.05 g, 51.01 umol, 1.0 eq) were added. The mixture was stirred at 25° C. for 30 min. LC-MS indicated 20% of compound 5 remained, several new peaks formed, and 60% of the reaction mixture was desired product ([M+2H+]/2=1040.4). The reaction mixture was directly purified by prep-HPLC (neutral condition). Compound 6 (0.07 g, 33.66 umol, 66.0% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 7
To a solution of compound 6 (0.07 g, 33.66 umol, 1.0 eq) in DMF (4 mL), piperidine (2.9 mg, 33.66 umol, 3.3 μL, 1.0 eq) was added. The mixture was stirred at 25° C. for 15 min. LC-MS showed compound 6 was consumed completely and one main peak with desired MS was detected ([M+2H+]/2=929.1, while m/z=264.2 corresponded to Fmoc-piperidine adduct). The reaction mixture was directly purified by prep-HPLC (neutral condition). Compound 7 (0.045 g, 24.23 umol, 72.0% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 8
To a solution of compound 7 (0.04 g, 21.54 umol, 1.0 eq) in DMA (1 mL), DIEA (8.3 mg, 64.61 umol, 11.2 μL, 3.0 eq) and tetrahydropyran-2,6-dione (7.4 mg, 64.61 umol, 3.0 eq) were added. The mixture was stirred at 25° C. for 1 hr. LC-MS showed compound 7 was consumed completely and one main peak with desired MS was detected ([M+2H+]/2=986.4). The reaction mixture was then directly purified by prep-HPLC (neutral condition). Compound 8 (0.035 g, 17.75 umol, 82.4% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 9
To a solution of compound 8 (0.035 g, 17.75 umol, 1.0 eq), 1-hydroxypyrrolidine-2,5-dione (6.1 mg, 53.26 umol, 3.0 eq) in DMA (3 mL) and DCM (1 mL), EDCI (10.2 mg, 53.26 umol, 3.0 eq) was added. The mixture was stirred at 25° C. for 16 hr. LC-MS showed compound 8 was partially remained and one peak with desired MS was detected ([M+2H+]/2=1034.7). DCM was then removed, following by mixture being purified by prep-HPLC (neutral condition). Compound 9 (0.03 g, 14.50 umol, 81.7% yield) was obtained as a white solid.
General Procedure for Preparation of Compound 10
To a solution of BCY6014 (52.9 mg, 17.40 umol, 1.59 μL, 1.2 eq) in DMF (2 mL), DIEA (5.6 mg, 43.51 umol, 7.6 μL, 3.0 eq) and compound 9 (0.03 g, 14.50 umol, 1.0 eq) were added. The mixture was stirred at 25° C. for 16 hr. LC-MS showed one main peak with desired MS ([M+4H+]/4=1249.2, [M+5H+]/5=999.3). The solvent was then removed and the resulting crude product 10 (0.06 g, crude) was used into the next step without further purification.
General Procedure for Preparation of BCY6107
To a solution of compound 10 (0.055 g, 11.01 umol, 1.0 eq) in DCM (1 mL), 1 mL TFA was added. The mixture was stirred at 0° C. for 15 min. LC-MS showed compound 10 was consumed completely and one main peak with desired MS was detected ([M+4H+]/4=1221.0, [M+5H+]/5=977.0). The reaction mixture was concentrated under reduced pressure to remove solvent, resulting a residue which was then directly purified by prep-HPLC (TFA condition). Compound BCY6107 (20.4 mg, 4.03 umol, 36.6% yield, 96.36% purity) 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 2.5 nM in PBS with 0.01% tween 20 or 50 mM HEPES with 100 mM NaCl 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 25 μL in a black walled and bottomed low bind low volume 384 well plates, typically 5 μL assay buffer, 10 μL protein (Table 1) then 10 μL fluorescent peptide. One in two serial dilutions were used to give 12 different concentrations with top concentrations ranging from 500 nM for known high affinity binders to 10 μM 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 485 nm and detects parallel and perpendicular emission at 520 nm. 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){circumflex over ( )}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 FI-G-Sar5-ACPWGPAWCPVNRPGCA (FI-G-Sar5-(SEQ ID NO: 94)). Reference Compound B has the sequence FI-G-Sar5-ACPWGPFWCPVNRPGCA (FI-G-Sar5-(SEQ ID NO: 95)). Reference Compound C has the sequence FI-G-Sar5-ADVTCPWGPFWCPVNRPGCA (FI-G-Sar5-(SEQ ID NO: 96). 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 μL of diluted peptide was added to the plate followed by 10 μL of human or mouse EphA2 (Table 1) at a fixed concentration which was dependent on the fluorescent peptide used (Table 2), then 10 μL 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=y min+(y max−y min)/Lig*((Lig*((2*((Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflex over ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}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){circumflex over ( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflex over ( )}3){circumflex over ( )}0.5)))/3))−(Klig+Kcomp+Lig+Comp−Prot*c)))/((3*Klig)+((2*((Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflex over ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}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){circumflex over ( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflex over ( )}3){circumflex over ( )}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.
Certain peptide ligands of the invention were tested in the above mentioned assays and the results are shown in Tables 3-7:
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 μL of increasing (2 fold) concentrations of test compound was added to the plate followed by 10 μL of EphA2 protein (Table 8) at a fixed concentration which was dependent on the fluorescent peptide used (Table 9), then 10 μL 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 485 nm and detects parallel and perpendicular emission at 520 nm. 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=y min+(y max−y min)/Lig*((Lig*((2*((Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflex over ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}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){circumflex over ( )}2-3*(Kcomp*(Lig−Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)){circumflex over ( )}3){circumflex over ( )}0.5)))/3))−(Klig+Kcomp+Lig+Comp−Prot*c)))/((3*Klig)+((2*((Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflex over ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over ( )}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){circumflex over ( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)){circumflex over ( )}3){circumflex over ( )}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.
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 11:
The results from the competition binding assay in Table 10 show that Bicycle peptides targeting human EphA2 (BCY6014 and BCY6099) bind with high affinity to mouse and rat EphA2. Similarly, BCY6019 binds to both human and mouse EphA2. These results show that certain peptides of the invention can be used in in vivo mouse and rat efficacy and toxicology models.
Table 11 shows that certain Bicycle Drug Conjugates of the invention exhibit excellent cross reactivity between human, mouse and rodent EphA2. Peptides 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 4 mM sodium acetate, 100 mM NaCl, pH 5.4 with a 3× 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 NaCl, 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 μl/min. For capture of streptavidin, the protein was diluted to 0.2 mg/ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μl 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 0.2 μM 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 100 nM and 6 further 2-fold dilutions. The SPR analysis was run at 25° C. at a flow rate of 90 μl/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 100 nM. 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 H10500) was diluted to 20 μg/ml in 10 mM 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 5-10 μg/ml protein in PBS/0.05% Tween 20.
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 13 to 15:
Table 13 details binding affinities and kinetic parameters (Koff and Kon) for binding of selected Bicycle Drug Conjugates to human EphA2 determined using the SPR assay.
Table 14 illustrates binding results with four Bicycle Drug Conjugates (BCY6033, BCY6082, 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.
The results in Table 15 show that certain Bicycle Drug Conjugates of the invention (BCY6033, BCY6082, 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
(i) Animals and Housing Condition
Animals
Housing Condition
(ii) Test and Positive Control Articles
1Full details of MEDI-547 (a fully human monoclonal antibody 1C1 (recognizing both human and murine EphA2) conjugated to MMAF via an mc linker) are described in Jackson et al (2008) Cancer Res 68, 9367-74.
(b) Experimental Methods and Procedures
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 mm3 using the formula: V=0.5 a×b2 where a and b are the long and short diameters of the tumor, respectively. The tumor size was then used for calculations of T/C value. The T/C value (in percent) is an indication of antitumor effectiveness; T and C are the mean volumes of the treated and control groups, respectively, on a given day. TGI was calculated for each group using the formula: TGI (%)=[1−(Ti−T0)/(Vi−V0)]×100; Ti is the average tumor volume of a treatment group on a given day, T0 is the average tumor volume of the treatment group on the day of treatment start, Vi is the average tumor volume of the vehicle control group on the same day with Ti, and V0 is the average tumor volume of the vehicle group on the day of treatment start.
(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: In Vivo Efficacy in the LU-01-0046 PDX Model
Cancer cell lines (CCL) are originally derived from patient tumors, but acquire the ability to proliferate within in vitro cell cultures. As a result of in vitro manipulation, CCL that have been traditionally used in cancer research undergo genetic transformations that are not restored when cells are allowed to grow in vivo. Because of the cell culturing process cells that are better adapted to survive in culture are selected, tumor resident cells and proteins that interact with cancer cells are eliminated, and the culture becomes phenotypically homogeneous. Researchers are beginning to attribute the reason that only 5% of anti-cancer agents are approved by the Food and Drug Administration after pre-clinical testing to the lack of tumor heterogeneity and the absence of the human stromal microenvironment. Specifically, CCL-xenografts often are not predictive of the drug response in the primary tumors because CCL do not follow pathways of drug resistance or the effects of the microenvironment on drug response found in human primary tumors. To overcome these problems, the inventors have used PDX models to improve the predictive power of pre-clinical models.
PDX are created when cancerous tissue from a patient's primary tumor is implanted directly into an immunodeficient mouse. PDX can maintain patient histology, including the presence of non-tumor cells (eg stromal cells) and thus better mimic the tumor microenvironment. In general PDX are therefore more reflective of the heterogeneity and histology of primary tumors than CCL-xenografts.
BCY6031 was screened in a primary adenocarcinoma PDX xenograft (LU-01-0046) derived from a patient with non-small cell lung carcinomas (NSCLC). LU-01-0046 has been shown to express high levels of EphA2 using RNA sequencing. BCY6031 exhibited excellent efficacy in the LU-01-0046 model and is therefore a promising novel therapy for the treatment of non-small cell lung cancer.
(a) Treatment Arms
The experiment was designed to compare tumour growth in vehicle treated animals and animals treated with BCY6031 at 5 mg/kg qw for four weeks.
(b) Experimental Method
(i) PDX Information
(ii) Tumor Inoculation
Each mouse was inoculated subcutaneously in the right flank with an approximately 30 mm3 LU-01-0046 tumor fragment. Drug treatment was started when the average tumor volume reached 943 mm3. The test article, route of administration, dosing frequency and the animal numbers in each group are described above.
(iii) Testing Article Formulation Preparation
(c) Results
(i) Mortality, Morbidity, and Body Weight Gain or Loss
Animal body weight was monitored regularly as an indirect measure of toxicity. Body weight change in female Balb/C nude mice bearing LU-01-0046 tumor dosed with BCY6031 is shown in
(ii) Tumor Growth Curve
The tumor growth curve is shown in
(iii) Tumor Growth Inhibition Analysis
Tumor growth inhibition rate for BCY6031 in the PDX model LU-01-0046 was calculated based on tumor volume measurements on day 7 after the start of treatment.
aMean ± SEM.
bTumor 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) Discussion
The study evaluated the therapeutic efficacy of BCY6031 in the LU-01-0046 PDX model. The measured body weights are shown in the
The mean tumor size of vehicle treated mice reached 2191 mm3 on day 7. BCY6031 at 5 mg/kg produced potent antitumor activity with tumor measured as 463 mm3 (TGI=138.6%, p<0.05) by day 7. Furthermore, the BCY6031 treatment completely eradicated the tumors from day 32 and no tumour regrowth occurred following dosing suspension on day 28. BCY6031 gave rise to no significant body weight loss (
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 mm3. Tumour measurements and statistical analysis were performed as described above. Tumour bearing animals were treated once weekly with BCY6136 or vehicle.
(b) Discussion
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 (
The NCI-H1975 NSCLC Model:
Complete regression of tumour growth in the NCI-H1975 model was observed by around day 28 following 2 and 3 mg/kg once weekly dosing with BCY6136 (
The MDA-MB-231 Breast Model:
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 (
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 Fibrinogen 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 Cynomolgous Monkeys
Twenty eight day toxicology studies with BCY6136 we conducted in cynomolgous 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 20). No in-life bleeding events were reported and no evidence of internal bleeding was detected following pathology examination.
Study 7: In Vivo Efficacy Study of BCY6033 and 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 BCY6033 and 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
Each mouse will be inoculated subcutaneously at the right flank with PC-3 (10*106) tumor cells for tumor development. The animals will be randomized and treatment will be started when the average tumor volume reaches approximately 150 mm3. 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
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curve are shown in
(ii) Tumor Volume Trace
Mean tumor volume over time in female Balb/c nude mice bearing PC-3 xenograft is shown in Table 21.
(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.
aMean ± SEM.
(e) Results Summary and Discussion
In this study, the therapeutic efficacy of test articles in the PC-3 xenograft model was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the
The mean tumor size of vehicle treated mice reached 2070 mm3 on day 16. BCY6136 at 1 mg/kg, qw (TV=107 mm3, TGI=102.2%, p<0.001), BCY6136 at 2 mg/kg, qw (TV=79 mm3, TGI=103.6%, p<0.001) and BCY6136 at 3 mg/kg, qw (TV=70 mm3, TGI=104.1%, p<0.001) showed potent anti-tumor effect.
BCY6033 at 3 mg/kg, qw (TV=116 mm3, TGI=101.8%, p<0.001) and ADC at 3 mg/kg, qw (TV=124 mm3, TGI=101.4%, p<0.001) showed comparable 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 of BCY6136 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
aN, the number of animals in each group.
bAfter 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.
cDue 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
(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% 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 PC-3 tumor cells (10×106) in 0.2 ml of PBS for tumor development. 52 animals were randomized when the average tumor volume reached 454 mm3. The test article administration and the animal numbers in each group were shown in the experimental design table.
(iii) Testing Article Formulation Preparation
150 mM Acetate 10% sucrose pH 5 3.25 mM Histidine pH 5.5
(c) Results
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curve is shown in
(ii) Tumor Volume Trace
Mean tumor volume over time in male Balb/c nude mice bearing PC-3 xenograft is shown in Table 23.
(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 the start of the treatment.
aMean ± SEM.
bTumor 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 body weights and tumor volumes of all treatment groups at various time points are shown in the
The mean tumor size of vehicle treated mice reached 2364 mm3 on day 20. BCY6136 at 0.167 mg/kg, qw (TV=1188 mm3, TGI=61.4%, p<0.001), 0.5 mg/kg, q2w (TV=530 mm3, TGI=96.0%, p<0.001), 0.5 mg/kg, qw (TV=234 mm3, TGI=111.4%, p<0.001) and 1.5 mg/kg, qw (TV=131 mm3, TGI=117.2%, p<0.001) produced significant anti-tumor activity in dose or dose-frequency dependent manner on day 20. BCY6136 at 1.5 mg/kg, q2w (TV=128 mm3, TGI=117.0%, p<0.001) 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, 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 mm3, TGI=38.1%, p<0.001), 1 mg/kg, qw (TV=981 mm3, TGI=72.2%, p<0.001) and 3 mg/kg, qw (TV=184 mm3, 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 mm3, 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 BCY6033, BCY6136 and BCY6082 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 BCY6033, BCY6136 and BCY6082 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{circumflex over ( )}6) in 0.2 ml of PBS for tumor development. 36 animals were randomized when the average tumor volume reached 149 mm3. 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-D23, we fixed the tumors of Group 1 for FFPE.
On PG-D44, we fixed the tumors of Group 2 and 5 for FFPE.
At the end of study, we the tumors of Group 6 for FFPE.
(d) Results
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth are shown in
(ii) Tumor Volume Trace
Mean tumor volume over time in female Balb/c nude mice bearing NCI-H1975 xenograft is shown in Table 25 to 29.
(iii) Tumor Growth Inhibition Analysis
Tumor growth inhibition rate for BCY6033, BCY6136 and BCY6082 in the NCI-H1975 xenograft model was calculated based on tumor volume measurements at day 21 after the start of treatment.
aMean ± SEM.
bTumor 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 BCY6033, BCY6136 and BCY6082 in the NCI-H1975 xenograft model was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the
The mean tumor size of vehicle treated mice reached 2371 mm3 on day 21. BCY6033 at 1 mg/kg (TV=306 mm3, TGI=92.9%, p<0.001), 2 mg/kg (TV=95 mm3, TGI=102.5%, p<0.001) and 3 mg/kg (TV=29 mm3, TGI=105.4%, p<0.001) produced dose-dependent antitumor activity. BCY6033 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 didn't show obvious re-growth in following 5-6 weeks monitoring.
BCY6136 at 1 mg/kg (TV=205 mm3, TGI=97.5%, p<0.001), 2 mg/kg (TV=99 mm3, TGI=102.3%, p<0.001) and 3 mg/kg (TV=94 mm3, 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.
BCY6082 at 2 mg/kg (TV=1528 mm3, TGI=37.9%, p>0.05) didn't show obvious antitumor activity, BCY6082 at 5 mg/kg (TV=390 mm3, TGI=89.1%, p<0.001) produced significant antitumor activity.
In this study, one mouse treated with BCY60333 mg/kg lost over 15% bodyweight during the monitoring, other 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
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 mm3) for tumor development. The treatment was started when the average tumor volume reached 174 mm3 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
1Acetate buffer: 50 mM Acetate 10% sucrose pH 5
2ADC buffer: 20 mM Histidine pH 5.5
(d) Results
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curve are shown in
(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 31.
(iii) Tumor 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.
aMean ± SEM;
bTumor 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 body weight and tumor volume of all treatment groups at various time points are shown in the
In this study, the mean tumor volume of vehicle treated mice reached 2208 mm3 on day 28 after the start of treatment. BCY6136 at 1 mg/kg, qw (TV=499 mm3, TGI=84.0%, p<0.001), 2 mg/kg, qw (TV=32 mm3, TGI=107.0%, p<0.001) and 3 mg/kg, qw (TV=0 mm3, TGI=108.6%, p<0.001) produced dose-dependent anti-tumor activity. ADC at 3 mg/kg, qw (TV=39 mm3, 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
(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 mm3) for tumor development. The treatment was started when the average tumor volume reached 960 mm3 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
1His-buffer: 25 mM Histidine 10% sucrose pH 7
2ADC-buffer: 20 mM Histidine pH 5.5
(iii) 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) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curve are shown in
(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 33.
(iii) Tumor 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.
aMean ± SEM;
bTumor 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 body weight and tumor volume of all treatment groups at various time points are shown in the
In this study, the treatment was started when the average tumor volume reached 960 mm3.
On day 17 after the start of treatment, the mean tumor volume of vehicle treated mice reached 2301 mm3. BCY6136 at 1 mg/kg qw (TV=1672 mm3, TGI=47.0%, p>0.05) didn't show obvious antitumor activity; BCY6136 at 2 mg/kg qw (TV=398 mm3, TGI=142.1%, p<0.001) and 3 mg/kg qw (TV=216 mm3, 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 mm3, 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 BCY6033, BCY6136, BCY6082 and BCY6031 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 BCY6033, BCY6136, BCY6082 and BCY6031 in large LU-01-0046 PDX tumors 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 mm3) for tumor development. The treatment was started when the average tumor volume reaches 955 mm3 for BT17BDCs study and 1039 mm3 for BCYs study. The test article administration and the animal numbers in each group are shown in the experimental design table.
(ii) Testing Article Formulation Preparation
1Acetate buffer: 50 mM Acetate 10% sucrose pH 5
2Dissolve 0.419 g His. hydrochloride in 100 ml water, use 1M HCl adjust PH to 5.5
(d) Results
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curve are shown in
(ii) Tumor Volume Trace
Mean tumor volume over time in female Balb/c nude mice bearing LU-01-0046 is shown in Table 35.
(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.
aMean ± SEM;
bTumor 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).
(e) Results Summary and Discussion
In this study, the therapeutic efficacy of test articles in large LU-01-0046 tumors was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the
In BCYs study, the mean tumor size of vehicle treated mice was calculated as 6186 mm3 on day 22. BCY6082, BCY6033, BCY6136 at 1 mg/kg and ADC at 3 mg/kg didn't show obvious anti-tumor activity when starting treatment from tumor size of 1000 mm3.
BCY6082 (TV=1999 mm3, TGI=81.2%, p<0.01), BCY6033 (TV=88 mm3, TGI=118.6%, p<0.001), BCY6136 (TV=233 mm3, TGI=115.6%, p<0.001) and BCY6031 (TV=115 mm3, TGI=110.2%, p<0.001) at 3 mg/kg produced significant anti-tumor antitumor activity. Among them, BCY6033 and 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
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
(c) Experimental Methods and Procedures
(i) Tumor Inoculation
Each mouse was inoculated subcutaneously at the right flank with certain kind of tumor fragment (˜30 mm3) for tumor development. The treatments were started when the average tumor volume reached approximately 198 mm3. The test article administration and the animal numbers in each group are shown in the experimental design table.
(ii) Testing Article Formulation Preparation
(iii) Sample Collection
Groups were terminated when average tumor volume reached over 2000 mm3 and tumors were harvested for FFPE after the last measurement: Group 1 on PG-D14, group 5 on PG-D18, group 2 & 6 on PG-D21 and group 3 & 4 on PG-D31.
(d) Results
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curve are shown in
(ii) Tumor Volume Trace
Mean tumor volume over time in female Balb/c nude mice bearing LU-01-0046 NSCLC PDX model is shown in Table 37.
(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.
aMean ± SEM.
bTumor 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).
cTGI was calculated for each group using the formula: TGI (%) = [1 − (Ti − T0)/(Vi − V0)] × 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 body weights and tumor volumes of all treatment groups at various time points were shown in the
The mean tumor size of vehicle treated mice reached 2307 mm3 on PG-D14. BCY6136 at 1 mg/kg (TV=1058 mm3, TGI=59.1%, p<0.05), at 2 mg/kg (TV=264 mm3, TGI=97.0%, p<0.001) and at 3 mg/kg (TV=26 mm3, 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).
In this study, all of the group's animals maintained the body weight well.
Study 14: In Vivo Efficacy Study of BCY6033, BCY6136, BCY6082, BCY6173, BCY6175 and BCY6031 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 mm3) for tumor development. The treatment was started when the average tumor volume reaches 200 mm3 for part 1 study and 192 mm3 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
1Acetate buffer: 50 mM Acetate 10% sucrose pH 5
(d) Results
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curve are shown in
(ii) 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 39 and 40.
(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 21 after the start of the treatment.
aMean ± SEM;
bTumor 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).
aMean ± SEM;
bTumor 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 body weights and tumor volumes of all treatment groups at various time points are shown in the
In part 1 study, the mean tumor size of vehicle treated mice reached 2551 mm3 on day 21 after the start of treatment.
BCY6033 at ½ mg/kg, qw (TV=1285 mm3, TGI=53.9%, p<0.001) and BCY6136 at ½ mg/kg, qw (TV=1285 mm3, TGI=53.9%, p<0.001) produced significant anti-tumor activity, but didn't exhibit any tumor regression. BCY6033 at 3 mg/kg, qw (TV=0 mm3, TGI=108.6%, p<0.001) and BCY6136 at 3 mg/kg, qw (TV=0 mm3, TGI=108.5%, p<0.001) completely eradicated the tumors, 1 of 5 tumors respectively in BCY6033 and BCY6136 3 mg/kg groups showed regrowth after the dosing suspension and the tumors were resistant to BCY6033 or BICY6136 treatment when resuming the dosing. The remaining tumors in the BCY6033 and BCY6136 groups (4/5 for each group) showed no regrowth after 80 days of dosing suspension. BCY6082 at 1 mg/kg, qw (TV=1826 mm3, TGI=30.8%, p<0.05) and 3 mg/kg, qw (TV=1042 mm3, TGI=64.2%, p<0.001) produced dose-dependent anti-tumor activity, but didn't show tumor regression.
In part 2 study, the mean tumor size of vehicle treated mice reached 2342 mm3 on day 21 after the start of treatment. BCY6173 at 1 mg/kg, qw (TV=2151 mm3, TGI=8.9%, p>0.05) did not show anti-tumor antitumor activity. BCY6173 at 3 mg/kg, qw (TV=890 mm3, TGI=67.5%, p<0.05) produced obvious anti-tumor activity.
BCY6175 at 3 mg/kg, qw (TV=0 mm3, TGI=108.9%, p<0.001) completely eradicated 4/5 tumors on day 14. BCY6031 at 3 mg/kg, qw (TV=874 mm3, TGI=68.2%, p<0.05) produced obvious anti-tumor activity, but didn't show any tumor regression.
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
(c) Experimental Methods and Procedures
(i) Tumor Inoculation
Each mouse was inoculated subcutaneously at the right flank with LU-01-0412 tumor fragment (˜30 mm3) for tumor development. Animals were randomized when the average tumor volume reached 159 mm3. The test article administration and the animal numbers in each group were shown in the experimental design table.
(ii) Testing Article Formulation Preparation
(iii) 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
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curves are shown in
(ii) Tumor Volume Trace
Mean tumor volume over time in female BALB/c nude mice bearing LU-01-0412 xenograft is shown in Table 43.
(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.
aMean ± SEM;
bTumor 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 body weight and tumor volume of all treatment groups at various time points are shown in
The mean tumor volume of vehicle treated mice reached 1104 mm3 on day 32 after the start of treatment. BCY6136 at 1 mg/kg, qw*4 (TV=758 mm3, TGI=36.7%, p<0.05) and 3 mg/kg, qw*4 (TV=416 mm3, 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 mm3, TGI=116.8%, p<0.001) and BCY8781 at 3 mg/kg, qw*4 (TV=12 mm3, 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.
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
(i) Tumor Inoculation
Each mouse was inoculated subcutaneously at the right flank with LU-01-0486 of tumor fragment (˜30 mm3) for tumor development. The treatment was started when the average tumor volume reached 180 mm3 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
1Acetate buffer: 50 mM Acetate 10% sucrose pH 5
(d) Results
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curve are shown in
(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 45.
(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.
aMean ± SEM;
bTumor 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 body weight and tumor volume of all treatment groups at various time points are shown in the
In this study, the mean tumor volume of vehicle treated mice reached 651 mm3 on day 14 after the start of treatment. BCY6136 at 1 mg/kg, qw (TV=638 mm3, TGI=3.0%, p>0.05) and 2 mg/kg, qw (TV=645 mm3, TGI=1.2%, p>0.05) didn't show any anti-tumor activity. BCY6136 at 3 mg/kg, qw (TV=449 mm3, TGI=43.1%, p>0.05) produced slight anti-tumor activity without statistical significance.
Study 17: In Vivo Efficacy Test of BCY6033, BCY6136 and BCY6082 in Treatment of MDA-MB-231-Luc Xenograft in Balb/c Nude Mice
(a) Study Objective
The objective of the research was to evaluate the in vivo anti-tumor efficacy of BCY6033, BCY6136 and BCY6082 in treatment of MDA-MB-231-luc 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 MDA-MB-231-luc tumor cells (10×10{circumflex over ( )}6) in 0.1 ml of PBS with 0.1 ml matrigel for tumor development. 36 animals were randomized when the average tumor volume reached 159 mm3. 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-D24, we collected and fixed the tumors of Group 1, 8 and 9 for FFPE.
On PG-D33, we collected and fixed the tumors of Group 2 and 5 for FFPE.
At the end of study, we collected and fixed the tumors of Group 3, 4, 6 and 7 for FFPE.
(d) Results
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth are shown in
(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 47 to 49.
(iii) Tumor Growth Inhibition Analysis
Tumor growth inhibition rate for BCY6033, BCY6136 and BCY6082 in the MDA-MB-231-luc xenograft model was calculated based on tumor volume measurements at day 21 after the start of treatment.
aMean ± SEM.
bTumor 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 BCY6033, BCY6136 and BCY6082 in the MDA-MB-231-luc xenograft model was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the
The mean tumor size of vehicle treated mice reached 1661 mm3 on day 21. BCY6033 at 1 mg/kg (TV=880 mm3, TGI=52.0%, p<0.001), 2 mg/kg (TV=67 mm3, TGI=106.1%, p<0.001) and 3 mg/kg (TV=22 mm3, TGI=109.1%, p<0.001) produced dose-dependent antitumor activity. BCY6033 at 2 mg/kg and 3 mg/kg regressed the tumors potently, but the tumors showed obvious re-growth from day 21.
BCY6136 at 1 mg/kg (TV=994 mm3, TGI=44.4%, p<0.01) showed moderate antitumor activity, BCY6136 at 2 mg/kg (TV=33 mm3, TGI=108.4%, p<0.001) and 3 mg/kg (TV=132 mm3, TGI=101.1%, p<0.001) produced potent antitumor activity, but the tumors showed obvious re-growth from day 28.
BCY6082 at 2 mg/kg (TV=1287 mm3, TGI=24.9%, p>0.05) didn't show obvious antitumor activity, BCY6082 at 5 mg/kg (TV=218 mm3, TGI=96.1%, p<0.001) produced significant antitumor activity.
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 BALB/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
aThe injection volume of each mouse is 10 ml/kg.
bThe 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.
(ii) Tumor Inoculation
Each mouse was inoculated subcutaneously at the right flank with EMT-6 tumor cells (5×106) in 0.1 ml of PBS for tumor development. 44 animals were randomized when the average tumor volume reached 75 mm3. 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
3 tumors from spare mice were collected for FACS on day 11. The data was supplied by biology team.
(d) Results
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curve are shown in
(ii) Tumor Volume Trace
Mean tumor volume over time in female BALB/c mice bearing EMT-6 syngeneic is shown in Table 51.
The dosage of group 3 and group 4 was changed to 5 mpk and 3 mpk from Day 14.
(iii) Tumor 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.
aMean ± SEM.
bTumor 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).
cThe 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 body weights and tumor volumes of all treatment groups at various time points are shown in the
The mean tumor size of vehicle treated mice reached 1499 mm3 on day 21. BCY6136 at 3 mg/kg, qw (TV=561 mm3, TGI=66.2%, p<0.05) showed obvious antitumor activity. BCY6136 at ⅕ mg/kg, qw (TV=1272 mm3, TGI=16.1%, p>0.05) and BCY6136 at 0.3/3 mg/kg, qw (TV=1394 mm3, 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.
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.
(ii) Tumor Inoculation
Each mouse was inoculated subcutaneously at the right flank with NCI-N87 tumor cells (10×106) 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 mm3. The test article administration and the animal number in each group are shown in the experimental design table.
(iii) Testing Article Formulation Preparation
1Acetate buffer: 50 mM Acetate 10% sucrose pH 5
(d) Results
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curve is shown in
(ii) Tumor Volume Trace
Mean tumor volume over time in female Balb/c nude mice bearing NCI-N87 xenograft is shown in Table 53.
(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.
aMean ± SEM.
bTumor 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 body weight and tumor volume of all treatment groups at various time points are shown in the
The mean tumor size of vehicle treated mice reached 1465 mm3 on day 30. BCY6136 at 1 mg/kg, qw (TV=425 mm3, TGI=80.7%, p<0.001) and 2 mg/kg, qw (TV=210 mm3, TGI=97.4%, p<0.001) produced significant antitumor activity in a dose-dependent manner, BCY6136 at 3 mg/kg, qw (TV=201 mm3, 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
(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 (10×106) 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 mm3. The test article administration and the animal number in each group are shown in the experimental design table.
(iii) Testing Article Formulation Preparation
1Acetate buffer: 50 mM Acetate 10% sucrose pH 5
2ADC buffer: 25 mM Histidine 10% sucrose pH 5.5
(d) Results
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curve is shown in
(ii) Tumor Volume Trace
Mean tumor volume over time in female Balb/c nude mice bearing SK-OV-3 xenograft is shown in Table 55.
(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.
aMean ± SEM.
bTumor 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 SK-OV-3 model was evaluated. The measured body weight and tumor volume of all treatment groups at various time points are shown in the
The mean tumor size of vehicle treated mice reached 1560 mm3 on day 28. ADC at 3 mg/kg, qw (TV=684 mm3, TGI=63.8%, p<0.01) showed moderate anti-tumor efficacy. BCY6136 at 1 mg/kg, qw (TV=1035 mm3, TGI=38.1%, p>0.05) didn't show obvious anti-tumor activity. BCY6136 at 2 mg/kg, qw (TV=277 mm3, TGI=93.3%, p<0.001) and 3 mg/kg, qw (TV=254 mm3, TGI=95.0%, 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 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
(i) 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% 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 OE21 tumor cells (5×106) 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 mm3. The test article administration and the animal number in each group are shown in the experimental design table.
(iii) Testing Article Formulation Preparation
1Acetate buffer: 50 mM Acetate 10% sucrose pH 5
(d) Results
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curve is shown in
(ii) Tumor Volume Trace
Mean tumor volume over time in female Balb/c nude mice bearing OE21 xenograft is shown in Table 57.
(iii) Tumor Growth Inhibition Analysis
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.
aMean ± SEM.
bTumor 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 body weight and tumor volume of all treatment groups at various time points are shown in the
The mean tumor size of vehicle treated mice reached 1586 mm3 on day 23. BCY6136 at 1 mg/kg, qw (TV=1155 mm3, TGI=30.4% p<0.05) showed slight anti-tumor activity. BCY6136 at 2 mg/kg, qw (TV=537 mm3, TGI=73.4%, p<0.001) and 3 mg/kg, qw (TV=489 mm3, 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 and BCY6082 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 and BCY6082 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 (10×106) in 0.2 ml PBS with 50% matrigel for tumor development. 36 animals were randomized when the average tumor volume reached 141 mm3. 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) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curve are shown in
(ii) Tumor Volume Trace
Mean tumor volume over time in female CB17-SCID mice bearing MOLP-8 xenograft is shown in Table 59.
(iii) Tumor Growth Inhibition Analysis
Tumor growth inhibition rate for BCY6136 and BCY6082 in the MOLP-8 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment.
aMean ± SEM.
bTumor 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 BCY6082 in the MOLP-8 xenograft model was evaluated. The measured body weights and tumor volumes of all treatment groups at various time points are shown in the
The mean tumor size of vehicle treated mice reached 2528 mm3 on day 14. BCY6136 at 1 mg/kg (TV=1146 mm3, TGI=45.5%, p>0.05), 2 mg/kg (TV=1218 mm3, TGI=54.9%, p<0.05) and 3 mg/kg (TV=938 mm3, TGI=66.7%, p<0.01) produced dose-dependent antitumor activity, but all of dosage didn't regress the tumors in MOLP-8 xenografts.
BCY6082 at 1 mg/kg (TV=2296 mm3, TGI=9.8%, p>0.05) and 2 mg/kg (TV=1554 mm3, TGI=40.8%, p>0.05) didn't show obvious anti-tumor activity. BCY6082 at 3 mg/kg inhibited the tumor growth significantly (TV=1321 mm3, TGI=50.6%, p<0.05), but 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
(c) Experimental Methods and Procedures
(i) 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% 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
Each mouse will be inoculated subcutaneously at the right flank with HT1080 tumor cells (5*106) for tumor development. The animals will be randomized and treatment will be started when the average tumor volume reaches approximately 150-200 mm3. 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
(i) Body Weight change and Tumor Growth Curve
Body weight and tumor growth curve are shown in
(ii) Tumor Volume Trace
Mean tumor volume over time in female Balb/c nude mice bearing HT1080 xenograft is shown in Table 61.
(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.
aMean ± SEM.
bTumor 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 BCYs in the HT1080 xenograft model was evaluated.
The measured body weights and tumor volumes of all treatment groups at various time points are shown in the
The mean tumor size of vehicle treated mice reached 1737 mm3 on day 14.
BCY6082 at 2 mg/kg, qw (TV=5 mm3, TGI=111.1%, p<0.01) and BCY6031 at 2 mg/kg qw (TV=7 mm3, TGI=106.8%, p<0.01) showed potent anti-tumor activity.
BCY6173 at 1 mg/kg, qw (TV=1074 mm3, TGI=42.5%, p>0.05), 2 mg/kg, qw (TV=480 mm3, TGI=80.6%, p<0.05) and 3 mg/kg, qw (TV=7 mm3, TGI=108.4%, p<0.01) produced dose-dependent antitumor activity.
BCY6135 at 1 mg/kg, qw (TV=871 mm3, TGI=55.5%, p<0.01), 2 mg/kg, qw (TV=62 mm3, TGI=107.5%, p<0.001) and 3 mg/kg, qw (TV=44 mm3, TGI=108.6%, p<0.001) produced dose-dependent antitumor activity.
BCY6033 at 3 mg/kg, qw (TV=9 mm3, TGI=110.8%, p<0.001) and 5 mg/kg, qw (TV=0 mm3, TGI=111.4%, p<0.001) showed potent anti-tumor activity, and completely eradicated the tumors by day 14 at 5 mg/kg.
BCY6136 at 2 mg/kg, qw (TV=345 mm3, TGI=95.7%, p<0.001), 3 mg/kg, qw (TV=3 mm3, TGI=111.2%, p<0.001) and 5 mg/kg, qw (TV=2 mm3, TGI=111.3%, p<0.001) showed potent anti-tumor activity.
BCY6174 at 1 mg/kg, qw (TV=1066 mm3, TGI=43.1%, p<0.05), 2 mg/kg, qw (TV=532 mm3, TGI=77.3%, p<0.01) and 3 mg/kg, qw (TV=11 mm3, TGI=110.7%, p<0.001) produced dose-dependent antitumor activity.
BCY6175 at 1 mg/kg, qw (TV=769 mm3, TGI=62.1%, p<0.01), 2 mg/kg, qw (TV=295 mm3, TGI=92.5%, p<0.001) and 3 mg/kg, qw (TV=11 mm3, TGI=110.8%, p<0.001) produced dose-dependent antitumor activity.
ADC at 3 mg/kg, qw (TV=0 mm3, TGI=111.5%) completely eradicated the tumors by day 14.
Study 24: Investigation of Association Between Copy Number Variation (CNV) and Gene Expression for EphA2 from Multiple Tumour Types
Methods
1. Select all studies in cBioPortal (cbioportal.org) and search for EPHA2.
2. Export CNV and RNA expression data from cBioPortal.
3. Test if CNVs are statistically significantly associated with changes in mRNA expression for EphA2 (log 2 not applied).
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).
Results
The results are shown in Table 64 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:
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 TOGA 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 (TOGA, 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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1721259 | Dec 2017 | GB | national |
| 1804102 | Mar 2018 | GB | national |
| 1818603 | Nov 2018 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2018/053678 | 12/19/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2019/122863 | 6/27/2019 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 8685890 | Winter et al. | Apr 2014 | B2 |
| 9868767 | Pei et al. | Jan 2018 | B2 |
| 10919937 | Beswick et al. | Feb 2021 | B2 |
| 11306123 | Mudd et al. | Apr 2022 | B2 |
| 11312749 | Mudd et al. | Apr 2022 | B2 |
| 11332500 | Mudd et al. | May 2022 | B2 |
| 20170190743 | Pei et al. | Jul 2017 | A1 |
| 20190184025 | Chen et al. | Jun 2019 | A1 |
| 20190307836 | Keen et al. | Oct 2019 | A1 |
| 20190389906 | Beswick | Dec 2019 | A1 |
| 20200338203 | Chen et al. | Oct 2020 | A1 |
| 20210040154 | Mudd et al. | Feb 2021 | A1 |
| 20210069287 | Mudd et al. | Mar 2021 | A1 |
| 20210101937 | Mudd et al. | Apr 2021 | A1 |
| 20210147484 | Beswick et al. | May 2021 | A1 |
| 20210261620 | Teufel et al. | Aug 2021 | A1 |
| 20210299210 | Keen et al. | Sep 2021 | A2 |
| 20220184222 | Bennett et al. | Jun 2022 | A1 |
| Number | Date | Country |
|---|---|---|
| 2009098450 | Aug 2009 | WO |
| 2016067035 | May 2016 | WO |
| WO-2016174103 | Nov 2016 | WO |
| 2017191460 | Nov 2017 | WO |
| 2018115203 | Jun 2018 | WO |
| 2019122860 | Jun 2019 | WO |
| 2019122861 | Jun 2019 | WO |
| 2019122863 | Jun 2019 | WO |
| 2019193328 | Oct 2019 | WO |
| 2019243313 | Dec 2019 | WO |
| 2020084305 | Apr 2020 | WO |
| 2020201753 | Oct 2020 | WO |
| 2021019243 | Feb 2021 | WO |
| 2021019245 | Feb 2021 | WO |
| 2021064428 | Apr 2021 | WO |
| 2021105694 | Jun 2021 | WO |
| 2021250418 | Dec 2021 | WO |
| Entry |
|---|
| Merritt et al., Analysis of EphA2 expression and mutant p53 in ovarian cancer, Cancer Biology and Therapy, pp. 1357-1360; 2006 (Year: 2006). |
| Center for Diseases, “What Can I Do to Reduce My Risk of Ovarian Cancer”, 2021 (Year: 2021). |
| Bennett et al., “Abstract 5855: Bicycle Drug Conjugates targeting EphA2 for the treatment of solid tumors: Discovery and selection of BT5528,” Cancer Research. 2018 <<http://cancerres.aacrjournals.org/content/78/13_Supplement/5855>>. |
| PCT International Search Report and Written Opinion for PCT/GB2018/05367 dated Mar. 20, 2018. |
| Bennett et al., “Abstract 5855: Bicycle Drug Conjugates Targeting EphA2 for the Treatment of Solid Tumors: Discovery and Selection of BT5528,” Cancer Research, 2018, 4 Pages. |
| Annunziata et al., “Phase 1, open-label study of MEDI-547 in patients with relapsed or refractory solid tumors,” Invest. New. Drugs. 2013;31(1):77-84. |
| Bennett et al., “Development of BT1718, a Bicycle Drug Conjugate (BDC) targeting MT1-MMP for treatment of solid tumours,” European Journal of Cancer. 2016;69(1):S21. |
| Bennett, “BT5528, an EphA2-Targeting Bicycle Toxin Conjugate (BTC): Profound Efficacy Without Bleeding and Coagulation Abnormalities in Animal Models,” Association for Cancer Research Annual Meeting, 2019, 11 Pages. |
| Chen et al., “Peptide Ligands Stabilized by Small Molecules,” Angewandte Chemie International Edition. 2014;56(6):1602-1606. |
| Deonarain et al., “Small-Format Drug Conjugates: A Viable Alternative to ADCs for Solid Tumours?” Antibodies (Basel). 2018;7(2):16. |
| Deyle et al., “Phage Selection of Cyclic Peptides for Application in Research and Drug Development,” Accounts of Chemical Research, 2017, vol. 50(8), pp. 1866-1874. |
| Harrison et al., “Abstract 5144: BT1718, a novel bicyclic peptide-maytansinoid conjugate targeting MT1-MMP for the treatment of solid tumors: Design of bicyclic peptide and linker selection,” Cancer Res. 2017;77(13):5144. |
| Heinis et al., “Phage-encoded combinatorial chemical libraries based on bicyclic peptides,” Nat Chem Biol. 2009;5(7):502-7. |
| Mitra et al., “Structure-Activity Relationship Analysis of Peptides Targeting the EphA2 Receptor,” Biochemistry. 2010;49(31):6687-95. |
| Mudd et al., “Identification and Optimization of EphA2-Selective Biccyles for the Delivery of Cytotoxic Payloads,” J Med Chem. 2020; 63(8) 4107-4116. |
| PCT International Search Report and Written Opinion for PCT/GB2018/053678 dated Mar. 20, 2019. |
| PCT International Search Report for PCT Application No. PCT/EP2019/065993, mailed by the European Patent Office dated Sep. 24, 2019, 5 Pages. |
| PCT International Search Report for PCT Application No. PCT/GB2020/051829, mailed by the European Patent Office dated Oct. 30, 2020, 5 Pages. |
| Shi et al., “One-Bead-Two-Compound Thioether Bridged Macrocyclic [gamma]-AApeptide Screening Library Against EphA2,” J. Med. Chem. 2017;60(22):9290-9298. |
| U.S. Appl. No. 16/771,186, filed Jun. 9, 2020. |
| U.S. Appl. No. 17/590,875, filed Feb. 2, 2022. |
| U.S. Appl. No. 17/592,966, filed Feb. 4, 2022. |
| U.S. Appl. No. 17/630,314, filed Jan. 26, 2022. |
| U.S. Appl. No. 17/630,747, filed Jan. 27, 2022. |
| U.S. Appl. No. 17/655,822, filed Mar. 22, 2022. |
| U.S. Appl. No. 17/663,169, filed May 12, 2022. |
| U.S. Appl. No. 17/779,226, filed May 24, 2022. |
| Wu et al., “Design and Characterization of Novel EphA2 Agonists for Targeted Delivery of Chemotherapy to Cancer Cells,” Chem. Biol. 2015;22(7):876-887. |
| Number | Date | Country | |
|---|---|---|---|
| 20220289792 A1 | Sep 2022 | US |
