Conjugate and use thereof

Abstract

To provide a new conjugate usable for photoimmunotherapy.SOLUTION: The conjugate of the present disclosure comprises an epidermal growth factor receptor (EGFR) peptide ligand and a photosensitive dye, wherein said photosensitive dye is conjugated to said EGFR peptide ligand via a linker, wherein the backbone length of said linker is between 14 and 55 atoms.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present disclosure relates to conjugates and uses thereof. [Background technology]

[0002] Photoimmunotherapy is a novel cancer treatment method. In photoimmunotherapy, a conjugate consisting of an antibody against a protein expressed in cancer cells and the photosensitive dye IRDye 700DX (IR700) is used as a therapeutic agent. When the conjugate is administered to a subject, it binds to the protein expressed in the cancer cells via the antibody. When irradiated with light at a wavelength of approximately 690 nm, the absorption wavelength of IR700, the IR700 is activated, damaging and killing the cancer cells. Therefore, IR700-antibody conjugates that can be used in photoimmunotherapy have been developed (Non-Patent Documents 1 and 2). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Kobayashi H, et al., “Near-infrared photoimmunotherapy of cancer.”, Acc Chem Res. 52,2332-2339 (2019). [Non-patent document 2] Maruoka Y, et al., “Near infrared photoimmunotherapy for cancers: A translational perspective”, EBioMedicine. 70,103501 (2021). Summary of the Invention [Problem to be solved by the invention]

[0004] However, the development of antibodies against such proteins, particularly antibodies that can be used as pharmaceuticals, requires time and costs.

[0005] Therefore, an object of the present disclosure is to provide a new conjugate that can be used in photoimmunotherapy. [Means for solving the problem]

[0006] To achieve the above object, the conjugate of the present disclosure comprises an epidermal growth factor receptor (EGFR) peptide ligand and a photosensitive dye, The photosensitive dye is conjugated to the EGFR peptide ligand.

[0007] The pharmaceutical compositions of the present disclosure include the conjugates of the present disclosure. [Effects of the Invention]

[0008] According to the present disclosure, a new conjugate that can be used in photoimmunotherapy can be provided. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows the binding of Compound 2 or Compound 6 to cells in Reference Example 1, where (A) is a phase contrast image and a fluorescence microscope image, and (B) is a graph showing the fluorescence intensity. [Figure 2] FIG. 2 is a micrograph showing the morphological changes of EGFR-positive cells caused by Compound 9 in Reference Example 1. [Figure 3] FIG. 3 is a graph showing the binding of Compound 9 (Comparative Example Compound) or Compound 12 (Example Compound) in Example 1 to EGFR-positive cells. [Figure 4] FIG. 4 is a microscopic photograph showing the morphological changes of cells caused by Compound 12 in Example 1. [Figure 5] FIG. 5 is a graph showing the cell viability in Example 1. [Figure 6]FIG. 6 is a graph showing the concentration-dependent cell viability of Compound 12 upon near-infrared light irradiation in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0010] <Definition> As used herein, "epidermal growth factor receptor" (EGFR) refers to a tyrosine kinase receptor that recognizes epidermal growth factor (EGF) and belongs to the ErbB family. EGFR is also called HER1 or ErbB1. EGFR derived from various animals can be found, for example, in information registered in existing databases. Specific examples of human EGFR include a protein consisting of the following amino acid sequence (SEQ ID NO: 1), registered in UniProt under accession number P00533. Furthermore, examples of mouse EGFR include a protein consisting of the following amino acid sequence (SEQ ID NO: 2), registered under Q01279.

[0011] Human EGFR (SEQ ID NO: 1)

[0012] Mouse EGFR (SEQ ID NO: 2)

[0013] As used herein, "epidermal growth factor" (EGF) is a ligand that binds to EGFR and is a protein known to induce cell proliferation and differentiation. For EGF derived from various animals, information registered in existing databases can be referenced, for example. Specific examples of human EGF include a protein consisting of the following amino acid sequence (SEQ ID NO: 3), registered in UniProt under accession number P01133. Furthermore, examples of mouse EGF include a protein consisting of the following amino acid sequence (SEQ ID NO: 4), registered under P01132. In the EGF, the region that binds to EGFR is, for example, a region containing amino acids 971 to 1013 in the amino acid sequence of SEQ ID NO: 3.

[0014] Human EGF (SEQ ID NO: 3)

[0015] Mouse EGF (SEQ ID NO: 4)

[0016] As used herein, a "cyclic peptide" is a peptide having a cyclic portion.

[0017] As used herein, "ligand" refers to a molecule that specifically binds to a target receptor.

[0018] As used herein, the term "photosensitive dye" refers to a compound that undergoes a chemical change in response to light.

[0019] As used herein, "phthalocyanine" or "phthalocyanine dye" refers to a compound containing a phthalocyanine ring. The phthalocyanine is an azaporphyrin (C) containing four phthalimide rings connected by nitrogen bridges in a 16-membered ring of alternating carbon and nitrogen atoms. 32 H 16 N8), a compound known to form stable chelates at its core with metal and non-metal cations.

[0020] As used herein, "conjugate" means one or more other molecules chemically linked, directly or indirectly.

[0021] As used herein, the term "pharmaceutical composition" refers to a preparation prepared to effectively exert the biological activity of the active ingredient contained in the composition.

[0022] As used herein, "positive" (+) means that a higher signal is detected by an analytical method such as flow cytometry, which utilizes an antigen-antibody reaction, compared to a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen. As used herein, "negative" (-) means that a signal equivalent to or lower than a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen is detected.

[0023] As used herein, the term "antibody" refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Examples of the antibody include polyclonal antibodies and monoclonal antibodies. Examples of the antibody isotype include IgG (e.g., IgG1, IgG2, IgG3, IgG4, etc.), IgM, IgA (e.g., IgA1, IgA2, etc.), IgE, IgD, IgY, etc. Examples of the origin of the antibody include antibodies derived from animals such as mammals such as mouse, rat, hamster, rabbit, goat, cow, horse, camel, and alpaca; birds such as chicken and ostrich; and cartilaginous fish such as shark. The antibody may be, for example, a camelid-derived heavy chain antibody (VHH antibody), a cartilaginous fish-derived immunoglobulin new antigen receptor (IgNAR), an antibody fragment (e.g., Fab, Fab', F(ab')2, single domain antibody (nanobody), etc.), a recombinant antibody (e.g., scFv, disulfide-linked Fv (dsFv), diabody, minibody, etc.). The antibody may also be an antibody-like molecule (e.g., affibody, anticalin, DARPins, monobody, etc.) produced by molecular biological techniques such as phage display and / or by protein engineering techniques using existing protein motifs.

[0024] As used herein, the term "subject" refers to an animal or a cell, tissue, or organ derived from an animal, and particularly includes humans. The term "animal" refers to both humans and non-human animals. Examples of non-human animals include mammals such as mice, rats, hamsters, rabbits, goats, cows, horses, dogs, cats, pigs, monkeys, dolphins, and sea lions.

[0025] As used herein, "treatment" means therapeutic treatment and / or prophylactic treatment. As used herein, "treatment" means treating, curing, preventing, suppressing, ameliorating, or improving a disease, pathology, or disorder, or halting, inhibiting, reducing, or delaying the progression of a disease, pathology, or disorder. As used herein, "prevention" means reducing the likelihood of developing a disease or pathology, or delaying the onset of a disease or pathology. The "treatment" may be, for example, treatment of a patient who develops a target disease, or treatment of an animal model of the target disease.

[0026] As used herein, "peptide" refers to a polymer composed of several unmodified (naturally occurring), modified, and / or artificial amino acids.

[0027] As used herein, "protein" or "polypeptide" refers to a polymer composed of unmodified (naturally occurring), modified, and / or artificial amino acids. The protein or polypeptide is a peptide having a length of more than 10 amino acids.

[0028] In this specification, "optionally substituted" means that the group in question is either unsubstituted or substituted.

[0029] As used herein, "isolated" or "purified" means identified and separated and / or recovered from components in their natural state. The "isolation" or "purification" can be achieved, for example, by obtaining at least one purification step.

[0030] As used herein, the term "kit" generally refers to a unit in which the components to be provided (e.g., conjugate, cover, instruction manual, etc.) are provided separately in two or more compartments. The kit can be suitably used to provide a composition that is not provided in a mixed state, but is preferably mixed immediately before use, for reasons of stability, etc. The kit preferably includes, for example, instructions or instructions on how to use the components to be provided (e.g., other components such as reagents, culture media, additives, etc.), or instructions or instructions describing how to treat the components. As used herein, when used as a pharmaceutical composition kit, the kit may also include instructions, etc., describing how to use the pharmaceutical composition and other components, etc.

[0031] As used herein, "instructions" or "manuals" refer to written instructions to a technician or other user on how to use the present disclosure. The instructions, for example, describe instructions on how to use the conjugate or pharmaceutical composition of the present disclosure. The instructions may be a package insert and are usually provided in paper form, but are not limited thereto, and may also be provided in the form of, for example, electronic media (e.g., a homepage provided on the Internet, e-mail).

[0032] Sequence information for the proteins described herein or the nucleic acids (e.g., DNA or RNA) encoding them is available from Protein Data Bank, UniProt, GenBank, etc. RNA nucleic acid sequences can also be obtained from the corresponding DNA base sequences using appropriate sequence conversion software, etc.

[0033] The present disclosure will be described below using examples, but the present disclosure is not limited to the following examples and can be implemented with any modifications. Furthermore, the descriptions in this disclosure are mutually applicable unless otherwise specified. In this specification, the expression "to" is used to include the numerical or physical values ​​before and after it. In this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0034] <Conjugate> In one aspect, the present disclosure provides a new conjugate that can be used in photoimmunotherapy. The conjugate of the present disclosure comprises an epidermal growth factor receptor (EGFR) peptide ligand and a photosensitive dye, wherein the photosensitive dye is conjugated to the EGFR peptide ligand. The conjugate of the present disclosure can be used in photoimmunotherapy.

[0035] The present inventors conceived the idea that photoimmunotherapy, in which a conjugate of an antibody against a target and a photosensitive dye is administered to a subject and then irradiated with light to activate the photosensitive dye and damage cells expressing the target bound by the antibody, could be used for subjects other than the antibody by using a ligand for the target instead of the antibody. As a result of further research, the present inventors discovered that using a ligand for the target EGFR and a photosensitive dye can damage cells such as EGFR-positive cancer cells. In particular, for EGFR peptide ligands, linking the photosensitive dye to the EGFR peptide ligand via a linker of a certain length or longer can effectively damage cells, thereby establishing the present disclosure. The conjugates of the present disclosure can be used to suitably perform photoimmunotherapy, for example, targeting EGFR-positive cancer.

[0036] The EGFR peptide ligand is a peptide that binds to EGFR, and is preferably a cyclic peptide. The number of amino acids constituting the EGFR peptide ligand is, for example, 5 to 30 amino acids, 5 to 25 amino acids, or 5 to 20 amino acids. The number of amino acids is, for example, the number of amino acids per peptide unit (one molecule) that binds to EGFR.

[0037] Examples of the EGFR peptide ligand include the EGFR-binding peptides (SEQ ID NOs: 5 to 8) described in Japanese Patent Application Laid-Open No. 2014-005235 and the following References 1 to 4. Reference 1: Li Z, et.al., “Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics. FASEB J. 2005;19:1978-85. Reference 2: Song S et.al., “Novel peptide ligand directs liposomes toward EGF-R high-expressing cancer cells in vitro and in vivo.”, FASEB J. 2009;23:1396-404. Reference 3: Xue EY et.al., “Synthesis and biological evaluation of an epidermal growth factor receptor-targeted peptide-conjugated phthalocyanine-based photosensitiser.”, RSC Adv. 2019;9:20652-20662. Reference 4: Kim MH et.al., “A novel dual-labeled small peptide as a multimodal imaging agent for targeting wild-type EGFR in tumors.”, PLoS One. 2022;17:e0263474. Sequence number 5: YHWYGYTPQNVI (Reference 1) SEQ ID NO: 6: LARLLT (Reference 2) SEQ ID NO: 7: QRHKPRE (Reference 3) SEQ ID NO: 8: SYPIPDT-GHEG-ECG-K-tetramethylrhodamine (Reference 4)

[0038] When the EGFR peptide ligand is a cyclic peptide, examples of the EGFR peptide ligand include cyclic peptides composed of the amino acid sequences of SEQ ID NOs: 9 to 15 below. In the cyclic peptide of SEQ ID NO: 9 below, the N-terminal lysine and the C-terminal glutamic acid form an amide bond between the amino group of the main chain and the carboxyl group of the side chain. The peptide of SEQ ID NO: 9 below is, for example, a cyclic peptide represented by the following formula (1). In the cyclic peptide of SEQ ID NO: 10 below, the N-terminal cysteine ​​and the C-terminal cysteine ​​form a disulfide bond between the thiol group of the side chain. For the EGFR peptide ligand, reference can be made to the EGFR-binding peptides described in JP 2010-154842 A and the following References 5 to 8. Reference 5: Toyama K et.al., “Inhibition of EGFR activation by bivalent ligands based on a cyclic peptide mimicking the dimerization arm structure of EGFR.”, Chem Pharm Bull. 2018;66:1083-1089. Reference 6: Mizuguchi T et.al., “Inhibitory effect of a dimerization-arm-mimetic peptide on EGF receptor activation.', Bioorg Med Chem Lett. 2009;19:3279-3282. https: / / doi.org / 10.1016 / j.bmcl.2009.04.080 Reference 7: Mizuguchi T et.al, “Evaluation of dimerization-inhibitory activities of cyclic peptides containing a β-hairpin loop sequence of the EGF receptor.”, Bioorg Med Chem. 2012;20:5730-5737. Reference 8: Toyama K et.al., “Functional evaluation of fluorescein-labeled derivatives of a peptide inhibitor of the EGF receptor dimerization.”, Bioorg Med Chem 2016;24:3406-3412. SEQ ID NO: 9: KQTPYYMNTE (Reference 5) SEQ ID NO: 10: CYNPTTYQMC (Reference 6) SEQ ID NO: 11: CPPLMLYNPTTYQMDVPEGC (cyclized by disulfide bonds via N- and C-terminal cysteine ​​side chains, Reference 7) SEQ ID NO: 12: MCYNPTTYQMC (cyclized by a disulfide bond between the second residue and the C-terminal cysteine ​​side chain, Reference 7) SEQ ID NO: 13: CMQYTTPNYC (all D amino acids, cyclized by disulfide bonds via N- and C-terminal cysteine ​​side chains, Reference 7) SEQ ID NO: 14: CQTPYYMNTC (cyclized by disulfide bonds via N- and C-terminal cysteine ​​side chains, Reference 8) SEQ ID NO: 15: CMQYTTPNYC (cyclized by disulfide bonds via N- and C-terminal cysteine ​​side chains, Reference 8) [ka]

[0039] The EGFR peptide ligand may be designed based on the amino acid sequence of the EGFR-binding region of the aforementioned EGF. When the EGFR peptide ligand is a cyclic peptide, the EGFR-binding cyclic peptide can be screened, for example, by referring to WO 2015 / 030014, WO 2018 / 225864, and the aforementioned Reference 6.

[0040] The photosensitive dye may be, for example, a phthalocyanine (dye), porphyrin, benzoporphyrin, corrin, chlorin, bacteriochlorophyll, cholphin, pyropheophorbide-a, pheophorbide, chlorin e6, purpurin, purpurinimide, verteporfin, hematoporphyrin, photofrin porfimer, rostaporfin, tarporfin, or temoporfin, or a derivative thereof. A specific example of a phthalocyanine dye derivative is IRDye 700DX (IR700). An example of a hematoporphyrin derivative is Photofrin (registered trademark). An example of a temoporfin is meta-tetrahydroxyphenyl chlorin (Foscan (registered trademark)). An example of a chlorin e6 derivative is mono-L-aspartyl chlorin e6 (NPe6) (Laserphyrin (registered trademark)).

[0041] The photosensitive dye preferably has a maximum absorption wavelength in the range of 600 to 1200 nm, 650 to 740 nm, or 680 to 700 nm. Examples of the photosensitive dye include the phthalocyanine dyes and their derivatives. The phthalocyanine dye is preferably IR700.

[0042] The phthalocyanine dye or its derivative includes, for example, a phthalocyanine dye represented by the following formula (2) or its derivative. [ka] In the formula (2), L is a linker; Q is a reactive group or a bond to a target molecule (EGFR peptide ligand) for attaching the dye to the target molecule or its linker; R 2 , R 3 , R 7 , and R 8 are each independently an optionally substituted alkyl group or an optionally substituted aryl group; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkanoyl group, an optionally substituted alkoxycarbonyl group, an optionally substituted alkylcarbamoyl group, or a chelating ligand, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of which contains a water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently a hydrogen atom, a halogen atom, an optionally substituted alkylthio group, an optionally substituted alkylamino group, or an optionally substituted alkoxy group; and X 2 and X 3each independently represents a C-C group optionally interrupted by a heteroatom; 10 It is an alkylene group.

[0043] In the L, the linker is an atomic group bonded by a covalent bond. The atomic group may be, for example, a linear or branched, cyclic or heterocyclic, saturated or unsaturated atomic group having 1 to 60 atoms, 1 to 45 atoms, or 1 to 25 atoms. The atoms may be selected from C, N, P, O, and S. In the L, for example, the atomic group satisfies the above number of atoms and may have an additional hydrogen atom (for example, in addition to the above 1 to 60 atoms). The covalent bond may contain, for example, an ether, thioether, amine, ester, carbamate, urea, thiourea, oxy, or amide bond; or a single bond, double bond, triple bond, or aromatic carbon-carbon bond; or a phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bond; or any combination of aromatic or heteroaromatic bonds.

[0044] wherein L is -R 1 -YX 1 -Y 1 The R 1 is a divalent radical or a direct bond (bond). 1 are each independently a direct bond, oxygen, optionally substituted nitrogen, or sulfur. 1 is a direct bond or a C1-C bond which may be separated by an atom 10 The divalent radical is, for example, an optionally substituted alkylene group, an optionally substituted alkyleneoxycarbonyl group, an optionally substituted alkylenecarbamoyl group, an optionally substituted alkylenesulfonyl group, or an optionally substituted arylene group.

[0045] R 1Examples of the alkyl group include an optionally substituted alkylene group, an optionally substituted alkyleneoxycarbonyl group, an optionally substituted alkylenecarbamoyl group, an optionally substituted alkylenesulfonyl group, an optionally substituted alkylenesulfonylcarbamoyl group, an optionally substituted arylene group, an optionally substituted arylenesulfonyl group, an optionally substituted aryleneoxycarbonyl group, an optionally substituted arylenecarbamoyl group, an optionally substituted arylenesulfonylcarbamoyl group, an optionally substituted carboxyalkyl group, an optionally substituted carbamoyl group, an optionally substituted carbonyl group, an optionally substituted heteroarylene group, an optionally substituted heteroaryleneoxycarbonyl group, an optionally substituted heteroarylenecarbamoyl group, an optionally substituted heteroarylenesulfonylcarbamoyl group, an optionally substituted sulfonylcarbamoyl group, an optionally substituted thiocarbonyl group, an optionally substituted sulfonyl group, and an optionally substituted sulfinyl group.

[0046] Q contains a reactive group for attachment to a target molecule, such as a peptide or protein. The term "reactive group" or "reactive chemical group" refers to a moiety on a compound that can chemically react with a functional group on a different material (e.g., a target molecule) to form a covalent bond, or a moiety on a compound that can bind by interacting with a functional group on a different material (e.g., a target molecule). The reactive group is typically an electrophile or nucleophile that can form a covalent bond via reaction with a corresponding functional group that is a nucleophile or electrophile, or a photoactivatable group that becomes chemically reactive after irradiation with light of an appropriate wavelength. When Q reacts with the target molecule, the resulting reaction product typically incorporates one or more atoms of the reactive group Q between the conjugated reactive dye and the target molecule via a conjugation reaction between the dye and the conjugated target molecule.

[0047] Q contains a reactive group that is reactive with, for example, a carboxyl group, an amine, or a thiol group on a target molecule. Examples of the reactive group include amine-reactive chemical groups, sulfhydryl-reactive chemical groups, activated esters, acyl halides, alkyl halides, anhydrides, carboxylic acids, carbodiimides, carbonates, carbamates, haloacetamides (e.g., iodoacetamide), isocyanates, isothiocyanates, maleimides, NHS esters, phosphoramidites, platinum complexes, sulfonate esters, and thiocyanates, for optional binding to a target molecule. Examples of the reactive group include a group reactive with, for example, a carboxyl group, an amine, or a thiol group on a target molecule. Examples of the reactive group include sulfhydryl-reactive chemical groups such as maleimides, haloacetyls, and pyridyl disulfides. Examples of the reactive group include amine-reactive groups. The reactive group is preferably an NHS ester.

[0048] R 2 , R 3 , R 7 , and R 8 is preferably an optionally substituted alkyl group, more preferably an optionally substituted methyl group, ethyl group, or isopropyl group.

[0049] R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of the groups "containing a water-soluble group" means, for example, 4 , R 5 , R 6 , R 9 , R 10 , and R 11 The alkyl group portion of R is substituted with a water-soluble substituent. As used herein, the "water-soluble group" refers to a group containing one or more polar and / or ionic substituents that improve the solubility of the entire molecule in an aqueous medium. 4 , R 5 , R 6 , R9 , R 10 , and R 11 At least two or three or more of the above may contain a water-soluble group. The water-soluble group is not particularly limited, and examples thereof include a carboxylate (-CO2-) group, a sulfonate (-SO3-) group, a sulfonyl (-SO2-) group, a sulfate (-SO4 2- ) group, hydroxyl (-OH) group, phosphate (-OPO3 2- ) group, phosphonate (-PO3 2- ) group, an amine (—NH 2 ) group, and an optionally substituted quaternary nitrogen (each with an optional counterion).

[0050] The counter ions include, for example, sodium ions, potassium ions, calcium ions, ammonium ions, organic amino salts, or magnesium salts, or similar salts. The counter ions are preferably biologically acceptable counter ions.

[0051] R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 The nitrogen atom to which is attached may be, for example, trivalent or tetravalent.

[0052] R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each preferably a hydrogen atom.

[0053] X 2 and X 3 are each independently a C1-C bond optionally interrupted by an atom. 10 The X is an alkylene group. 2 and / or X 3The nitrogen attached to may, for example, be quaternized.

[0054] The phthalocyanine dye or its derivative includes, for example, a phthalocyanine dye represented by the following formula (3) or its derivative. [ka] In the formula (3), X 1 and X 4 each independently represents a C-C group optionally interrupted by a heteroatom; 10 It is an alkylene group. In the formula (3), R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 16 , R 17 , R 18 , R 19 , X 2 and X 3 is the same as the above formula (2).

[0055] In the formula (3), the reactive group is an NHS ester. The reactivity of the NHS ester is determined by the X between the NHS ester and the carbamate functional group. 4 The length of the alkylene group in X between the NHS ester and the carbamate functional group can be adjusted. 4 The length of the alkylene group in X is inversely proportional to the reactivity of the NHS ester. 4 is, for example, a C3-alkylene group or a C5-alkylene group. 1 is, for example, a C3-alkylene group or a C6-alkylene group.

[0056] The phthalocyanine dye is preferably IR700 NHS ester represented by the following formula (4): [ka]

[0057] When the phthalocyanine dyes of the formulas (2) to (4) or their derivatives are bound to the EGFR, the reactive group such as NHS ester of the phthalocyanine dyes of the formulas (2) to (4) or their derivatives is eliminated, and the phthalocyanine dyes or their derivatives are bound to the EGFR peptide ligand.

[0058] The photosensitive dye may be, for example, a commercially available product or may be self-synthesized. Specifically, the phthalocyanine dye can be synthesized using commercially available starting materials, and its core structure can be synthesized by condensation of two or more different diiminoisoindolines. The synthesis method using different dinitriles or diiminoisoindolines allows the synthesis of phthalocyanines with various degrees of substitution and / or positional isomers with various degrees of distribution. For a synthesis scheme of the phthalocyanine dye, see, for example, U.S. Pat. No. 7,005,518.

[0059] The number of the EGFR peptide ligands conjugated to the photosensitive dye may be one or more, for example, one to five, or one to three, and preferably one to three.

[0060] The binding position of the photosensitive dye in the EGFR peptide ligand can be, for example, the N-terminus (e.g., amino group) or side chain of an amino acid constituting the EGFR peptide ligand. The photosensitive dye is preferably bound to an N-terminal amino group or an amino group or thiol group of an amino acid residue having an amino group or thiol group in its side chain, because this can suppress the influence on the binding between the ligand and the EGFR.

[0061] The photosensitive dye is bound to the EGFR peptide ligand via a linker. The same explanation as for L can be applied to the linker. The linker may include, for example, a peptide linker in addition to L. The length of the peptide linker may be within a range that allows the conjugate to bind to EGFR and exhibit cytotoxic activity, for example, 2 to 8 amino acid residues.

[0062] The length of the linker may be, for example, within a range that allows the conjugate to bind to EGFR and exhibit cytotoxic activity. The length of the linker can be represented, for example, by the shortest main chain length from the EGFR peptide ligand to the photosensitive dye. Specific examples of the main chain length of the linker are 13 to 55 atoms, 23 to 55 atoms, or 24 to 55 atoms, and preferably 16 to 50 atoms, 18 to 40 atoms, 21 to 29 atoms, 23 to 25 atoms, 31 to 43 atoms, 35 to 39 atoms, or 36 to 38 atoms. In the linker, the atom at the end on the photosensitive dye side is the atom bonded to the skeleton of the photosensitive dye.

[0063] An example of the conjugate of the present disclosure is a compound represented by the following formula (5) or a salt thereof. In the compound represented by the following formula (5), the main chain length of the linker is 37 atoms. In the compound represented by the following formula (5), the main chain length of the linker is the length from the oxygen atom (O) bonded to the phthalocyanine dye, shown circled in formula (5), to the carbon atom (C) bonded to the amino group of the arginine side chain of the cyclic peptide. [ka]

[0064] In the conjugate of the present disclosure, the salt is not particularly limited and may be, for example, a pharmaceutically acceptable salt. The conjugate of the present disclosure forms, for example, an acid addition salt or a salt with a base depending on the type of substituent. The pharmaceutically acceptable salt is not particularly limited and may, for example, be an alkali metal salt such as a sodium salt or a potassium salt; an alkaline earth metal salt such as a calcium salt or a magnesium salt; an ammonium salt; an aliphatic amine salt such as a trimethylamine salt, a triethylamine salt, a dicyclohexylamine salt, an ethanolamine salt, a diethanolamine salt, or a triethanolamine salt; an aralkylamine salt such as N,N-dibenzylethylenediamine; a heterocyclic aromatic amine salt such as a pyridine salt, a picoline salt, a quinoline salt, or an isoquinoline salt; a quaternary ammonium salt such as a tetramethylammonium salt, a tetraethylammonium salt, a benzyltrimethylammonium salt, a benzyltributylammonium salt, a methyltrioctylammonium salt, or a tetrabutylammonium salt; an arginine salt, a lysine salt, an aspartate salt, or a glutamic acid salt. inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, bicarbonate, and perchlorate; aliphatic organic acid or aromatic organic acid salts such as acetate, propionate, succinate, glycolate, lactate, maleate, fumarate, tartrate, malate, citrate, ascorbate, hydroxymaleate, pyruvate, phenylacetate, benzoate, 4-aminobenzoate, anthranilate, 4-hydroxybenzoate, salicylate, 4-aminosalicylate, pamoate, gluconate, and nicotinate; sulfonates such as methanesulfonate, isethionate, ethanesulfonate, benzenesulfonate, halobenzenesulfonate, p-toluenesulfonate, toluenesulfonate, naphthalenesulfonate, sulfanilate, and cyclohexylsulfamate; and the like.

[0065] The conjugate of the present disclosure can be prepared by reacting a linker-added EGFR peptide ligand with a linker-added photosensitive dye, for example, as described in the Examples below. The reaction conditions can be determined depending on the type of reactive group of the photosensitive dye.

[0066] The conjugate of the present disclosure contains the photosensitive dye and is therefore activatable, for example, by light irradiation, and exhibits cytotoxic activity against cells to which the EGFR peptide ligand is bound. The light irradiation, for example, activates the photosensitive dye, resulting in cytotoxicity against cells to which the conjugate is bound. The number of light irradiations in the activation can be one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. The wavelength and dose used for the light irradiation can be determined, for example, by referring to the extinction coefficient and absorption wavelength of the photosensitive dye, particularly the maximum absorption wavelength. The extinction coefficient of Photofrin® at 630 nm is 1.2 x 10 3 M -1 cm -1 The extinction coefficient of the Foscan® is 2.2 x 10 at 652 nm. 4 M -1 cm -1 The extinction coefficient of Laserphyrin® at 654 nm is 4.0 × 10 4 M -1 cm -1 The absorption coefficient of the IR700 is 2.1 × 10 at 689 nm (maximum absorption wavelength). 5 M -1 cm -1 is.

[0067] When the photosensitive dye is IR700, the wavelength of the light irradiation is, for example, 650 to 1200 nm, 600 to 800 nm, 600 to 740 nm, 540 to 760 nm, 660 to 740 nm, 650 to 740 nm, 650 to 690 nm, 680 to 720 nm, or 690 to 710 nm. The dose of the light irradiation is, for example, 1 J / cm. 2 More than 10J / cm 2 Above, 30J / cm 2 Above, 50J / cm 2 More than 100J / cm 2 or more than 500J / cm 2 The dose of the light irradiation is, for example, 1 to 1000 J / cm2 , 1~500J / cm 2 , 1~100J / cm 2 , 1~50J / cm 2 , or 10 to 50 J / cm 2 When the photosensitive dye is IR700, the wavelength and dose of the light irradiation are, for example, 600 to 850 nm and 1 to 1000 J / cm. 2 is.

[0068] The subject to which the conjugate of the present disclosure is administered (administration subject) can be, for example, a human or a non-human animal. Examples of the non-human animal include mammals such as mice, rats, hamsters, rabbits, goats, cows, horses, dogs, cats, pigs, monkeys, dolphins, and sea lions, as well as birds.

[0069] The administration route of the conjugate of the present disclosure is preferably one that is effective for the treatment, and examples thereof include intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration. The dosage form of the pharmaceutical composition may be, for example, an injection, infusion, capsule, tablet, or granule. The dosage form is effectively used as an injection. The aqueous solution for injection may be stored in a container such as a vial or a stainless steel container. The aqueous solution for injection may also be blended with, for example, physiological saline, sugar (e.g., trehalose), NaCl, or NaOH. The conjugate may also be blended with, for example, an effective amount of a buffer (e.g., phosphate buffer), a pH adjuster, a stabilizer, or the like.

[0070] The dose of the conjugate of the present disclosure is, for example, a therapeutically effective amount, specifically, 0.01 to 200 mg / kg body weight per administration. The administration interval is not particularly limited, and may be, for example, once or twice every 1 to 28 days. The dose, administration interval, and administration method may be appropriately selected depending on, for example, the age, body weight, symptoms, target organ, etc. of the subject.

[0071] The conjugate of the present disclosure is preferably housed in a container having a cover that absorbs light in the wavelength range absorbed by the photosensitive dye, and the cover has a transmittance of, for example, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or 5% or less at wavelengths of 600 to 850 nm.

[0072] The conjugate of the present disclosure may be configured as a kit including other components such as the cover.

[0073] The conjugates of the present disclosure may further be accompanied by instructions or directions.

[0074] The conjugates of the present disclosure can be suitably used in the treatment methods of the present disclosure described below.

[0075] <Pharmaceutical Composition> In some embodiments, the present disclosure provides a novel pharmaceutical composition that can be used for photoimmunotherapy. The pharmaceutical composition of the present disclosure comprises a conjugate of the present disclosure. The pharmaceutical composition of the present disclosure can be used, for example, for photoimmunotherapy of EGFR-positive cancer.

[0076] The pharmaceutical composition of the present disclosure may further comprise, for example, a pharmaceutically acceptable carrier, such as a buffer, excipient, stabilizer, pH adjuster, or preservative.

[0077] The pharmaceutical composition of the present disclosure can be used, for example, to treat EGFR-positive cancers, such as head and neck cancer, esophageal cancer, gastric cancer, lung cancer, colon cancer, breast cancer, bladder cancer, and cervical cancer.

[0078] The pharmaceutical composition of the present disclosure can be suitably used in the treatment method of the present disclosure described below.

[0079] <Treatment method> In another embodiment, the present disclosure provides a method for treating cancer.The method for treating cancer of the present disclosure comprises using the conjugate and / or pharmaceutical composition of the present disclosure.According to the present disclosure, for example, photoimmunotherapy can be performed for EGFR-positive cancer.

[0080] The treatment method of the present disclosure includes, for example, an administration step of administering the conjugate and / or pharmaceutical composition of the present disclosure to a subject. The treatment method of the present disclosure can be used to treat diseases associated with EGFR positivity.

[0081] The treatment method of the present disclosure may include an irradiation step of irradiating one or more cancers in the subject with light after the administration. The light irradiation can be carried out, for example, by guiding light from a light source to the cancer in the subject using a light-guiding means such as an optical fiber and irradiating the light. The light source may be, for example, a light source that emits light of a single wavelength, such as a laser light source, or a light source that emits light of multiple wavelengths. The light source may be, for example, an irradiation device. The wavelength of the light source can be determined, for example, by referring to the extinction coefficient, absorption wavelength, particularly the maximum absorption wavelength, of the photosensitive dye of the conjugate.

[0082] The wavelength of light in the irradiation step can be determined, for example, with reference to the extinction coefficient and absorption wavelength, particularly the maximum absorption wavelength, of the photosensitive dye in the conjugate. The irradiation time in the irradiation step can be set, for example, according to the dose to be irradiated to the subject or the cancer. As a specific example, the dose to be irradiated to the subject or the cancer in the irradiation step can be set, for example, to 1 J / cm. 2 More than 10J / cm 2 Above, 30J / cm 2 Above, 50J / cm 2 More than 100J / cm 2 or more than 500J / cm 2 The dose of irradiation to the subject or the cancer is, for example, 1 to 1000 J / cm 2 , 1~500J / cm 2 , 1~100J / cm 2 , 1~50J / cm 2, or 10 to 50 J / cm 2 is.

[0083] The irradiation step may be performed once or multiple times, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. When the irradiation step is performed multiple times, each irradiation may be performed on the same day or on consecutive days. Furthermore, each irradiation may be performed, for example, every 1 to 3 days, every 3 to 7 days, every 1 to 2 weeks, every 2 to 4 weeks, every 1 to 2 months, or at even longer intervals.

[0084] The interval between administration of the conjugate and the light irradiation can be set, for example, depending on the time it takes for the conjugate to be delivered to the cancer, and examples of the interval include 30 minutes to 48 hours, 1 to 24 hours, 1 to 12 hours, and 6 to 12 hours.

[0085] In the treatment methods of the present disclosure, the use comprises: in In vitro It can also be used in in vivo It may also be used in.

[0086] <Use> The present disclosure is a conjugate of the present disclosure for use in a method of treating cancer. The present disclosure is a conjugate of the present disclosure, or the use (application) of a conjugate of the present disclosure, in the manufacture of a pharmaceutical composition for use in a method of treating cancer. [Example]

[0087] The present disclosure will be described in detail below using examples, but the present invention is not limited to the embodiments described in the examples. Unless otherwise specified, commercially available reagents and kits were used according to the attached protocols. In the following description, "mol / l" may also be abbreviated as "M."

[0088] [Reference example 1] Monovalent peptide-fluorescein (monovalent peptide-fluorescein) or bivalent peptide-fluorescein (bivalent peptide-fluorescein) was prepared and confirmed to bind to EGFR-positive cells.

[0089] (1) Synthesis of monovalent peptide-fluorescein conjugates Compound 2, a monovalent peptide-fluorescein conjugate (monovalent peptide-fluorescein), was synthesized from compound 1 according to Scheme 1. Peptide 1 was prepared as described in reference 9 (Kei Toyama et al., “Inhibition of EGFR activation by bivalent ligands based on a cyclic peptide mimicking the dimerization arm structure of EGFR,” Chem.Pharm.Bull. (Tokyo). 2018, 66(11), 1083-1089). Compound 1 (5.0 mg, 4.0 μmol) was mixed with 30 μL of DMF (Fujifilm Wako Pure Chemical Industries, Ltd., 043-32361). To the resulting mixture, 1.3 mg (4.0 μmol) of 6-[(tert-butoxycarbonyl)amino]hexanoic acid N-succinimidyl ester (Tokyo Chemical Industry Co., Ltd., B5684) and 0.69 μL (4.0 μmol) of N,N-diisopropylethylamine (DIPEA, Fujifilm Wako Pure Chemical Industries, Ltd., 051-05351) were added and stirred at room temperature (approximately 25°C, the same applies below) for 24 hours. After stirring, the mixture was diluted with 10% acetonitrile (Fujifilm Wako Pure Chemical Industries, Ltd., 014-00386). The resulting diluted solution was purified using a preparative RP-HPLC (Shimadzu, LC-20AR system) under the following HPLC conditions. The resulting purified product was a mixture (3.7 mg) of compounds containing and not containing a Boc group. [ka]

[0090] (HPLC conditions) Column: COSMOSIL 5C18-ARII column 20 mm ID x 250 mm (Nacalai Tesque, 38150-41) mobile phase Other than compounds 9 and 12: 0.1% TFA aqueous solution and 0.1% TFA (Fujifilm Wako Pure Chemical Industries, Ltd., 208-02746) containing acetonitrile (Fujifilm Wako Pure Chemical Industries, Ltd., 017-13907) Compounds 9 and 12: 0.1 M triethylammonium acetate aqueous solution (Fujifilm Wako Pure Chemical Industries, Ltd., 202-13131) and acetonitrile Linear Gradient Flow rate 9.0 mL / min Detection: OD 220 nm (except for compounds 9 and 12) or 348 nm (compounds 9 and 12)

[0091] The mixture was added to 1.0 mL of TFA / 1,2-ethanedithiol (EDT, Tokyo Chemical Industry Co., Ltd., E0032) / triisopropylsilane (TIS, Tokyo Chemical Industry Co., Ltd., T1533) / HO (92.5:2.5:2.5:2.5 (v / v / v / v)) and reacted at room temperature for 30 minutes. The Boc group was deprotected by this reaction. The reaction solution was concentrated under reduced pressure, HO was added, and the mixture was lyophilized. The dried product, 2.2 mg (3.8 μmol) of 5-SFX (6-(fluorescein-5-carboxamido)hexanoic acid succinimidyl ester, Funakoshi Co., Ltd., 6934), and 0.44 μL (2.5 μmol) of DIPEA were mixed with 480 μL of DMF. The resulting mixture was stirred at room temperature for 19 hours to react. The reaction mixture was diluted with 10% acetonitrile to obtain a diluted solution. The diluted solution was purified using preparative RP-HPLC under the HPLC conditions. The purified product was lyophilized to obtain 2.7 mg of lyophilized powder. The yield of the lyophilized powder was 58%. The lyophilized powder was analyzed using MALDI-TOF MS (Shimadzu Corporation, MALDI-8030), and a signal at 1840.6 m / z was confirmed, confirming that the powder was Compound 2, a monovalent peptide-fluorescein conjugate. MALDI-TOF MS used α-cyano-4-hydroxycinnamic acid as a matrix.

[0092] (2) Synthesis of divalent peptide-fluorescein conjugates Compound 6, a conjugate of divalent peptide and fluorescein (divalent peptide-fluorescein), was synthesized using Rink Amide ProTide Resin (manufactured by CEM, R003-A) according to Scheme 2 below. [ka]

[0093] (2-1) Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis using a microwave-assisted peptide synthesizer Peptide synthesis using Fmoc solid-phase synthesis was performed using a microwave peptide synthesizer (CEM, Liberty Blue). Fmoc amino acid derivatives (Watanabe Chemical Co., Ltd.) were used. A 0.2 M DMF solution of the amino acid was added to a 0.5 M DMF solution of DIC (N,N'-diisopropylcarbodiimide, Fujifilm Wako Pure Chemical Industries, Ltd., 044-32891) and a 0.5 M DMF solution of OxymaPure (CEM, S001-C). Coupling was carried out at 90 °C for 2 minutes. After washing the reaction mixture, 20% (v / v) piperidine (Fujifilm Wako Pure Chemical Industries, Ltd., 160-02776) in DMF was added to the mixture and reacted at 90 °C for 1 minute to perform Fmoc deprotection. All steps were washed four times with DMF.

[0094] (2-2) Synthesis of Compound 4 Compound 4 was synthesized by Fmoc solid-phase peptide synthesis (Fmoc-SPPS) using the microwave peptide synthesizer described above. The resin peptide was synthesized from Rink Amide ProTide Resin. To cleave and deprotect the resin peptide from the resin, the resin peptide was mixed with 2.0 mL of TFA / TIS / HO (10:0.25:0.1 (v / v / v)) and reacted at room temperature (25 °C) for 2 hours. The mixture was filtered and concentrated under reduced pressure. Ice-cold diethyl ether was added to the concentrated residue. The added solution was centrifuged (Kubota Shoji Co., Ltd., Micro Refrigerated Centrifuge 3740, 4000 rpm, 5 minutes, 4 °C) to obtain a powder. The powder was washed three times with ice-cold diethyl ether to obtain a crude product. The crude product was purified using preparative RP-HPLC under the HPLC conditions described above. The purified product was lyophilized to obtain 25 mg of lyophilized powder. The yield of the freeze-dried powder was 27%. The dried powder was analyzed by MALDI-TOF MS, and a signal of 1895.2 m / z was confirmed, confirming that it was Compound 4. The measurement conditions for MALDI-TOF MS were the same as those in Reference Example 1(1).

[0095] (2-3) Synthesis of Compound 5 Compound 3 was prepared according to the method described in Reference 9. Compound 4 (2.1 mg (1.1 μmol)) and compound 3 (2.9 mg (2.2 μmol)) were mixed in 50 μL of DMF. To the mixture, 0.6 mg (2.2 μmol) of CuSO4·5H2O (Fujifilm Wako Pure Chemical Industries, Ltd., 039-04412) was added. 50 μL of aqueous sodium ascorbate solution (4.4 μmol) was added and reacted at room temperature (25 °C) for 24 hours. The mixture was diluted with 50% acetonitrile. The diluted solution was purified using preparative RP-HPLC under the HPLC conditions described above. The purified product was lyophilized to obtain 2.6 mg of lyophilized powder. The yield of the lyophilized powder was 51%. The dried powder was analyzed using MALDI-TOF MS, and a signal at 4532.1 m / z was confirmed, confirming that it was compound 5. The measurement conditions for MALDI-TOF MS were the same as those in Reference Example 1(1).

[0096] (2-4) Synthesis of Compound 6 Compound 5 (1.1 mg (0.24 μmol)) and 5-SFX (0.2 mg (0.35 μmol)) were mixed in 50 μL of DMF. 0.041 μL (0.24 μmol) of DIPEA was added to the mixture, and the mixture was allowed to react at room temperature (25°C) for 19 hours. The reaction solution was diluted with 50% acetonitrile and purified using preparative RP-HPLC under the HPLC conditions. The purified product was lyophilized to obtain 0.7 mg of lyophilized powder. The yield of the lyophilized powder was 57%. The dried powder was analyzed by MALDI-TOF MS, and a signal at 5003.1 m / z was confirmed, confirming that the powder was compound 6. The MALDI-TOF MS measurement conditions were the same as those in Reference Example 1(1).

[0097] (3) Binding to A431 cells To examine the binding of compounds 2 and 6 to A431 cells (provided by RikenBRC) that highly express EGFR, the fluorescence intensity was evaluated using a fluorescence microscope. A431 cells were cultured at approximately 2 × 10 5The cells were seeded onto glass-bottom dishes (Matsunami, D11130H) at 1 cell per dish and cultured overnight (approximately 10 hours, hereinafter the same) at 37°C under 5% CO2 conditions. The medium used was Dulbecco's modified Eagle's medium (DMEM, Fujifilm Wako Pure Chemical Industries, Ltd., 044-29765) supplemented with 10% fetal bovine serum (FBS, Biowest, S1580-500) and 2% penicillin-streptomycin solution (Fujifilm Wako Pure Chemical Industries, Ltd., 168-23191). After the culture, the medium was removed and washed with Roswell Park Memorial Institute (RPMI) medium (Fujifilm Wako Pure Chemical Industries, Ltd., 186-02155). After the wash, a solution of compound 2 or compound 6 in RPMI medium (1 mL, 2 μM) was added to the dish and cultured at 37°C for 10 minutes. After removing the medium, the dish was washed with phosphate-buffered saline (PBS, Fujifilm Wako Pure Chemical Industries, Ltd., 045-29795). Cell imaging was performed using a microscope (Olympus, IX83). Fluorescein was excited at 470-495 nm and monitored at 510-550 nm. Fluorescence intensity was analyzed using Olympus IX83 software (Olympus, cell Sens Dimension). The measurements were performed three times, with calculations performed on five cells each, for a total of 15 cells. The measurement results and fluorescence intensity results are shown in Figure 1.

[0098] Figure 1 shows the binding of Compound 2 or Compound 6 to cells. (A) is a photograph of a phase-contrast image and a fluorescence microscope image, and (B) is a graph showing fluorescence intensity. In Figure 1(A), each photograph, from left to right, shows a phase-contrast image and a microscope image of fluorescein, respectively. Also, in Figure 1(A), each photograph, from top to bottom, shows the results for monovalent peptide-fluorescein (Compound 2) and bivalent peptide-fluorescein (Compound 6), respectively. In Figure 1(B), the horizontal axis indicates the type of peptide added, and the vertical axis indicates fluorescence intensity. As shown in Figures 1(A) and (B), the bivalent peptide-fluorescein exhibited higher fluorescence intensity than the monovalent peptide-fluorescein (p<0.01 vs. monovalent peptide, Student's t-test). This indicates that the bivalent peptide-fluorescein exhibited higher cell binding, i.e., EGFR binding, than the monovalent peptide-fluorescein.

[0099] [Comparative Example 1] We prepared a conjugate with a short linker connecting the bivalent peptide and IR700, added it to cells, and confirmed that it did not exhibit cell cytotoxicity when irradiated with near-infrared light.

[0100] (1) Synthesis of a conjugate of IR700 and a bivalent peptide with a short linker Compound 9, a conjugate of a bivalent peptide and IR700, was synthesized from Rink Amide ProTide Resin according to Scheme 3 below. [ka]

[0101] (1-1) Synthesis of Compound 7 Compound 7 was prepared in the same manner as in Reference Example 1 (2-2). 18 mg of lyophilized powder was obtained from 0.05 mmol of Rink Amide ProTide Resin. The yield of the lyophilized powder was 20%. The lyophilized powder was analyzed using MALDI-TOF MS, and a signal at 1781.7 m / z was confirmed, confirming that it was Compound 7. The measurement conditions for MALDI-TOF MS were the same as those in Reference Example 1 (1).

[0102] (1-2) Synthesis of Compound 8 Compound 8 was synthesized from Compound 7 in the same manner as in the synthesis of Compound 5 from Compound 4. 3.7 mg of a freeze-dried powder was obtained from Compound 7 (3.5 mg (2.0 μmol)). The yield of the freeze-dried powder was 42%. The freeze-dried powder was analyzed using MALDI-TOF MS, and a signal at 4418.7 m / z was confirmed, confirming that it was Compound 8. The measurement conditions for MALDI-TOF MS were the same as those in Reference Example 1 (1).

[0103] (1-3) Synthesis of Compound 9 Compound 8 (1.5 mg (0.34 μmol)) was added to 240 μL of dimethyl sulfoxide (Fujifilm Wako Pure Chemical Industries, Ltd., 048-32811), and 0.3 mg (0.17 μmol) of IR700 NHS ester (LI-COR, 929-70011) was added. 0.029 μL (0.17 μmol) of DIPEA was added to the added solution, and the mixture was reacted at 40°C for 19 hours. The mixture was diluted with HO. The diluted solution was purified using preparative RP-HPLC under the HPLC conditions described above. The purified product was passed through a Sep-Pak C18 cartridge (Waters Corporation, WAT023501). The purified product after passing through the cartridge was lyophilized to obtain 0.4 mg of lyophilized powder. The yield of the lyophilized powder was 41%. The freeze-dried powder was analyzed using ESI-MS (electrospray ionization mass spectrometry), and a signal at 1232.1 m / z was confirmed, confirming that it was Compound 9.

[0104] (2) Cell morphological changes caused by Compound 9 A431 cells were approximately 5 × 10 5 The cells were seeded onto glass-bottom dishes at 1 cell per dish and cultured overnight at 37°C under 5% CO2 conditions. DMEM medium was used. After the culture, the medium was removed, and each dish was washed with RPMI medium. Compound 9 was added to each dish to a final concentration of 5 μM (1 mL), and the dish was cultured at 37°C for 10 minutes. Compound 9 was prepared by adding it to RPMI medium. After the culture, the medium was washed with PBS, and the cells in each dish were visualized using a fluorescence microscope (Olympus IX83). Then, near-infrared light was irradiated through a microscope excitation filter (768.5-849.5 nm) to observe changes in cell morphology. The results are shown in Figure 2.

[0105] Figure 2 is a set of micrographs showing the morphological changes of EGFR-positive cells caused by Compound 9. In Figure 2, the photographs, from left to right, show micrographs taken before near-infrared light irradiation, 5 minutes after irradiation, 10 minutes after irradiation, 30 minutes after irradiation, and 60 minutes after irradiation, respectively. As shown in Figure 2, no change in cell morphology was observed between before and after irradiation. In other words, no cytotoxic activity was observed due to the addition of Compound 9.

[0106] [Example 1] Conjugates with long linkers connecting the bivalent peptide and IR700 were prepared, and it was confirmed that the conjugates with long linkers have higher cell binding and cytotoxicity than conjugates with short linkers.

[0107] (1) Synthesis of a conjugate of a bivalent peptide with a long linker and IR700 Compound 12, a conjugate of a bivalent peptide with a long linker and IR700, was synthesized from Rink Amide ProTide Resin (LL) according to Scheme 4 below. [ka]

[0108] (1-1) Synthesis of Compound 10 Compound 10 was obtained in the same manner as in Reference Example 1 (2-2). 14 mg of lyophilized powder was obtained from 0.25 mmol of Rink Amide ProTide Resin (LL) (CEM, R002-B). The yield of the lyophilized powder was 29%. The lyophilized powder was analyzed by MALDI-TOF MS, and a signal of 2008.4 m / z was confirmed, confirming that it was Compound 10. The measurement conditions for MALDI-TOF MS were the same as those in Reference Example 1 (1).

[0109] (1-2) Synthesis of Compound 11 Compound 11 was synthesized from compound 10 in the same manner as in the synthesis of compound 5 from compound 4. 4.2 mg of a freeze-dried powder was obtained from compound 10 (3.5 mg (1.7 μmol)). The yield of the freeze-dried powder was 52%. The freeze-dried powder was analyzed by MALDI-TOF MS, and a signal at 4623.2 m / z was confirmed, confirming that the powder was compound 11. The measurement conditions for MALDI-TOF MS were the same as those in Reference Example 1(1).

[0110] (1-3) Synthesis of Compound 12 Compound 12 was synthesized from IR700NHS ester in the same manner as compound 9. 0.3 mg of lyophilized powder was obtained from 0.3 mg (0.17 μmol) of IR700NHS ester. The yield of the lyophilized powder was 26%. The dried powder was analyzed by ESI-MS, and a signal at 1597.0 m / z was confirmed, confirming that it was compound 12.

[0111] (2) Binding of Compound 9 or Compound 12 to cells The binding of Compound 9 and Compound 12, which have different linker lengths connecting IR700 and the peptide, to cells was evaluated. A431 cells were cultured at approximately 2 × 10 3The cells were seeded into a 96-well plate at 100 μL per well and cultured overnight at 37°C and 5% CO2. DMEM was used as the culture medium. After the culture, the medium was removed. Compound 9 or Compound 12 was added to each well to a final concentration of 2 μM (100 μL). Compound 9 or Compound 12 was prepared by adding it to RPMI medium. After the culture, the medium was replaced with RPMI medium, and each well was analyzed using a microplate reader to evaluate the binding of each compound to the cells. A microplate reader (Promega, GloMaxDiscover) was used. IR700 was excited at 627 nm and fluorescence from 660 to 720 nm was monitored. After the evaluation, cell counts were measured using a Cell Count Normalization Kit (DOJINDO, C544). The binding rate to the cells was calculated by taking the fluorescence intensity immediately after addition of the compound solution as 100% and correcting it for the number of cells in each well. The results are shown in Figure 3.

[0112] FIG. 3 is a graph showing the binding of compound 9 (a comparative example compound) or compound 12 (an example compound) to EGFR-positive cells. In FIG. 3, the horizontal axis indicates the type of compound, and the vertical axis indicates the binding rate corrected for the number of cells. As shown in FIG. 3, compound 12 was confirmed to have a higher binding rate to EGFR-positive cells than compound 9 (p<0.01 vs. compound 9, Student's t-test). In other words, it was found that a longer linker connecting IR700 and the peptide had higher cell binding ability than a shorter linker. Specifically, it was found that a linker main chain length of 14 atoms or more is preferable.

[0113] (3) Cell morphological changes caused by Compound 12 The morphological changes in EGFR-positive cells caused by the addition of Compound 12 were examined in the same manner as in Comparative Example 1(2), except that Compound 12 was used instead of Compound 9. The results are shown in FIG.

[0114] Figure 4 shows microscopic images of cell morphology induced by compound 12. In Figure 4, the photographs, from left to right, show microscopic images taken before, 5 minutes after, 10 minutes after, 30 minutes after, and 60 minutes after near-infrared light irradiation, respectively. As shown in Figure 4, with compound 12, cell abscesses were observed 10 minutes after irradiation, and cell death was induced. In other words, compound 12, which has a long linker connecting IR700 to the peptide, was found to exhibit cytotoxic activity by activating IR700 through near-infrared light irradiation.

[0115] (4) Cytotoxic activity of Compound 9 or Compound 12 The cytotoxic activity of Compound 9 or Compound 12 against EGFR-positive cells was examined. A conjugate of cetuximab (anti-EGFR antibody) and IR700 (Cet-IR700) was used as a positive control. Cell viability was evaluated using a CCK-8 kit (DOJINDO, 343-07623). A431 cells were cultured at approximately 2 × 10 4 The cells were seeded onto a 24-well plate at 10 cells / well and cultured overnight at 37°C under 5% CO2 conditions. DMEM medium was used. After the culture, the medium was removed, and each well was washed with RPMI medium. Compound 9 or compound 12 was added to each well to a final concentration of 2 μM (500 μL), and cultured at 37°C for 10 minutes. Compound 9 and compound 12 were prepared by adding them to RPMI medium. Cet-IR700 was added to each well to a final concentration of 10 μg / mL (500 μL), and cultured at 37°C for 1 hour. Cet-IR700 was prepared by adding them to RPMI medium. After the culture, the cells in each well were irradiated with near-infrared light (dose of compound 9 and compound 12: 0, 5, 10, 15, or 20 J / cm 2 , Cet-IR700 dose: 0, 1, 2, 5, or 10 J / cm 2) was irradiated. After the irradiation, each medium was removed and washed with PBS. After the washing, DMEM medium was added to each well and cultured at 37°C for 24 hours. After the culture, the medium was removed and each well was washed with RPMI medium. Next, CCK-8 solution was added to each well and cultured at 37°C for 3 hours. After the culture, 100 μL of the culture supernatant was transferred to a 96-well plate and the absorbance was measured. The absorbance was measured at a wavelength of 450 nm using the microplate reader. As a control, the measurement was performed in the same manner except that compound 9, compound 12, or Cet-IR700 was not added. These results are shown in Figure 5.

[0116] FIG. 5 is a graph showing cell viability. FIG. 5(A) is a graph showing cell viability for Compound 9 and Compound 12 after near-infrared light irradiation. FIG. 5(B) is a graph showing cell viability for Cet-IR700 after near-infrared light irradiation. In FIGS. 5(A) and 5(B), the horizontal axis represents the dose of near-infrared light, and the vertical axis represents cell viability (cytotoxic activity). As shown in FIG. 5, Compound 9 did not change cell viability at any dose of near-infrared light. In contrast, Compound 12 decreased cell viability, i.e., increased cytotoxic activity, depending on the dose of near-infrared light.

[0117] (5) Concentration-dependent cytotoxic activity of compound 12 The concentration of compound 12 added was set to a predetermined concentration (0, 0.1, 0.5, 1, or 2 μM), and the dose was set to 20 J / cm 2 The concentration-dependent cytotoxic activity of Compound 12 was examined in the same manner as in Example 1(4), except that the following was used: The results are shown in FIG.

[0118] Figure 6 is a graph showing the concentration-dependent effect of compound 12 on cell viability upon near-infrared light irradiation. In Figure 6, the horizontal axis represents the concentration of compound 12, and the vertical axis represents cell viability (cytotoxic activity). As shown in Figure 6, it was found that the cell viability decreased in a concentration-dependent manner with compound 12, i.e., the cytotoxic activity increased.

[0119] Although the present disclosure has been described above with reference to embodiments and examples, the present disclosure is not limited to the above embodiments and examples. Various modifications that can be understood by a person skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0120] <Additional Notes> Some or all of the above-described embodiments and examples can be described as, but are not limited to, the following supplementary notes. (Appendix 1) comprising an epidermal growth factor receptor (EGFR) peptide ligand and a photosensitive dye; the EGFR peptide ligand is conjugated to a photosensitive dye via a linker; A conjugate wherein the main chain length of the linker is 14 to 55 atoms. (Appendix 2) The conjugate according to Appendix 1, wherein the EGFR peptide ligand is composed of 5 to 30 amino acid residues per molecule of the peptide ligand. (Appendix 3) 3. The conjugate of claim 1 or 2, wherein the EGFR peptide ligand comprises a cyclic peptide. (Appendix 4) The conjugate according to any one of claims 1 to 3, wherein the EGFR peptide ligand is represented by formula (1). (Appendix 5) 5. The conjugate of any one of claims 1 to 4, wherein the photosensitive dye comprises a phthalocyanine dye. (Appendix 6) 6. The conjugate of any one of claims 1 to 5, wherein the photosensitive dye comprises IR700. (Appendix 7) 7. The conjugate of any one of claims 1 to 6, wherein the photosensitive dye is conjugated to a lysine residue of the EGFR peptide ligand. (Appendix 8) 9. The conjugate according to any one of claims 1 to 8, wherein at least two EGFR-binding peptide ligands are conjugated to one molecule of the photosensitive dye. (Appendix 9) The conjugate according to any one of appendices 1 to 8, which is a compound represented by formula (5) or a salt thereof: (Appendix 10) 10. The conjugate of any one of claims 1 to 9, which is activatable by light irradiation and thereby exhibits cytotoxic activity against cells to which the EGFR peptide ligand is bound. (Appendix 11) 11. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 10. (Appendix 12) 12. The pharmaceutical composition of claim 11 for use in treating EGFR-positive cancer. (Appendix 13) 13. A method for treating cancer using a conjugate according to any one of appendices 1 to 10 and / or a pharmaceutical composition according to appendices 11 or 12. (Appendix 14) 14. The method of treatment described in Appendix 13, comprising administering to a subject a therapeutically effective amount of the conjugate and / or the pharmaceutical composition. (Appendix 15) The method of claim 14, further comprising, after the administration, irradiating the cancer in the subject with light. (Appendix 16) The dose of the light is 1 to 1000 J / cm 2 16. The method of treatment according to claim 14 or 15, (Appendix 17) 17. The method of any one of claims 14 to 16, wherein the wavelength of light is a wavelength capable of activating the photosensitive dye of the conjugate. (Appendix 18) the photosensitive dye is IR700, The treatment method according to any one of appendices 14 to 17, wherein the wavelength of the light is 600 to 850 nm. (Appendix 19) 19. The method of any one of claims 14 to 18, wherein the cancer is an EGFR-positive cancer. (Appendix 20) in In vitro or in vivo 20. The method of treatment according to any one of appendices 14 to 19, wherein (Appendix 21) 11. The conjugate of any one of appendices 1 to 10 for use in a method for treating cancer. (Appendix 22) 22. The conjugate of claim 21, wherein the cancer is an EGFR-positive cancer. [Industrial Applicability]

[0121] As described above, the present disclosure provides a conjugate that can be used in photoimmunotherapy, and is therefore extremely useful, for example, in the pharmaceutical field.

Claims

1. comprising an epidermal growth factor receptor (EGFR) peptide ligand and a photosensitive dye; the EGFR peptide ligand is conjugated to a photosensitive dye via a linker; A conjugate wherein the main chain length of said linker is 14 to 55 atoms.

2. The conjugate according to claim 1, wherein the EGFR peptide ligand is composed of 5 to 30 amino acid residues per molecule of the peptide ligand.

3. 3. The conjugate of claim 1 or 2, wherein the EGFR peptide ligand comprises a cyclic peptide.

4. The conjugate according to claim 1 , wherein the EGFR peptide ligand is represented by the following formula (1): 【Chemistry 1】

5. The conjugate of claim 1 , wherein the photosensitive dye comprises a phthalocyanine dye.

6. 6. The conjugate of claim 1, wherein the photosensitive dye comprises IR700.

7. 7. The conjugate of claim 1, wherein the photosensitive dye is conjugated to a lysine residue of the EGFR peptide ligand.

8. The conjugate according to claim 1 , wherein at least two EGFR-binding peptide ligands are conjugated to one molecule of the photosensitive dye.

9. The conjugate according to any one of claims 1 to 8, which is a compound represented by the following formula (5) or a salt thereof: 【Transformation 5】

10. The conjugate of claim 1 , which is activatable by light irradiation and thereby exhibits cytotoxic activity against cells to which the EGFR peptide ligand is bound.

11. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 10.

12. 12. The pharmaceutical composition of claim 11 for use in treating EGFR-positive cancer.

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