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) ligand and a photosensitive dye, wherein said photosensitive dye is conjugated to said EGFR ligand.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) ligand and a photosensitive dye, The photosensitive dye is conjugated to the EGFR 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 is a histogram showing the binding of panitumumab-IR700, AREG-IR700, and EGF-IR700 to EGFR-positive cells in Example 1. [Figure 2] FIG. 2 is a photograph of a micrograph showing the binding of AREG-IR700 or EGF-IR700 to EGFR-positive cells in Example 2. [Figure 3] FIG. 3 is a micrograph showing the morphological changes of cells caused by AREG-IR700 in Example 3. [Figure 4] Figure 4 is a graph showing the survival rate of EGFR-positive cells irradiated with near-infrared light after the addition of panitumumab-IR700, AREG-IR700, or EGF-IR700 in Example 4. [Figure 5]FIG. 5 shows the treatment of cancer model mice in Example 5, where (A) is a graph showing tumor size, and (B) is a photograph showing fluorescent images before and after near-infrared light irradiation. 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, "amphiregulin" (AREG) is a ligand that binds to EGFR and is a protein known to induce cell proliferation. For AREG derived from various animals, reference can be made to information registered in existing databases, for example. Specific examples of human AREG include a protein consisting of the following amino acid sequence (SEQ ID NO: 5), registered in UniProt under accession number P15514. Furthermore, examples of mouse AREG include a protein consisting of the following amino acid sequence (SEQ ID NO: 6), registered under P31955. In the AREG, the region that binds to EGFR is, for example, a region comprising the amino acid sequence of positions 101 to 198 or 142 to 182 in the amino acid sequence of SEQ ID NO: 5.

[0017] Human AREG (SEQ ID NO: 5) MRAPLLPPAPVVLSLLILGSGHYAAGLDLNDTYSGKREPFSGDHSADGFEVTSRSEMSSGSEISPVSEMPSSSEPSSGADYDYSEEYDNEPQIPGYIVDDSVRVEQVVKPPQNKTESENTSDKPKR KKKGGKNGKNRRNRKKKNPCNAEFQNFCIHGECKYIEHLEAVTCKCQQEYFGERCGEKSMKTHSMIDSSSLSKIALAAIAAFMSAVILTAVAVITVQLRRQYVRKYEGEAEERKKLRQENGNVHAIA

[0018] Mouse AREG (SEQ ID NO: 6) MRTPLLPLARSVLLLLVLGSGHYAAALELNDPSSGKGESLSGDHSAGGLELSVGREVSTISEMPSGSELSTGDYDYSEEYDNEPQISGYIIDDSVRVEQVIKPKKNKTEGEKSTEKPKRKKKGG KNGKGRRNKKKKNPCTAKFQNFCIHGECRYIENLEVVTCNCHQDYFGERCGEKSMKTHSEDDKDLSKIAVVAVTIFVSAIILAAIGIGIVITVHLWKRYFREYEGETEERRRLRQENGTVHAIA

[0019] As used herein, "ligand" refers to a molecule that specifically binds to a target receptor.

[0020] As used herein, the term "photosensitive dye" refers to a compound that undergoes a chemical change in response to light.

[0021] 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.

[0022] As used herein, "conjugate" means one or more other molecules chemically linked, directly or indirectly.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] As used herein, "subject" means an animal or a cell, tissue, or organ derived from an animal. The term "animal" is used to mean both human and non-human animals. The non-human animals include, for example, mice, rats, hamsters, rabbits, goats, cows, and horses. Examples of mammals include animals such as dogs, cats, pigs, monkeys, dolphins, and sea lions.

[0027] As used herein, "treatment" means therapeutic and / or prophylactic treatment. As used herein, "treatment" means the treatment, cure, prevention, suppression, amelioration, or alleviation of a disease, condition, or disorder. Relief, improvement, or arrest, arrest, reduce, or slow the progression of a disease, condition, or disorder As used herein, "prevention" means reducing the likelihood of developing a disease or condition, The term "treatment" refers to, for example, the treatment of a target disease. The treatment may be for patients who develop the disease, or for animal models of the target disease.

[0028] As used herein, "peptide" refers to a polymer composed of several unmodified (naturally occurring), modified, and / or artificial amino acids.

[0029] 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.

[0030] In this specification, "optionally substituted" means that the group in question is either unsubstituted or substituted.

[0031] 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.

[0032] 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.

[0033] 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).

[0034] 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.

[0035] 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."

[0036] <Conjugate> In one embodiment, 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) ligand and a photosensitive dye, and the photosensitive dye is conjugated to the EGFR ligand. The conjugate of the present disclosure can be used in photoimmunotherapy.

[0037] 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, leading to the establishment of the present disclosure. The conjugate of the present disclosure can be used to suitably perform photoimmunotherapy, for example, targeting EGFR-positive cancer.

[0038] The EGFR ligand is, for example, a protein that binds to EGFR or an EGFR-binding fragment thereof. The EGFR-binding protein is, for example, EGF, AREG, TGF-α, EPGN (epigen), BTC (betacellulin), EREG (epiregulin), HB-EGF (heparin-binding EGF), or an EGFR-binding fragment thereof, and is preferably AREG or an EGFR-binding fragment thereof, since when bound to the EGFR, it can remain on the surface of the EGFR-expressing cells for a longer period of time, thereby improving the effect of the conjugate on the cells. The EGFR does not include, for example, an antibody against the EGFR.

[0039] 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)).

[0040] 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.

[0041] The phthalocyanine dye or its derivative includes, for example, a phthalocyanine dye represented by the following formula (1) or its derivative. [ka] In the formula (1), L is a linker; Q is a reactive group for binding the dye to a target molecule (EGFR ligand) or a bond to the target molecule; 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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).

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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), 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 (2), 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 (1).

[0054] In the formula (2), 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.

[0055] The phthalocyanine dye is preferably IR700 NHS ester represented by the following formula (3): [ka]

[0056] When the phthalocyanine dyes of the formulas (1) to (3) or their derivatives are bound to the EGFR, the reactive group such as NHS ester is eliminated from the phthalocyanine dyes of the formulas (1) to (3) or their derivatives, and the phthalocyanine dyes of the formulas (1) to (3) or their derivatives are bound to the EGFR ligand.

[0057] 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.

[0058] The number of the photosensitive dyes conjugated to the EGFR ligand may be 1 or more, for example, 1 to 2, or 1 to 3. The number of the photosensitive dyes is the average number of photosensitive dyes bound to one molecule of the EGFR ligand.

[0059] The binding position of the photosensitive dye in the EGFR ligand can be, for example, the N-terminus (e.g., amino group) or side chain of an amino acid constituting the EGFR ligand. The photosensitive dye is preferably bound to the N-terminus amino group or to the amino or thiol group of an amino acid residue having an amino or thiol group in its side chain, since this can suppress the influence on the binding between the ligand and the EGFR.

[0060] The photosensitive dye is directly or indirectly bound to the EGFR ligand. The direct bond is, for example, a covalent bond. The indirect bond is, for example, a bond via a linker. The linker can be the same as described above for L. The linker may be a peptide linker.

[0061] The conjugate of the present disclosure can be produced, for example, by reacting the EGFR ligand with the photosensitive dye. The reaction can be determined, for example, depending on the type of reactive group of the photosensitive dye.

[0062] The conjugate of the present disclosure contains the photosensitive dye and can be activated, for example, by light irradiation, to exhibit cytotoxic activity against cells to which the EGFR ligand is bound. The light irradiation, for example, activates the photosensitive dye, resulting in the conjugate exhibiting cytotoxicity against cells to which the conjugate is bound. The number of light irradiations in the activation can be one or more times, 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.

[0063] 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 / cm 2 , 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.

[0064] 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.

[0065] 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, intratumoral and tumor surface, or oral administration. The dosage form of the pharmaceutical composition may be, for example, an injection, infusion, capsule, tablet, granule, or the like. 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, NaOH, or the like. 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.

[0066] 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.

[0067] 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.

[0068] The conjugate of the present disclosure may be configured as a kit including other components such as the cover.

[0069] The conjugates of the present disclosure may further be accompanied by instructions or directions.

[0070] The conjugates of the present disclosure can be suitably used in the treatment methods of the present disclosure described below.

[0071] <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.

[0072] 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.

[0073] 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.

[0074] The pharmaceutical composition of the present disclosure can be suitably used in the treatment method of the present disclosure described below.

[0075] <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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] <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]

[0083] 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."

[0084] [Example 1] An AREG-IR700 conjugate (AREG-IR700) or an EGF-IR700 conjugate (EGF-IR700) was prepared and confirmed to bind to EGFR-positive cells.

[0085] (1) Preparation of panitumumab-IR700 conjugate (panitumumab-IR700) To evaluate the binding of the conjugates of the present disclosure to EGFR-positive cells, a conjugate of panitumumab (anti-EGFR antibody) and IR700 was prepared as a positive control. 1 mg (6.8 nmol) of panitumumab (Vectibix IV Infusion 100 mg, Takeda Pharmaceutical Co., Ltd.) and 60.2 μg (30.8 nmol) of IR700 NHS ester (Li-COR Biosciences, 929-70011) were mixed in a 0.1 M NaHPO aqueous solution (pH 8.5, Fujifilm Wako Co., Ltd., 196-02835) and incubated at room temperature (approximately 25°C) for 24 hours. The mixture was purified using a Sephadex G25 column (PD-10, GE Healthcare). The protein concentration in the purified product was calculated by measuring absorbance at 562 nm using a protein assay BCA reagent (Fujifilm Wako Pure Chemical Industries, Ltd., 06385-00) and a spectrophotometer (JASCO, V-730Bio). The IR700 concentration in the purified product was calculated by measuring absorbance at 689 nm using the spectrophotometer. It was confirmed that an average of three IR700 molecules bound to one panitumumab molecule in the purified product.

[0086] (2) Preparation of AREG-IR700 and EGF-IR700 AREG-IR700 and EGF-IR700 were prepared in the same manner as in Example 1(1) above, except that human AREG (PEPROTECH, 100-55B) or human EGF (PEPROTECH, AF-100-15) was used instead of the panitumumab. Similarly to Example 1(1) above, it was confirmed that an average of 1 to 2 molecules of IR700 and 1 molecule of IR700 bound to one AREG molecule and one molecule of EGF, respectively.

[0087] (3)Cell culture The human epithelial cell carcinoma-derived cell line A431 (provided by RikenBRC) was cultured in DMEM medium (Fujifilm Wako Co., Ltd., 044-29765) supplemented with 10% FBS (BIOWEST, S1580-500) and 100 μg / ml of antibiotics (penicillin / streptomycin, Fujifilm Wako Co., Ltd., 168-23191). The cells were cultured at 37°C and 5% CO2. The A431 cell line is EGFR-positive.

[0088] (4) Cell binding The cells were collected and washed twice with ice-cold staining buffer. The staining buffer consisted of PBS (Fujifilm Wako Co., Ltd., 045-29795) containing 10% FBS and 1% sodium azide (Fujifilm Wako Co., Ltd., 195-11092). After washing, the cells were collected at a concentration of 1 × 10 6 The cells were prepared so that the cell count was 100 μl / 100 μl. Panitumumab-IR700, AREG-IR700, or EGF-IR700 was added to the cells at a final concentration of 10 μg / ml, and the cells were cultured at 4°C for 30 minutes. The cells were washed twice with ice-cold staining buffer and then analyzed using a BD FACSymphony™ A1 flow cytometer (BD Biosciences) to evaluate the binding of each conjugate to the cells. A control was performed in the same manner, except that the staining buffer was used instead of the conjugate. The results are shown in Figure 1.

[0089] Figure 1 is a histogram showing the binding of panitumumab-IR700 (Pan-IR700, hereinafter the same), AREG-IR700, and EGF-IR700 to EGFR-positive cells. In Figure 1, the horizontal axis represents fluorescence intensity, and the vertical axis represents counts. As shown in Figure 1, it was confirmed that AREG-IR700 and EGF-IR700 bind to EGFR-positive cells.

[0090] [Example 2] AREG-IR700 or EGF-IR700 was added to EGFR-positive cells, and it was confirmed that AREG-IR700 was internalized more slowly than EGF-IR700 and remained localized on the cell membrane for a longer period of time.

[0091] The EGFR cells used were the same as those used in Example 1(3) (A431 cell line). The cells were seeded on a glass-bottom dish (D11130H, manufactured by Matsunami Glass Industry Co., Ltd.) and cultured overnight (approximately 12 hours; the same applies below). AREG-IR700 or EGF-IR700 was then added to the cells to a final concentration of 10 μg / ml. Phenol red-free DMEM medium was used for the cell culture. After the addition of the conjugate, the cells were cultured at 37°C for 1 hour. After the culture, the cells were washed with PBS, and then new medium not containing the conjugate was added and the cells were cultured at 37°C for 1 hour. The resulting cells were then observed for internalization of the conjugate. Specifically, the presence of the conjugate on the cell membrane of the cells was visualized using a confocal laser microscope (FV3000, manufactured by Olympus). The cell membrane was stained with PlasMem Bright Green (DOJINDO, P504). The colocalization of each signal obtained by the confocal microscope was quantified using the Coloc 2 plugin in ImageJ Fiji software and calculated using Pearson's correlation coefficient. The results are shown in Figure 2.

[0092] Figure 2 shows micrographs of the binding of AREG-IR700 or EGF-IR700 to EGFR-positive cells. In Figure 2, the photographs, from left to right, respectively show a phase-contrast image, a micrograph of the cell membrane, a micrograph of IR700, and a superimposed micrograph of the cell membrane and the IR700. Furthermore, in Figure 2, the photographs, from top to bottom, show micrographs taken 0 hours and 1 hour after the addition and removal of AREG-IR700, and 0 hours and 1 hour after the addition and removal of EGF-IR700. As shown in Figure 2, no difference in IR700 fluorescence intensity was observed immediately (0 h) and 1 hour after removal of AREG-IR700. Furthermore, IR700 in AREG-IR700 was mainly distributed on the cell membrane. On the other hand, the fluorescence intensity of EGF-IR700 was weaker 1 hour after removal compared to immediately after removal. Furthermore, compared with AREG-IR700, EGF-IR700 showed a relatively reduced amount of conjugates localized on the cell membrane 1 hour after removal, confirming its rapid internalization into cells. These findings indicate that, of the EGFR ligands, AREG-IR700 localizes on the cell membrane for a longer period of time.

[0093] [Example 3] After adding AREG-IR700 to EGFR-positive cells, the morphological changes of the cells were observed, and it was confirmed that AREG-IR700 induces the formation of cell nodules and cell rupture.

[0094] The EGFR cells used were the same as those used in Example 1(3) (A431 cell line). The cells were seeded on a glass-bottom dish and cultured overnight. Then, panitumumab-IR700 or AREG-IR700 was added to the cells to a final concentration of 10 μg / ml. Phenol red-free DMEM medium was used as the cell culture medium. After the addition of the conjugate, the cells were cultured at 37°C for 1 hour. Next, the cells were irradiated with near-infrared light (670.5 to 745.5 nm), and the changes in cell shape were observed for 10 minutes using an inverted microscope (Olympus IX83). A control was performed in the same manner except for the addition of the DMEM medium. These results are shown in Figure 3.

[0095] Figure 3 is a set of micrographs showing changes in cell morphology due to AREG-IR700. In Figure 3, the photographs, from left to right, show micrographs of a control, panitumumab-IR700, and AREG-IR700, respectively. Furthermore, in Figure 3, the photographs, from top to bottom, show micrographs taken before and 10 minutes after irradiation with near-infrared light. As shown in Figure 3, after irradiation with AREG-IR700, cell aneurysms and cell rupture were confirmed, as shown in the figure. These results demonstrate that AREG-IR700 exhibits cytotoxic activity when irradiated with near-infrared light to activate IR700.

[0096] [Example 4] It was confirmed that cytotoxic activity could be induced by adding AREG-IR700 or EGF-IR700 to EGFR-positive cells and irradiating them with near-infrared light.

[0097] The EGFR cells used were the same as those used in Example 1(3) (A431 cell line). The cells were seeded onto a 24-well plate and cultured overnight. After the culture, panitumumab-IR700, AREG-IR700, or EGF-IR700 was added to the cells to a final concentration of 10 μg / ml. The cell culture medium used was a DMEM medium containing no phenol red. After the addition of the conjugate, the cells were cultured at 37°C for 1 hour. After the culture, the cells were illuminated with a light-emitting diode (Ushio Epitex, 690-66-60) at a wavelength of 690 nm and a power density of 50 mW / cm. 2 The cells were irradiated with near-infrared light at 450 nm. The light irradiation dose was measured at 690 nm using an optical power meter (PM100, manufactured by Thorlabs). After culturing the cells for 24 hours, the cell viability (cytotoxic activity) was determined by measuring the absorbance at 450 nm using Cell Counting Kit-8 (CK04, manufactured by Dojindo) and a microplate reader (GloMax Discover, manufactured by Promega). A control was performed in the same manner except that the conjugate was not added. These results are shown in Figure 4.

[0098] Figure 4 is a graph showing the viability of EGFR-positive cells irradiated with near-infrared light after the addition of panitumumab-IR700, AREG-IR700, or EGF-IR700. In Figure 4, the horizontal axis represents the dose of near-infrared light, and the vertical axis represents cell viability (cytotoxic activity). As shown in Figure 4, it was confirmed that AREG-IR700 reduces cell viability, i.e., increases cytotoxic activity, depending on the dose of near-infrared light. Similarly to AREG-IR700, it was confirmed that EGF-IR700 also reduces cell viability depending on the dose of near-infrared light. Furthermore, AREG-IR700 reduced cell viability at lower doses than EGF-IR700. In other words, it was found that AREG-IR700 has higher cytotoxic activity than EGF-IR700.

[0099] [Example 5] AREG-IR700 was administered to EGFR-positive cancer model mice and irradiated with near-infrared light, and it was confirmed that cancer growth could be suppressed.

[0100] The following animal experiments were conducted in accordance with the guidelines of the "Act on the Protection and Welfare of Animals in Japan" and were approved by the Kansai Medical University Animal Experiment Committee. A minimum of five mice were used in the animal experiments. Six-week-old female BALB / C nu / nu mice (The Jackson Laboratory) were used. The suspension containing A431 cells was prepared at a concentration of 5 x 10 per 100 μl. 6 The suspension was adjusted with PBS to a volume of 100 μl. 100 μl of the suspension was injected subcutaneously into both flanks of the mice. After the injection, the tumor volume was 150 mm 3 When the tumor size exceeded 100 μg, the mice were divided into two groups. Group 1 was the untreated group, and Group 2 was the treated group, which received an intravenous injection of 50 μg of AREG-IR700 per mouse. In the treated group, near-infrared light was irradiated only on the right side of the tumor 30 minutes after injection of AREG-IR700. The near-infrared light was irradiated using an ML7710 laser system (manufactured by Modulight) and a frontal diffuser (FD1, manufactured by Medlight). The dose for each irradiation was 100 J / cm. 2 The administration and irradiation were carried out twice in total, with an interval of 24 hours. Fluorescence imaging of IR700 before and after the irradiation was carried out using Pearl Trilogy (LI-COR). The tumor volume was calculated by measuring the length and width using a caliper and using the following formula (tumor volume (mm 3 ) = length (mm) × width (mm) × width (mm) × 0.5). These results are shown in Figure 5.

[0101] Figure 5 shows the treatment of cancer model mice. (A) is a graph showing tumor size, and (B) is a photograph showing fluorescence images before and after near-infrared light irradiation. In Figure 5(A), the horizontal axis indicates the number of days after administration of the conjugate, and the vertical axis indicates tumor size. In Figure 5, the area surrounded by the white line is the area where cancer is distributed. As shown in Figure 5(A), administration of AREG-IR700 alone did not change tumor size compared to the untreated group. In contrast, the photoimmunotherapy group (administration of AREG-IR700 and irradiation with near-infrared light) had significantly smaller tumor size than the untreated group. This confirms that AREG-IR700 administration and irradiation with near-infrared light can suppress tumor size growth. Furthermore, as shown in Figure 5(B), the near-infrared light irradiation eliminated the fluorescence in the irradiated right flank, while the fluorescence in the non-irradiated left flank remained, indicating that IR700 was chemically altered specifically in the near-infrared light-irradiated region. This suggests that IR700 was altered by light irradiation, resulting in tumor damage. These findings demonstrate that EGFR-positive cancer can be treated by administering AREG-IR700 and irradiating it with near-infrared light.

[0102] 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.

[0103] <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) ligand and a photosensitive dye; A conjugate wherein the photosensitive dye is conjugated to the EGFR ligand. (Appendix 2) 2. The conjugate of claim 1, wherein the EGFR ligand comprises amphiregulin (AREG) or epidermal growth factor (EGF). (Appendix 3) 3. The conjugate of claim 1 or 2, wherein the photosensitive dye comprises a phthalocyanine dye. (Appendix 4) 4. The conjugate of any one of claims 1 to 3, wherein the photosensitive dye comprises IR700. (Appendix 5) 5. The conjugate of any one of appendixes 1 to 4, wherein the EGFR ligand is conjugated to the photosensitive dye via a linker. (Appendix 6) 6. The conjugate of any one of claims 1 to 5, wherein the photosensitive dye is conjugated to a lysine residue of the EGFR ligand. (Appendix 7) The conjugate according to any one of appendix 1 to 6, wherein one or two photosensitive dyes are conjugated to one molecule of the EGFR ligand. (Appendix 8) 8. The conjugate of any one of claims 1 to 7, which is activatable by light irradiation and thereby exhibits cytotoxic activity against cells to which the EGFR ligand is bound. (Appendix 9) 9. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 8. (Appendix 10) 10. The pharmaceutical composition of claim 9 for use in treating EGFR-positive cancer. (Appendix 11) 11. A method for treating cancer using a conjugate according to any one of appendices 1 to 8 and / or a pharmaceutical composition according to appendices 9 or 10. (Appendix 12) 12. The method of treatment described in Appendix 11, comprising administering to a subject a therapeutically effective amount of the conjugate and / or the pharmaceutical composition. (Appendix 13) 13. The method of claim 12, further comprising, after the administration, irradiating the cancer in the subject with light. (Appendix 14) The dose of the light is 1 to 1000 J / cm 2 14. The method of treatment according to claim 13, wherein (Appendix 15) 15. The method of claim 13 or 14, wherein the wavelength of light is a wavelength capable of activating the photosensitive dye of the conjugate. (Appendix 16) the photosensitive dye is IR700, 16. The treatment method according to claim 15, wherein the wavelength of the light is 600 to 850 nm. (Appendix 17) 17. The method of any one of claims 11 to 16, wherein the cancer is an EGFR-positive cancer. (Appendix 18) in In vitro or in vivo 18. The method of treatment according to any one of appendices 11 to 17, wherein (Appendix 19) 10. The conjugate of any one of appendices 1 to 8 for use in a method for treating cancer. (Appendix 20) 20. The conjugate of claim 19, wherein the cancer is an EGFR-positive cancer. [Industrial Applicability]

[0104] 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) ligand and a photosensitive dye; A conjugate wherein the photosensitive dye is conjugated to the EGFR ligand.

2. 2. The conjugate of claim 1, wherein the EGFR ligand comprises amphiregulin (AREG) or epidermal growth factor (EGF).

3. The conjugate of claim 1 or 2, wherein the photosensitive dye comprises a phthalocyanine dye.

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

5. 5. The conjugate of claim 1, wherein the EGFR ligand is conjugated to a photosensitive dye via a linker.

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

7. The conjugate according to any one of claims 1 to 6, wherein one to two photosensitive dyes are conjugated to one molecule of the EGFR ligand.

8. The conjugate according to any one of claims 1 to 7, which is activatable by light irradiation, thereby exhibiting cytotoxic activity against cells to which the EGFR ligand is bound.

9. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 8.

10. 10. The pharmaceutical composition of claim 9 for use in treating EGFR-positive cancer.

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