Conjugates of biotin-modified dimers with phthalocyanine dyes

Abstract

To provide a conjugate of a biotin modified dimer and a phthalocyanine dye for use in photoimmunotherapy.SOLUTION: The present disclosure provides a compound having a specific chemical structure containing a conjugate compound represented by formula 10 or a salt thereof. A therapeutic agent contains the conjugate.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a conjugate of a biotin-modified dimer and a phthalocyanine dye, and its use. [Background technology]

[0002] The affinity between avidin and biotin or between streptavidin and biotin is very high (Kd=10 -15 From 10 -14 M), which is one of the strongest interactions between two biological molecules. Currently, the avidin / streptavidin-biotin interaction is widely applied in the fields of biochemistry, molecular biology, and medicine. Drug delivery methods and pretargeting methods have been devised that combine the high binding ability of avidin / streptavidin and biotin with antibody molecules. In relation to these studies, Patent Document 1 reports a streptavidin mutant with reduced affinity for natural biotin, as well as a biotin-modified dimer that has high affinity for this streptavidin mutant with low affinity for natural biotin.

[0003] On the other hand, photoimmunotherapy is a treatment that uses a photosensitizer and irradiated light to destroy specific cells in the body. When exposed to light of a specific wavelength, the photosensitizer generates cytotoxic reactive oxygen species that can induce apoptosis, necrosis, and / or autophagy in nearby cells. For example, Patent Document 2 describes a method for killing cells, comprising the steps of contacting cells containing a cell surface protein with a therapeutically effective amount of one or more antibody-IR700 molecules, where the antibody specifically binds to the cell surface protein; and exposing the cells to light at a wavelength of 660-740 nm and at least 1 Jcm. -2and about 0-8 hours after the cells are irradiated, contacting the cells with one or more therapeutic agents, thereby killing the cells. Patent Document 3 describes a method of inducing cytotoxicity in a subject suffering from a disease or condition, comprising: (a) administering to the subject a therapeutically effective agent comprising a phthalocyanine dye, such as IRDye® 700DX, conjugated to a probe that specifically binds to the cells of the subject; and (b) irradiating the cells with a suitable excitation light in an amount effective to induce cell death. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication WO2015 / 125820 [Patent Document 2] Patent No. 6127045 [Patent Document 3] Special Publication No. 2017-524659 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide a conjugate of a biotin-modified dimer and a phthalocyanine dye for use in photoimmunotherapy. A further aim of the present invention is to provide a treatment kit using the above-mentioned conjugate of a biotin-modified dimer and a phthalocyanine dye, and a combination of a streptavidin mutant-molecular probe conjugate. [Means for solving the problem]

[0006] As a result of intensive research aimed at solving the above problems, the present inventors discovered that the proliferation of cancer cells can be suppressed by photoimmunotherapy using a conjugate of a biotin-modified dimer and a phthalocyanine dye, and thus completed the present invention.

[0007] That is, according to the present invention, the following inventions are provided. [1] A compound represented by the following formula (1) or a salt thereof: [ka] (In the formula, X is a hydrophilic group, a cationic group or a substituent having an amino group at the end, or -OH. X1a, X1b, X2a and X2b each independently represent O or NH; Y 1 and Y 2 each independently represents C or S, Z 1 and Z 2 each independently represents O, S or NH, V 1 and V 2 are each independently S or S + -O - n1 and n2 each independently represent an integer of 0 or 1; L1 and L2 each independently represent a divalent linking group. L3 represents a trivalent linking group, L4, L5, and L6 each independently represent a divalent linking group. [2] The compound according to [1], represented by the following formula (2), or a salt thereof: [ka] (The meaning of each symbol in the formula is the same as in [1].) [3] X1a, X1b, X2a and X2b represent NH; Y 1 and Y 2 indicates C, and Z 1 and Z 2 indicates NH, and V 1 and V 2 The conjugate according to [1] or [2], wherein represents S. [4] The conjugate according to any one of [1] to [3], wherein L1 and L2 are each independently a divalent linking group consisting of a combination of groups selected from -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, and an alkylene group having 1 to 10 carbon atoms. [5] The conjugate according to any one of [1] to [4], wherein L4 is a group consisting of a combination of groups selected from -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, -NH-, and an alkylene group having 1 to 10 carbon atoms. [6] L5, [ka] The conjugate according to any one of [1] to [5], which is a triazole group, -S-, -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, -NH-, an alkylene group having 1 to 10 carbon atoms, or a group consisting of a combination thereof. [7] L6 is -Si(R 1 )(R 2 ) -O-, -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, -NH-, an alkylene group having 1 to 10 carbon atoms, or a group consisting of a combination thereof. [8] The conjugate according to any one of [1] to [7], wherein the substituent having a hydrophilic group, a cationic group or an amino group at its terminal, represented by X, is any one of the following: [ka] [9] Any of the following compounds: [ka] TIFF0007625188000006.tif198170TIFF0007625188000007.tif200170

[10] A therapeutic agent comprising a conjugate described in any one of [1] to [9].

[11] A therapeutic kit comprising: (1) a conjugate according to any one of [1] to [9]; and (b) a conjugate of a streptavidin mutant having the amino acid sequence set forth in SEQ ID NO: 1 (with the proviso that part or all of the C-terminal histidine tag may be deleted) and a molecular probe.

[12] The treatment kit described in

[11] , wherein the molecular probe is an anti-EREG antibody, an anti-CEA antibody, or an anti-HER2 antibody. Effect of the Invention

[0008] Photoimmunotherapy using the conjugate of the biotin-modified dimer and the phthalocyanine dye according to the present invention can suppress the proliferation of cancer cells. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows a structural diagram of CEA-V2122. [Diagram 2] FIG. 2 shows an electrophoretic profile of the purified product. [Diagram 3] FIG. 3 shows the evaluation of the binding ability of CEA-Cupid with an antigen (CEACAM5). [Figure 4] FIG. 4 shows the evaluation of binding ability of CEA-Cupid to biotin-modified forms. [Diagram 5] FIG. 5 shows an image of cells stained with FITC-labeled CEA-V2122. [Figure 6] FIG. 6 shows time series data of cell staining images (MKN45 cells) using FITC-labeled CEA-Cupid. [Figure 7] FIG. 7 shows the results of an in vitro cytotoxicity test using CEA-V2122 and a photoactivatable compound-labeled biotin modified product. [Figure 8] FIG. 8 shows the results of an in vitro cytotoxicity test using CEA-V2122 and a photoactivatable compound-labeled biotin modified product. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will now be described in further detail. (1) Biotin-modified dimer The present invention relates to a conjugate of a biotin-modified dimer and a phthalocyanine dye, which is a compound represented by the following formula (1) or a salt thereof, and preferably a compound represented by the following formula (2) or a salt thereof. [ka]

[0011] [ka]

[0012] The biotin-modified dimer portion is a compound represented by the following formula (11) or a salt thereof, and preferably a compound represented by the following formula (12) or a salt thereof. The biotin-modified dimer may be a compound described in International Publication WO2015 / 125820.

[0013] [ka]

[0014] [ka]

[0015] In the formula, X1a, X1b, X2a and X2b each independently represent O or NH. Y 1 and Y 2 each independently represents C or S, Z 1 and Z 2 each independently represents O, S or NH, V 1 and V 2 are each independently S or S + -O - n1 and n2 each independently represent an integer of 0 or 1; L1 and L2 each independently represent a divalent linking group. L3 represents a trivalent linking group, L4 represents a divalent linking group.

[0016] In the above formula, the following structure: [ka] The moiety represented by is preferably [ka] However, the present invention is not limited to these.

[0017] It is preferred that X1a, X1b, X2a and X2b represent NH, and Y 1 and Y 2 preferably represents C, Z 1 and Z 2 preferably denotes NH, V 1 and V 2 It is preferred that S is represented.

[0018] It is preferable that L1 and L2 each independently represent a divalent linking group consisting of a combination of groups selected from -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, and an alkylene group having 1 to 10 carbon atoms. It is preferable that L1 and L2 each independently represent a divalent linking group consisting of a combination of groups selected from -CONH-, -NHCO-, -O-, and an alkylene group having 1 to 10 carbon atoms. It is preferable that L1 and L2 each independently represent a divalent linking group consisting of a combination of groups selected from -CONH-, -NHCO-, and an alkylene group having 1 to 10 carbon atoms.

[0019] L3 represents a trivalent linking group, preferably [ka] Or, [ka] (a trivalent linking group derived from benzene or a nitrogen atom).

[0020] L4 is preferably a group consisting of a combination of groups selected from -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, -NH-, and an alkylene group having 1 to 10 carbon atoms.

[0021] L5 is preferably [ka] The alkylene group is a triazole group, -S-, -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, -NH-, an alkylene group having 1 to 10 carbon atoms, or a combination thereof.

[0022] L6 is preferably -Si(R 1 )(R 2 ) -O-, -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, -NH-, an alkylene group having 1 to 10 carbon atoms, or a combination thereof.

[0023] The substituent having a hydrophilic group, a cationic group or an amino group at its terminal, represented by X, is preferably any one of the following: [ka]

[0024] (2) Phthalocyanine dyes The phthalocyanine dye is preferably a silicon phthalocyanine dye. Specific examples of phthalocyanine dyes such as IRDye (registered trademark) 700DX are described in, for example, U.S. Patent No. 7,005,518. As the phthalocyanine dye, a commercially available product such as IRDye (registered trademark) 700DX can be used. As the phthalocyanine dye, a dye represented by the following formula can be used: The asterisk indicates the binding site with the linking group for linking the biotin-modified dimer.

[0025] [ka]

[0026] The compounds of the present invention can be synthesized according to the methods described in Preparation Examples 1 to 5 of the Examples.

[0027] (3) Therapeutic kit using conjugate of biotin-modified dimer and phthalocyanine dye According to the present invention, there is provided a therapeutic kit which combines a conjugate of the biotin-modified dimer and a phthalocyanine dye of the present invention with a mutant streptavidin-molecular probe conjugate.

[0028] As the streptavidin mutant, the streptavidin mutants described in International Publication WO2014 / 129446 and International Publication WO2015 / 125820 can be used. Particularly preferably, the streptavidin mutant LISA314-V2122 described in Example 3 of International Publication WO2015 / 125820 (SEQ ID NO: 4 of International Publication WO2015 / 125820) (SEQ ID NO: 1 of the present specification) can be used.

[0029] Examples of molecular probes include antibodies, peptides, nucleic acids, aptamers, etc. Specifically, antibodies, peptides, nucleic acids, aptamers, etc. that target the following antigens that are specifically expressed in cancer can be used.

[0030] Epiregulin (EREG), ROBO1,2,3,4, 1-40-β-amyloid, 4-1BB, 5AC, 5T4, ACVR2B, adenocarcinoma antigen, α-fetoprotein, angiopoietin 2, anthrax toxin, AOC3 (VAP-1), B-lymphoma cells, B7-H3, BAFF, β-amyloid, C242 antigen, C5, CA-125, carbonic anhydrase 9 (CA-IX), cardiac myosin, CCL11 (eotaxin-1), CCR4, CCR5, CD11, CD18, CD125, CD140a, CD147 (basigin), CD147 (basigin), CD15, CD152, CD154 (CD40L), CD154, CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD25 (α chain of IL-2 receptor), CD28, CD3, CD30 (TNFRSF8), CD33, CD37, CD38 (cyclic ADP-ribose hydrolase), CD4, CD40, CD41 (integrin α-IIb), CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CFD, ch4D5, CLDN18.2, Clostridium difficile, clumping factor A, CSF2, CTLA-4, cytomegalovirus, cytomegalovirus glycoprotein B, DLL4, DR5, E. coli Shiga toxin type 1, E. coli Shiga toxin type 2, EGFL7, EGFR, endotoxin, EpCAM, episialin, ERBB3, Escherichia coli, F protein of respiratory syncytial virus, FAP, fibrin II beta chain, fibronectin extra domain-B, folate receptor 1, Frizzled receptor, GD2, GD3 ganglioside, GMCSF receptor alpha chain, GPNMB, hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, HGF,HIV-1, HLA-DRβ, HNGF, Hsp90, human beta amyloid, human scatter factor receptor kinase, human TNF, ICAM-1 (CD54), IFN-α, IFN-γ, IgE, IgE Fc region, IGF-1 receptor, IGF-I, IgG4, IGHE, IL-1β, IL-12, IL-13, IL-17, IL-17A, IL-22, IL-23, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, ILGF2, influenza A hemagglutinin, insulin-like growth factor I receptor, integrin α4, integrin α4β7, integrin α5β1, integrin α7β7, integrin αIIbβ3, integrin αvβ3, Integrin gamma-inducing protein, interferon receptor, interferon alpha / beta receptor, ITGA2, ITGB2 (CD18), KIR2D, L-selectin (CD62L), Lewis-Y antigen, LFA-1 (CD11a), lipoteichoic acid, LOXL2, LTA, MCP-1, mesothelin, MS4A1, MUC1, mucin CanAg, myostatin, N-glycolylneuraminic acid, NARP-1, NCA-90 (granulocyte antigen), NGF, NOGO-A, NRP1, Oryctolagus cuniculus, OX-40, oxLDL, PCSK9, PD-1, PDCD1, PDGF-R alpha, phosphatidylserine, prostate cancer cells, Pseudomonas aeruginosa, rabies virus glycoprotein, RANKL, Respiratory syncytial virus, RHD, Rh (Rhesus) factor, RON, RTN4, sclerostin, SDC1, selectin P, SLAMF7, SOST, sphingosine-1-phosphate, TAG-72, TEM1, tenascin-C, TFPI, TGFβ1, TGFβ2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, tumor-specific glycosylation of MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), VEGF-A, VEGFR-1,VEGFR2, Vimentin, VWF, Among the above, epiregulin (EREG), CEA, and HER2 are preferred.

[0031] A fusion of a molecular probe such as a cancer antigen-specific antibody molecule with a streptavidin mutant is prepared and administered to a patient, thereby enabling the streptavidin mutant to accumulate specifically in cancer cells. Next, a conjugate of a phthalocyanine dye and a biotin-modified dimer having affinity for the streptavidin mutant is administered to the patient, enabling the phthalocyanine dye to accumulate precisely in the cancer cells.

[0032] Alternatively, in the present invention, a complex can be prepared by combining a conjugate of a molecular probe such as a cancer antigen-specific antibody molecule with a streptavidin mutant and a conjugate of a biotin-modified dimer with a phthalocyanine dye, and administering the complex to a patient.

[0033] Various molecules can be used as the antibody to be bound to the streptavidin mutant. Both polyclonal and monoclonal antibodies can be used. The subclass of the antibody is not particularly limited, but IgG, particularly IgG1, is preferably used. In addition, the term "antibody" includes all modified antibodies and antibody fragments. Examples of the antibody include humanized antibodies, human-type antibodies, human antibodies, antibodies derived from various animals such as mice, rabbits, rats, guinea pigs, and monkeys, chimeric antibodies of human antibodies and antibodies derived from various animals, diabodies, scFv, Fd, Fab, Fab', and F(ab)'2, but are not limited to these.

[0034] A conjugate of a streptavidin mutant and an antibody can be obtained by a method known to those skilled in the art. For example, it can be obtained by a chemical binding method (US Pat. No. 5,608,060), or it can be obtained as a fusion protein by linking a DNA encoding a streptavidin mutant with a DNA encoding an antibody and expressing it in a host cell using an expression vector or the like. The link between the DNA encoding a streptavidin mutant and the DNA encoding an antibody may be mediated by a DNA encoding a suitable peptide called a linker. It is desirable that a streptavidin mutant-antibody conjugate is prepared while retaining the specific binding ability between the antibody and the target molecule.

[0035] (4) Photoimmunotherapy By administering to a subject a conjugate of a biotin-modified dimer and a phthalocyanine dye according to the present invention and irradiating the cells with excitation light in an amount effective to inhibit cell proliferation or induce cell death, inhibition of cell proliferation or induction of cell death can be induced and the subject can be treated.

[0036] Preferably, a complex of a conjugate of a biotin-modified dimer and a phthalocyanine dye according to the present invention and a streptavidin mutant-molecular probe conjugate is administered to a subject, and the cells are irradiated with excitation light in an amount effective for inducing inhibition of cell proliferation or cell death, thereby inducing inhibition of cell proliferation or cell death and treating the subject.

[0037] The subject includes humans and non-human mammals, and may be a human or an experimental animal such as a mouse. The subject is preferably a subject suffering from a disease in which inhibition of cell proliferation or induction of cell death is desired, and may include, for example, a subject having cancer or a solid tumor.

[0038] "Cancer" includes carcinoma, lymphoma, blastoma, sarcoma, and leukemia or malignant lymphoma. Specific examples of cancer include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric corpus or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal region cancer, prostate cancer, vulva cancer, thyroid cancer, hepatocellular carcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.

[0039] A solid tumor refers to an abnormal mass of cells, either benign or malignant, that usually does not contain a cyst. Solid tumors include glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0040] In photoimmunotherapy, a subject can be treated by administering to the subject a "conjugate of a biotin-modified dimer and a phthalocyanine dye" or a "complex of a conjugate of a biotin-modified dimer and a phthalocyanine dye and a streptavidin mutant-molecular probe conjugate" and then irradiating the subject with light.

[0041] Methods of administration to a subject include, but are not limited to, topical routes, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous injections), oral routes, ocular routes, sublingual routes, rectal routes, transdermal routes, intranasal routes, vaginal routes, and inhalation routes.

[0042] The "conjugate of biotin-modified dimer and phthalocyanine dye" or the "complex of the conjugate of biotin-modified dimer and phthalocyanine dye and the streptavidin mutant-molecular probe conjugate" is preferably administered in a therapeutically effective amount. The therapeutically effective amount is at least 0.5 milligrams per 60 kilograms (mg / 60kg), at least 5 mg / 60kg, at least 10 mg / 60kg, at least 20 mg / 60kg, at least 30 mg / 60kg, at least 50 mg / 60kg. For example, when administered intravenously, the dose is 1 mg / 60kg, 2 mg / 60kg, 5 mg / 60kg, 20 mg / 60kg, or 50 mg / 60kg, for example, 0.5 to 50 mg / 60kg. In another example, the therapeutically effective amount is at least 10 μg / kg, such as at least 100 μg / kg, at least 500 μg / kg or at least 500 μg / kg, for example, 100 μg / kg, 250 μg / kg, about 500 μg / kg, 750 μg / kg, or 1000 μg / kg doses when administered intratumorally or intraperitoneally, for example, 10 μg / kg to 1000 μg / kg. In one example, the therapeutically effective amount is at least 1 μg / ml, such as at least 500 μg / ml, such as between 20 μg / ml and 100 μg / ml, such as 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, or 100 μg / ml administered in a topical solution.

[0043] The above-mentioned dosages may be administered in one or more divided doses (such as 2, 3, or 4 doses) or in a single formulation.

[0044] The "conjugate of a biotin-modified dimer and a phthalocyanine dye" or the "complex of a conjugate of a biotin-modified dimer and a phthalocyanine dye and a streptavidin mutant-molecular probe conjugate" may be administered alone, in the presence of a pharma- ceutically acceptable carrier, or in the presence of other therapeutic agents (such as other anticancer agents).

[0045] The "biotin modified dimer-phthalocyanine dye conjugate" or the "complex of the biotin modified dimer-phthalocyanine dye conjugate and the streptavidin mutant-molecular probe conjugate" can bind to a target cell or tissue, such as a circulating tumor cell or a cell of a solid tumor. When subsequently irradiated with light, the conjugate or complex can absorb the light and damage or destroy the target cell or tissue.

[0046] In photoimmunotherapy, the wavelength of the irradiated light is preferably 660 to 740 nm, for example, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, or 740 nm. The light irradiation may be performed by using a device equipped with a near-infrared (NIR) light-emitting diode.

[0047] The light dose must be at least 1 J / cm 2 , e.g., at least 4 J / cm 2 , at least 10 J / cm 2 , at least 15 J / cm 2 , at least 20 J / cm 2 , at least 50 J / cm 2 , or at least 100 J / cm 2 , e.g., 1 to 500 J / cm 2 The light irradiation may be carried out multiple times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times).

[0048] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. EXAMPLES

[0049] <Production Example 1: Synthesis of Compound 10> [ka]

[0050] Psyche J 1 (4.5 mg, 3.4 μmol) was added with (1R,8S,9S)-Bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate 2 (1.0 mg, 3.4 μmol), Et3N (1.7 mg, 17 μmol), and dehydrated DMF (500 μL) and stirred at room temperature for 15 h. The mixture was diluted with water to 2000 μL and purified by reversed-phase HPLC (gradient: 10% for 5 min; 10-60% for 20 min CH3CN in 0.1% trifluoroacetic acid aqueous solution, retention time = 19.0 min, YMC-Triart C18, flow rate = 3.5 mL / min) to give the target compound 3 (3.2 mg, 75%, colorless).

[0051] 1 H NMR (400 MHz, CD3OD):δ 4.72 (m, 2H), 4.52 (m, 2H), 4.12 (d, J = 8.2 Hz, 2H), 3.97 (s, 4H), 3.79 (t, J = 5.0 Hz, 2H), 3.62 (s, 4H), 3.53 (t, J = 6.0 Hz, 2H), 3.39 (brs, 2H), 3.30-3.23 (m, 8H), 3.17 (t, J = 6.0 Hz, 4H), 2.98 (dd, J= 13.7, 4.6 Hz, 2H), 2.81 (d, J = 13.7 Hz, 2H), 2.25-2.10 (m, 10H), 1.81-1.30 (m, 23H), 0.92 (t, J = 10.1 Hz, 2H). LRMS (ESI): m / z 1031.55 [M+H] + , 516.35 [M+2H] 2+ , 344.75 [M+3H] 3+

[0052] [ka]

[0053] Silicon phthalocyanine dihydride 4 (50 mg, 87 μmol) was added with (3-aminopropyl)dimethylethoxysilane (113 mg, 700 μmol) and dehydrated pyridine (30 mL), and the mixture was stirred and heated under reflux for 5 hours. The solvent was removed under reduced pressure, and the mixture was subjected to column chromatography (gradient: 1% for 3 min; 1-10% for 15 min; 10-20% for 10 min CH3OH in CH2Cl2, Yamazen Corporation Universal TM The residue was purified using Column Amino 40 mm 60Å 2.3×12.3 cm, 16 g, flow rate = 10 mL / min) to give the target compound 5 (62 mg, 77 μmol, 88%, deep blue color).

[0054] 1 H NMR (500 MHz, CDCl3): δ 9.65 (dd, J = 2.8 Hz, 5.7 Hz, 8H), 8.34 (dd, J = 2.8 Hz, 5.7 Hz, 8H), 1.18 (t, J= 7.6 Hz, 4H), -1.23 (m, 4H), -2.30 (m, 4H), -2.86 (s, 12H). LRMS (ESI): m / z 805.30 [M+H] +

[0055] [ka]

[0056] Compound 5 (29 mg, 36 μmol) in dehydrated dichloromethane (3 mL) was dissolved and stirred, and compound 6 (12 mg, 38 μmol) in dehydrated dichloromethane (2 mL) was added little by little at room temperature. The mixture was shielded from light with aluminum foil and stirred at room temperature for 16 hours. The solvent was removed under reduced pressure, and the mixture was subjected to column chromatography (gradient: 1% for 3 min; 1-5% for 15 min CH3OH in CH2Cl2, Yamazen Corporation Universal TM The residue was purified using Column Amino 40 mm 60Å 2.3×12.3 cm, 16 g, flow rate = 10 mL / min) to give the target compound 7 (12 mg, 12 μmol, 33%, deep blue color).

[0057] 1 H NMR (500 MHz, CDCl3): δ 9.65 (dd, J = 2.8 Hz, 5.7 Hz, 8H), 8.35 (d, J = 2.8 Hz, 5.7 Hz, 8H), 4.08 (t, J = 4.8 Hz, 2H), 3.73-3.60 (m, 8H), 3.39 (t, J = 5.7 Hz, 2H), 1.76 (m, 2H), 1.18 (t, J = 6.7 Hz, 2H), -1.23 (m, 4H), -2.30 (m, 4H), -2.86 (s, 12H). LRMS (ESI): m / z 1006.85 [M+H] +

[0058] [ka]

[0059] Compound 7 (4.8 mg, 4.8 μmol) was mixed with 1,3-Propanesultone (19 mg, 0.16 μmol), DIPEA (30 mg, 0.23 μmol) and methanol (500 μL), and the mixture was shielded from light with aluminum foil and stirred at 50 °C for 60 hours. After removing the solvent under reduced pressure, the mixture was diluted with water / acetonitrile (1:1) to 3000 μL and purified by reversed-phase HPLC (gradient: 50% for 5 min; 50-80% for 25 min; 80-100% for 10 min CH3CN in 50 mM triethylammonium acetate (pH 7.0), retention time = 15.8 (compound 8), 23.0 (compound 9) min, YMC-Triart C18, flow rate = 3.5 mL / min) to give the target compounds 8 (1.3 mg, 0.94 μmol, 20%, deep blue) and 9 (2.4 mg, 1.9 μmol, 40%, deep blue).

[0060] Compound 8: 1 H NMR (500 MHz, CD3OD): δ 9.72 (dd, J = 2.8 Hz, 5.7 Hz, 8H), 8.46 (dd, J = 2.8 Hz, 5.7 Hz, 8H), 3.97 (t, J = 4.8 Hz, 2H), 3.63-3.58 (m, 8H), 3.32 (m, 2H), 2.80-2.70 (m, 12H), 1.99 (t, J= 6.7 Hz, 2H), 1.70 (m, 6H), 1.60 (t, J= 6.7 Hz, 2H), -1.07 (m, 2H), -1.14 (m, 2H), -2.12 (m, 2H), -2.28 (m, 2H), -2.82 (s, 6H), -2.89 (s, 6H). LRMS (ESI): m / z 1372.55 [M+H] + Compound 9: 1H NMR (500 MHz, CD3OD): δ 9.70 (dd, J = 2.8 Hz, 5.7 Hz, 8H), 8.46 (dd, J = 2.8 Hz, 5.7 Hz, 8H), 3.97 (t, J = 4.8 Hz, 2H), 3.63-3.58 (m, 8H), 3.32 (m, 2H), 2.75 (t, J = 6.7 Hz, 4H), 2.56 (brs, 4H), 1.71 (m, 6H), 1.60 (t, J= 6.7 Hz, 2H), -1.00 (m, 2H), -1.14 (m, 2H), -2.15 (m, 2H), -2.27 (m, 2H), -2.83 (s, 6H), -2.89 (s, 6H). LRMS (ESI): m / z 1249.80 [M+H] +

[0061] [ka]

[0062] Compound 8 (1.6 mg, 1.0 μmol) was mixed with compound 3 (1.3 mg, 1.0 μmol) and methanol (500 μL), and the mixture was stirred at room temperature for 5 hours while shielding from light with aluminum foil. The mixture was diluted to 3000 μL with water / acetonitrile 3:1 and purified by reversed-phase HPLC (gradient: 30% for 5 min; 30-80% for 30 min; 80-100% for 8 min CH3CN in 50 mM triethylammonium acetate aqueous solution (pH 7.0), retention time = 26.5 min, YMC-Triart C18, flow rate = 3.5 mL / min) to obtain the target compound 10 (1.3 mg, 0.55 μmol, 55%, deep blue). Compound 10 is also referred to as compound 1. LRMS (ESI): m / z 1202.50 [M+2H] 2+

[0063] <Production Example 2: Synthesis of Compound 11> [ka]

[0064] Compound 9 (1.5 mg, 1.1 μmol) was mixed with compound 3 (1.5 mg, 1.1 μmol) and methanol (500 μL), and the mixture was stirred at room temperature for 5 hours while shielding from light with aluminum foil. The mixture was diluted to 3000 μL with water / acetonitrile 3:1 and purified by reversed-phase HPLC (gradient: 30% for 5 min; 30-80% for 30 min; 80-100% for 8 min CH3CN in 50 mM triethylammonium acetate aqueous solution (pH 7.0), retention time = 28.8 min, YMC-Triart C18, flow rate = 3.5 mL / min) to obtain the target compound 11 (1.6 mg, 0.68 μmol, 62%, deep blue). Compound 11 is also called compound 2.

[0065] LRMS (ESI): m / z 1141.65 [M+2H] 2+ , 760.90 [M+3H] 3+

[0066] <Production Example 3: Synthesis of Compound 13> [ka]

[0067] Compound 7 (2.8 mg, 2.8 μmol) was added with methyl iodide (22.8 mg, 161 μmol), K2CO3 (22.2 mg, 161 μmol), and dimethylformamide (500 μL), and the mixture was shielded from light with aluminum foil and stirred at 50 °C for 60 h. After removing the solvent under reduced pressure, the mixture was diluted with water / acetonitrile 1:1 to 1000 μL and purified by reversed-phase HPLC (gradient: 70% for 5 min; 70-100% for 25 min; CH3CN in 50 mM triethylammonium acetate aqueous solution (pH 7.0), retention time = 21.5 min, YMC-Triart C18, flow rate = 3.5 mL / min) to obtain the target compound 12 (2.1 mg, 2.0 μmol, 71%, deep blue).

[0068] 1 H NMR (400 MHz, CD3OD): δ 9.68 (dd, J = 2.8 Hz, 5.7 Hz, 8H), 8.44 (dd, J = 2.8 Hz, 5.7 Hz, 8H), 3.96 (t, J = 4.6 Hz, 2H), 3.63-3.58 (m, 8H), 3.31 (m, 2H), 2.44 (s, 9H), 1.94 (t, J = 8.8 Hz, 2H), 1.59 (t, J = 6.7 Hz, 2H), -1.16 (m, 4H), -2.14 (m, 2H), -2.28 (m, 2H), -2.84 (s, 6H), -2.89 (s, 6H). LRMS (ESI): m / z 1048.30 [M] +

[0069] [ka]

[0070] Compound 12 (1.0 mg, 1.0 μmol) was mixed with compound 3 (1.3 mg, 1.0 μmol) and methanol (500 μL) and stirred at room temperature for 5 hours while shielding from light with aluminum foil. The solvent was removed under reduced pressure, and the mixture was subjected to column chromatography (gradient: 25% for 5 min; 50-100% for 20 min CH3OH in CH2Cl2, Yamazen Corporation Universal TM The mixture was purified using Column Amino 40 mm 60Å 1.8×11.4 cm, 7 g, flow rate = 8 mL / min) to give the target compound 13 (0.92 mg, 0.43 μmol, 43%, deep blue color). Compound 13 is also referred to as compound 4.

[0071] LRMS (ESI): m / z 694.05 [M+2H] 3+ , 520.90 [M+3H] 4+

[0072] <Production Example 4: Synthesis of Compound 14> [ka]

[0073] Compound 7 (2.7 mg, 2.7 μmol) was mixed with compound 3 (3.6 mg, 2.7 μmol) and methanol (500 μL), and the mixture was stirred at room temperature for 5 hours while shielding from light with aluminum foil. The solvent was removed under reduced pressure, and the mixture was subjected to column chromatography (gradient: 25% for 5 min; 50-100% for 20 min CH3OH in CH2Cl2, Yamazen Corporation Universal TM The target compound 14 (3.5 mg, 1.7 μmol, 63%, deep blue color) was obtained by purification using Column Amino 40 mm 60Å 1.8×11.4 cm, 7 g, flow rate = 8 mL / min). Compound 10 is also referred to as compound 5. LRMS (ESI): m / z 1019.25 [M+2H] 2+ , 679.65 [M+3H] 3+ , 510.00 [M+4H] 4+

[0074] <Production Example 5: Synthesis of Compound 17> [ka]

[0075] Compound 5 (19.0 mg, 23.0 μmol) was mixed with trifluoroacetic acid (18.4 mg, 161 μmol) and dichloromethane (1.0 mL), and the mixture was stirred at room temperature for 4 hours while shielding from light with aluminum foil. Dichloromethane (10 mL), pyridine (1.0 mL) and water (10 mL) were added, and the organic layer was extracted. The solvent was distilled off under reduced pressure, and the extract was purified by column chromatography (2% CH3OH in CH2Cl2, Yamazen Corporation Universal TM The mixture was purified using Column Amino 40 mm 60Å 1.8×11.4 cm, 7 g, flow rate = 8 mL / min) to give the target compound 15 (6.1 mg, 6.1 μmol, 39%, deep blue color). 1 H NMR (500 MHz, CDCl3): δ 9.16 (m, 8H), 8.22 (d, J = 1.9 Hz, 4.7 Hz, 8H), 0.98 (t, J = 7.6 Hz, 2H), -1.47 (m, 2H), -2.51 (t, J = 8.6 Hz, 2H), -2.86 (s, 6H).

[0076] [ka]

[0077] Compound 15 (2.6 mg, 3.8 μmol) was mixed with compound 6 (1.2 mg, 3.8 μmol) and dehydrated dichloromethane (1.0 mL), and the mixture was stirred at room temperature for 4 hours while shielding from light with aluminum foil. The solvent was removed under reduced pressure, and the mixture was purified by column chromatography (gradient: 50-100% AcOEt in n-hexane, Yamazen Corporation Universal TM The mixture was purified using Premium Silica Gel 30 mm 60Å 1.8×11.4 cm, 7 g, flow rate = 8 mL / min) to give the target compound 16 (3.3 mg, 3.7 μmol, 98%, deep blue color).

[0078] 1 H NMR (500 MHz, CDCl3): δ 9.28 (brs, 8H), 8.25 (m, 8H), 4.00 (m, 2H), 3.67 (m, 8H), 3.37 (t, J = 4.8 Hz, 2H), 1.73 (m, 2H), -1.38 (m, 2H), -2.30 (m, 2H), -2.86 (s, 6H).

[0079] [ka]

[0080] Compound 16 (1.9 mg, 2.1 μmol) was mixed with compound 3 (2.5 mg, 1.9 μmol) and methanol (500 μL), and the mixture was stirred at room temperature for 5 hours while shielding from light with aluminum foil. The solvent was removed under reduced pressure, and the mixture was subjected to column chromatography (gradient: 10% for 5 min; 25% for 7 min CH3OH in CH2Cl2, Yamazen Corporation Universal TMThe target compound 17 (3.3 mg, 1.7 μmol, 89%, deep blue color) was obtained by purification using Column Amino 40 mm 60Å 1.8×11.4 cm, 7 g, flow rate = 8 mL / min). Compound 17 is also referred to as compound 3.

[0081] LRMS (ESI): m / z 952.55 [M-H2O+2H] 2+ , 635.85 [M-H2O+3H] 3+ , 476.80 [M-H2O+4H] 4+

[0082] <Example 1: Expression and purification of CEA-V2122 protein> V2122 is a streptavidin mutant described in Example 3 of International Publication WO2015 / 125820 (SEQ ID NO: 4 of International Publication WO2015 / 125820). The amino acid sequence of V2122 (sequence having a 6xHis tag at the C-terminus) is set forth in SEQ ID NO: 1 of the sequence listing.

[0083] scFv-V2122 is obtained by binding a single chain antibody (scFv) against CEACAM5 to the above V2122. This scFv-type anti-CEACAM5 antibody has an scFv sequence described in patent document US7626011B2. The amino acid sequence of the scFv-type anti-CEACAM5 antibody is shown in SEQ ID NO:2 in the sequence listing. The amino acid sequence of CEA-V2122, in which the scFv-type anti-CEACAM5 antibody and V2122 are bound with an amino acid linker (GGGGSGGGG) (SEQ ID NO:7), is shown in SEQ ID NO:3 in the sequence listing. To express the CEA-V2122 fusion protein, a pelB signal for secretion expression in E. coli was inserted at the N-terminus, and a 6xHis-Tag sequence was inserted at the C-terminus. The DNA codons of the CEA-V2122 gene sequence were optimized for E. coli, and an artificial gene was synthesized. The amino acid sequence is shown in SEQ ID NO: 4, and the DNA sequence is shown in SEQ ID NO: 5. The domain structure is shown in FIG. 1.

[0084] A specific protein expression vector used was a vector in which the chaperone skp gene was incorporated into MCS2 of the pETDuet1 vector. The skp gene was synthesized by artificial gene synthesis of DNA with codons optimized for E. coli based on the amino acid sequence described in SEQ ID NO:6 in the sequence listing. The synthesized skp gene was amplified by PCR using primers (AAGGAGATATACATATGGATAAAATTGCCATTGTTAATAT (SEQ ID NO:8), TTGAGATCTGCCATATGTTATTTCACTTGTTTCAGAACG (SEQ ID NO:9)), and cloned into MCS2 of the pETDue1 vector linearized with the restriction enzyme NdeI using the In-Fusion HD Cloning Kit to create pETDuet_skp. Next, the CEA-V2122 gene was incorporated into MCS1 of pETDuet_skp. Specifically, the artificially synthesized CEA-V2122 gene was amplified by PCR using primers (AGAAGGAGATATACCATGAAATATCTGCTGCCGAC (SEQ ID NO: 10), CGCCGAGCTCGAATTTTAATGATGGTGATGATGATG (SEQ ID NO: 11)). In addition, pETDuet_skp was linearized by PCR using primers (GGTATATCTCCTTCTTAAAGTTAAAC (SEQ ID NO: 12), AATTCGAGCTCGCGCCTGCAG (SEQ ID NO: 13)). The PCR-amplified CEA-V2122 and the linearized pETDuet_skp were cloned using the In-Fusion HD Cloning Kit. The cloned vector was sequenced to confirm the incorporated gene sequence, and named pETDuet_CEA-V2122_skp.

[0085] For protein expression, pETDuet_CEA-V2122_skp was transformed into BL21(DE3) (Nippon Gene) and pre-cultured in 2xYT medium (SIGMA-ADLRICH) at 37℃ overnight. The pre-cultured medium was added to fresh medium at a 100-fold dilution, and cultured at 37℃ until OD(600nm) = 0.5-2.0. IPTG was then added to a final concentration of 0.5mM, and cultured at 37℃ for 4 hours. The culture supernatant was collected and stored at 4℃.

[0086] CEA-V2122 protein was crudely purified by batch method using 6xHis-Tag added to the C-terminus. Specifically, cOmplete His-Tag Purification Resin equilibrated with Buffer A (50mM TrisHCl, 0.2M NaCl, 1mM EDTA, 5mM imidazole, pH8.0) was added to the culture supernatant stored at 4℃, and the protein was bound to the resin by stirring at 4℃ for 2 hours to overnight. The resin was then collected in a column and washed with 20 column volumes of Buffer A. The crude CEA-V2122 was then eluted with Buffer B (50mM TrisHCl, 0.2M NaCl, 1mM EDTA, 400mM imidazole, pH8.0) and collected.

[0087] Next, the crude product was purified using a Protein L column. Specifically, 1 mL of Capto L (GE Healthcare Life Sciences) was packed into a PD-10 column, and after equilibration with 10 column volumes of PBS, the crude product was applied and washed with 10 column volumes of PBS, eluted with 10 mM glycine hydrochloride pH 2.0, and centrifugal concentration was performed using a Vivaspin Turbo 15 (MWCO 100,000). The buffer was then replaced with PBS using a PD-10 (GE Healthcare Life Sciences), and centrifugal concentration was performed using a Vivaspin Turbo 4 (MWCO 100,000) to obtain the final purified product. After SDS-PAGE electrophoresis, the purity of the tetramer CEA-V2122 was examined by CBB staining, and the results are shown in Figure 2. The SDS-PAGE gel used was Mini-PROTEAN TGX 4-15% (Bio-Rad), and the CBB staining solution used was Bullet CBB Stain One (Ready To Use) (Nacalai Tesque). As shown in FIG. 2, it was confirmed that the purified CEA-V2122 was mainly composed of a tetramer of approximately 150 kDa.

[0088] <Example 2: Performance evaluation of CEA-V2122 in SPR> The affinity between CEA-V2122 and the antigen CEACAM5 was measured using a surface plasmon resonance (SPR) measurement device: Biacore T200 (GE Healthcare Life Sciences). Specifically, Recombinant Human CEACAM-5 / CD66e Protein, CF (R&D SYSTEMS) was immobilized on Sensor Chip CM5 (GE Healthcare Life Sciences) using an amine coupling kit (GE Healthcare Life Sciences), and the final immobilized amount of ligand was 279 RU. In addition, purified CEA-V2122 was adjusted as an analyte in a 2-fold dilution series from 1E-08 M to 6.25E-10 M. Data was obtained by Single-Cycle Kinetics analysis for interaction analysis. The obtained data was subjected to curve fitting using the Biacore T200 Evaluation Software, version 2.0 in the Bivalent Analyze mode, and the values ​​of ka1 = 3.208E+5 and kd1 = 3.461E-7 were obtained. D =kd1 / ka1, so K D The evaluation value obtained was = kd1 / ka1 = 3.461E-7 / 3.208E+5 = 1.078E-12. The results are shown in Figure 3. The sensorgrams shown in Fig. 3, K D The values ​​confirmed that CEA-V2122 strongly bound to CEACAM5.

[0089] In addition, the interaction analysis between CEA-V2122 and biotin modified products was also carried out using Biacore T200. The specific biotin modified product is the title compound 14 described in Example 1 of International Publication WO2018 / 07239. The specific analysis method is as follows. The purified CEA-V2122 was immobilized on Sensor Chip CM5 using an amine coupling kit with a target value of 5000RU. Five types of 2-fold dilution series of analyte concentrations from 1E-08 M to 6.25E-10 M were used. Data was acquired by Single-Cycle Kinetics analysis for the interaction analysis. Curve fitting was performed on the obtained data using the Bivalent Analyze mode in Biacore T200 Evaluation Software, version 2.0, and values ​​of ka1=3.792E+4 and kd1=4.424E-6 were obtained. In addition, in Bivalent Analyze, K D =kd1 / ka1, so K D The evaluation value obtained was = kd1 / ka1 = 3.792E+4 / 4.424E-6 = 1.167E-10. The results are shown in Figure 4. The sensorgram shown in FIG. D From these values, it was confirmed that CEA-V2122 strongly binds to the modified biotin.

[0090] <Example 3: Cell staining of CEACAM5-expressing cell line using FITC-labeled CEA-V2122> To stain cancer cell lines positive for CEACAM5 expression, 100 μg of purified CEA-V2122 protein was used for FITC labeling. Specifically, labeling was performed using the Fluorescein Labeling Kit - NH2 (Dojindo Laboratories) according to the dosage and use instructions in the operating instructions, and the resulting product was named CEA-V2122-FITC. Specific staining of cancer cell lines positive for CEACAM5 expression was as follows. 2.0x10 4CEACAM5-positive human gastric cancer MKN-45 cells and CEACAM5-negative human colon cancer DLD1 cells were seeded at 100 μL / well and cultured overnight. Next, 100 μL / well of culture medium containing 20 nM CEA-V2122-FITC and 1 μM Hoechist was added and incubated at 4°C for 30 minutes, after which images were taken using an In Cell Analyzer 6000 (GE Healthcare Life Sciences). The results are shown in Figures 5 and 6.

[0091] The results shown in Figure 5 confirmed that CEA-V2122-FITC specifically recognized CEACAM5 on the cell membrane surface, and the results shown in Figure 6 confirmed that CEACAM5 remained on the cell membrane surface after binding to CEA-V2122-FITC.

[0092] <Example 4: In vitro cytotoxicity test using CEA-V2122 and biotin modified with photoactivatable compound> Cytotoxicity tests were carried out using photoactivatable compound-labeled biotin variants (Compound 1 (Compound 10), Compound 2 (Compound 11), Compound 3 (Compound 17), Compound 4 (Compound 13) and Compound 5 (Compound 14)). Specifically, as shown in Figure 5, CEA-positive MKN45 cells and CEA-negative DLD1 cells were cultured in a 96-well cell culture plate at a cell number of 5x10 3 Cells were seeded at 100 μL / well and culture medium was added overnight. The complex solution of CEA-V2122 and biotin-modified photoactivatable compound was prepared by mixing CEA-V2122 and each compound at a molar ratio of 1:2 and incubating at room temperature for 10 minutes. The final concentration of CEA-V2122 was adjusted to 5 μg / mL with culture medium. The starting concentration was 5 μg / mL, and a 5-fold dilution series (5.0 μg / mL, 1.0 μg / mL, 0.2 μg / mL) was prepared. The control was medium without the complex.

[0093] Next, the culture medium of the cells cultured overnight was discarded, and the prepared dilution series of the complex (5.0 μg / mL, 1.0 μg / mL, 0.2 μg / mL) was added to the wells at 100 μL / well. 24 hours after the addition of the complex, the cells were exposed to 100 J / cm 2 light using an LED emitting light with a wavelength of 690 ± 10 nm. 2 The cells were irradiated with light so that the cell proliferation rate was 100%. After that, the cells were cultured for 24 hours and the number of live cells was compared using Cell Counting Kit-8 (Dojin Chemicals). The dosage was as per the instruction manual, and the reagent was added and incubated for 1.5 hours at 37°C in a CO2 incubator. The absorbance at 450 nm was measured, the average value was calculated, and after background correction, the percentage of cell proliferation was calculated for each condition relative to the control, with the control being set at 100%. The results for MKN45 cells are shown in Figure 7 and for DLD1 cells in Figure 8. The error bars in the figures indicate the standard deviation. These results confirmed that the reactions were antigen-specific and antibody concentration-dependent, except for the reaction with Compound 5 (5 μg / mL).

[0094] SEQ ID NO:1 AEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH

[0095] SEQ ID NO:2 (sm3E-scFv sequence) QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSS GGGGSGGGGSGGGGSENVLTQSPSSMSSVSVGDRVNIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIK

[0096] SEQ ID NO:3 QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSENVLTQSPSSMSVSVGDRVNIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASG VPSRFSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIKGGGGSGGGGAEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHHH

[0097] SEQ ID NO:4 MKYLLPTAAAGLLLLAAQPAMAQVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTA YLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSENVLTQSPSSMSVSVGDRVNIACSASSSVPYMHWLQQKPGKSPKL LIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIKGGGGSGGGGAEAGITGTWSDQLGDTFIVTAGADGALTGTYENAVGGAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH

[0098] SEQ ID NO:5

[0099] SEQ ID NO:6 MDKIAIVNMGSLFQQVAQKTGVSNTLENEFKGRASELQRMETDLQAKMKKLQSMKAGSDRTKLEKDVMAQRQTFAQKAQAFEQDRARRSNEERGKLVTRIQTAVKSVANSQDIDLVVDANAVAYNSSDVKDITADVLKQVK SEQ ID NO:7 GGGGSGGGG SEQ ID NO:8 AAGGAGATATACATATGGATAAAATTGCCATTGTTAATAT SEQ ID NO:9 TTGAGATCTGCCATATGTTATTTCACTTGTTTCAGAACG

[0100] SEQ ID NO:10 AGAAGGAGATATACCATGAAATATCTGCTGCCGAC SEQ ID NO:11 CGCCGAGCTCGAATTTTAATGATGGTGATGATGATG SEQ ID NO:12 GGTATTCTCCTTCTTAAAGTTAAAC SEQ ID NO:13 AATTCGAGCTCGGCGCGCCTGCAG

Claims

1. A compound represented by the following formula (2) or a salt thereof: 【Chemistry 1】 (In the formula, X is any one of the following hydrophilic groups: 【Chemistry 2】 X1a and X2a represent NH; Y １ indicates C, and Z １ represents NH, and V １ and V ２ indicates S, L １ and L ２ each independently represents a divalent linking group consisting of a combination of groups selected from -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, and an alkylene group having 1 to 10 carbon atoms, L ４ is a group consisting of a combination of groups selected from -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, -NH-, and an alkylene group having 1 to 10 carbon atoms, L ５ but, 【Chemistry 3】 a triazole group, -S-, -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, -NH-, an alkylene group having 1 to 10 carbon atoms, or a group consisting of a combination thereof, L ６ is -Si(CH 3 ) 2 -O-, -CONH-, -NHCO-, -COO-, -OCO-, -CO-, -O-, -NH-, an alkylene group having 1 to 10 carbon atoms, or a combination thereof.

2. Any of the following compounds: 【Chemistry 4】

3. A treatment kit for cancer or solid tumors comprising: (1) a conjugate according to claim 1 or 2; and (b) a conjugate of a streptavidin mutant having the amino acid sequence set forth in SEQ ID NO: 1 (although part or all of the C-terminal histidine tag may be deleted) and a molecular probe.

4. The treatment kit of claim 3 , wherein the molecular probe is an anti-EREG antibody, an anti-CEA antibody, or an anti-HER2 antibody.

Images