Photosensitizer-modified antibodies that reduce the side effects of photoimmunotherapy

Abstract

The objective is to provide a treatment method for diseases that utilizes antibodies conjugated with a photosensitizer, while reducing side effects. [Solution] An antibody modified with a photosensitizer and a quencher, characterized in that 1) the photosensitizer and quencher are bound to a peptide, and the peptide is bound to the N-terminus of the antibody; 2) the peptide contains a protease recognition sequence; 3) the photosensitizer is located closer to the antibody than the quencher, and the protease recognition sequence is located between the photosensitizer and the quencher; and 4) the peptide is encapsulated within the antibody when the antibody is not bound to an antigen, and is exposed from the antibody when the antibody is bound to an antigen.

Description

[Technical Field]

[0001] This invention relates to antibodies modified with a photosensitizer and a photoquenzylant, and therapeutic agents for photoimmunotherapy utilizing these antibodies. By using the antibodies of this invention, photoimmunotherapy with fewer side effects becomes possible. [Background technology]

[0002] Trastuzumab significantly improves treatment outcomes for HER2-positive breast cancer patients by inhibiting HER2 signaling and stimulating an immune response. However, many patients experience disease progression due to trastuzumab resistance to HER2-positive cells. To enhance treatment efficacy, trastuzumab conjugated with toxins such as emtansine and deruxtecan has been administered to patients with residual disease, significantly improving survival rates. However, normal, healthy cells also express a certain amount of antigen, and the binding of antibody-toxin complexes causes significant cytotoxicity. To produce immunoprobes with low toxicity to normal cells, trastuzumab has been conjugated with photosensitizers that are toxic only upon light irradiation. Photosensitizers are key components of photodynamic therapy, killing cancer cells by generating reactive oxygen species, particularly singlet oxygen, under light irradiation. Photosensitizers that absorb near-infrared light effectively treat deep tissue cancer, and trastuzumab conjugated with such photosensitizers selectively destroys cancer cells while minimizing adverse effects on normal cells in xenograft models, thus raising expectations for future clinical applications (Non-patent documents 1 and 2). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Kim, KS; Kim, J.; Kim, DH; Hwang, HS; Na, K. Multifunctional trastuzumab-chlorin e6 conjugate for the treatment of HER2-positive human breast cancer. Biomater. Sci. 2018, 6 (5), 1217-1226. DOI: 10.1039 / C7BM01084B [Non-Patent Document 2] Mitsunaga, M.; Ogawa, M.; Kosaka, N.; Rosenblum, LT; Choyke, PL; Kobayashi, H. Cancer cell-selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules. Nat. Med. 2011, 17 (12), 1685-1691. DOI: 10.1038 / nm.2554 [Overview of the project] [Problems that the invention aims to solve]

[0004] Antibodies conjugated with photosensitizers show promise in treating diseases such as cancer, but they are known to cause damage to normal cells and photosensitivity due to off-target effects caused by unbound or nonspecific binding. This invention was made against this backdrop and aims to provide a treatment method for diseases using antibodies conjugated with photosensitizers that reduces side effects. [Means for solving the problem]

[0005] The inventors of the present invention have conducted extensive research to solve the above problems and have found that by attaching a photosensitizer to the N-terminus of an antibody together with a quencher via a peptide that is cleaved only when it binds to an antigen, the side effects of the photosensitizer can be significantly reduced. Based on this finding, the present invention has been completed. That is, the present invention provides the following [1] to [7].

[0006] [1] An antibody modified with a photosensitizer and a quencher, characterized in that 1) the photosensitizer and quencher are bound to a peptide, the peptide is bound to the N-terminus of the antibody, 2) the peptide contains a protease recognition sequence, 3) the photosensitizer is located closer to the antibody than the quencher, and the protease recognition sequence is located between the photosensitizer and the quencher, and 4) the peptide is encapsulated within the antibody when the antibody is not bound to an antigen, and is exposed from the antibody when the antibody is bound to an antigen.

[0007] [2] The antibody according to [1], characterized in that the peptide comprises a trypsin recognition sequence and / or a matrix metalloproteinase recognition sequence.

[0008] [3] The antibody according to [1], characterized in that the peptide is a) a peptide containing the amino acid sequence described in SEQ ID NO: 1, b) a peptide containing an amino acid sequence in which one or more amino acid residues are substituted, added, or deleted in the amino acid sequence described in SEQ ID NO: 1, or c) a peptide containing an amino acid sequence having 90% or more homology to the amino acid sequence described in SEQ ID NO: 1.

[0009] [4] The antibody according to [1], characterized in that the photosensitizer is a porphyrin-based photosensitizer, a texaphyllin-based photosensitizer, a chlorin-based photosensitizer, a bacteriochlorin-based photosensitizer, a phthalocyanine-based photosensitizer, or a dye-based photosensitizer.

[0010] [5] The antibody according to [1], characterized in that the quenching agent is an azo quenching agent, a rhodamine quenching agent, a cyanine quenching agent, or DYQ quenchers.

[0011] [6] The antibody according to [1], wherein the antibody targets a protein that is highly expressed in cancer cells.

[0012] [7] A therapeutic agent for photodynamic immunotherapy, comprising the antibody according to any one of [1] to [6]. [Effects of the Invention]

[0013] The present invention provides a novel antibody modified with a photosensitizer and a quencher. The use of this antibody enables photodynamic immunotherapy with reduced side effects. [Brief Description of the Drawings]

[0014] [Figure 1] A diagram showing the fluorescence response and singlet oxygen generation of the antibody (BQ-MB-Traz) modified with a photosensitizer and a quencher. [Figure 2] A diagram showing the cytotoxicity against cancer cells of the antibody (BQ-MB-Traz) modified with a photosensitizer and a quencher in an experimental system mimicking the state where the antibody does not bind to cells and flows in the blood. [Figure 3] A diagram showing the cytotoxicity against cancer cells of the antibody (BQ-MB-Traz) modified with a photosensitizer and a quencher in an experimental system mimicking the state where the antibody binds to cancer cells presenting an antigen. [Modes for Carrying Out the Invention]

[0015] Hereinafter, the present invention will be described in detail. The antibody of the present invention is an antibody modified with a photosensitizer and a quencher, characterized by the following properties: 1) the photosensitizer and the quencher are bound to a peptide, and the peptide is bound to the N-terminus of the antibody; 2) the peptide comprises a protease recognition sequence; 3) the photosensitizer is located closer to the antibody than the quencher, and the protease recognition sequence is present between the photosensitizer and the quencher; 4) the peptide is encapsulated within the antibody when the antibody does not bind to an antigen, and is exposed from the antibody when the antibody binds to an antigen.

[0016] The antibody of the present invention has the following properties. A) It can generate reactive oxygen species only when the antibody binds to an antigen (antigen-binding-dependent reactive oxygen species generation). B) It can generate reactive oxygen species only in the tumor microenvironment (cancer cell-specific reactive oxygen species generation). C) It can generate reactive oxygen species only at the site irradiated with laser light.

[0017] Having the properties as described above, the antibody of the present invention can suppress damage to normal cells, kill only target cells (such as cancer cells), and can also suppress the onset of photosensitivity, which is a problem in therapeutic methods using photosensitizers.

[0018] The reason why the antibody of the present invention has the above property A) is explained below. When the antibody does not bind to an antigen, the peptide is encapsulated in the antibody. In this state, the peptide exhibits resistance to proteases, so even if a protease is present around the antibody, the peptide is not cleaved. For this reason, the quencher bound to the peptide suppresses the activation of the photosensitizer, and no reactive oxygen species are generated even when light is irradiated. On the other hand, when the antibody is bound to an antigen, the peptide is exposed from the antibody and loses protease resistance. As a result, if a protease is present, the peptide is cleaved at the protease recognition sequence, and the quencher is released. Thereby, the suppression of photosensitizer activation by the quencher is canceled, and when light is irradiated, the photosensitizer generates reactive oxygen species.

[0019] The antibody used is not particularly limited, but antibodies that target proteins highly expressed in cancer cells are preferred. Examples of proteins highly expressed in cancer cells include HER2, EGFR, CD20, CD19, PD-1, PD-L1, CTLA-4, VEGF, CD52, CD30, Claudin 18.2, CEA, and GPC3. Examples of antibodies that target proteins highly expressed in cancer cells include trastuzumab, cetuximab, rituximab, blinatumomab, nivolumab, atezolizumab, ipilimumab, bevacizumab, alemtuzumab, brentuximab, saltuzumab, rabetuzumab, and GC33.

[0020] The photosensitizer used is not particularly limited, and any photosensitizer commonly used in photoimmunotherapy or photodynamic therapy can be used. Specifically, porphyrin-based photosensitizers, texaphyrin-based photosensitizers, chlorin-based photosensitizers, bacteriochlorin-based photosensitizers, phthalocyanine-based photosensitizers, or dye-based photosensitizers can be used. Porphyrin-based photosensitizers are photosensitizers with porphyrin as their basic structure, such as ALA, M-ALA, Vertporfin, Metavix, HpD, PpIX, Texaphyrins, TLD1433, Purpurin, Tetraphenylporphyrin, tetrasulfonate TPPS4, amine-modified porphyrin, Core-modified porphyrin, and SnET2. Texaphyrin-based photosensitizers are photosensitizers with texaphyrin as their basic structure, such as Motexafin lutetium and Lutexaphyrin. Chlorin-based photosensitizers are photosensitizers with chlorin as their basic structure, such as Laserphyrin (Talaporfin Sodium), Temoporfin, Chlorin e6, mTHPC, HPPH, and chlorin e6 Trimethyl Ester. Bacteriochlorin-based photosensitizers are photosensitizers with bacteriochlorin as their basic structure, such as Padoporfin and LUZ11. Phthalocyanine-based photosensitizers are photosensitizers with phthalocyanine as their basic structure, such as AlPcSn, Pc4, ZnPc, and IR700. Dye-based photosensitizers are photosensitizers that also function as dyes, such as Indocyanine Green (ICG), BODIPY, Rose bengal, Toluidine blue, Methylene blue, Rh-123, AA1, and merocyanine 540.Preferred photosensitizers include ALA, M-ALA, Vertporfin, Metavix, HpD, Motexafin lutetium, Laserphyrin (Talaporfin Sodium), Temoporfin, Chlorin e6, Padoporfin, LUZ11, Indocyanine Green (ICG), and Methylene blue.

[0021] The quencher used is not particularly limited as long as it can suppress singlet oxygen generation from the photosensitizer, and can be selected according to the photosensitizer used. Specific examples of quenchers include azo quenchers, rhodamine quenchers, cyanine quenchers, or DYQ quenchers. Azo quenchers are quenchers whose basic skeleton is a compound containing an azo group, such as DABCYL, BHQ-0, BHQ-1, BHQ-2, BHQ-3, IQ4, BBQ-650, and Eclipse Dark Quencher. Rhodamine quenchers are quenchers whose basic skeleton is rhodamine, such as QSY7, QSY9, QSY21, and SINQ780. Cyanine quenchers are quenchers whose basic skeleton is cyanine, such as IRDye QC-1. DYQ quenchers include, for example, DYQ 1, DYQ 2, DYQ 3, and DYQ 4. As quenchers, BMN quenchers (e.g., BMN-Q460, BMN-Q535, BMN-Q590, BMN-Q620, BMN-Q651), Tide quenchers (e.g., TQ1, TQ2, TQ3, TQ4, TQ5, TQ6, TQ7), QXL quenchers (e.g., QXL490, QXL520, QXL570, QXL610, QXL670, QXL680), Deep dark quenchers (e.g., DDQ I, DDO II), Iowa Black Quenchers (e.g., IBFQ, IBRQ), and Onyx quenchers (e.g., OQ A, OQ B, OQ C, OQ D) may also be used.

[0022] The peptide used contains a protease recognition sequence. The protease recognition sequence is not particularly limited and can include trypsin recognition sequences, matrix metalloproteinase (MMP) recognition sequences, etc. Since it is preferable that the peptide be cleaved around cancer cells, it is preferable that it contains a recognition sequence for proteases that are abundant around cancer cells (e.g., MMP2 or MMP9).

[0023] Furthermore, the peptide used has the property of being encapsulated within the antibody when the antibody is not bound to the antigen, and being exposed from the antibody when the antibody is bound to the antigen. An example of a peptide having such properties is a peptide containing the amino acid sequence described in SEQ ID NO: 1 (Lys Pro Leu Gly Leu Ala Gly Arg Ser Lys Leu Pro Glu Thr Gly Gly). Here, "a peptide containing the amino acid sequence described in SEQ ID NO: 1" includes not only peptides consisting solely of the amino acid sequence described in SEQ ID NO: 1, but also peptides in which one or more amino acid residues are added to the N-terminus and / or C-terminus of the amino acid sequence described in SEQ ID NO: 1. The number of added amino acid residues is not particularly limited as long as it does not cause the above properties to be lost, but it is preferably 4 or less, more preferably 3 or less, even more preferably 2 or less, and particularly preferably 1.

[0024] Peptides containing amino acid sequences similar to the amino acid sequence described in SEQ ID NO: 1 are considered to have similar properties to peptides containing the amino acid sequence described in SEQ ID NO: 1, and such peptides may be used. Examples of amino acid sequences similar to the amino acid sequence described in SEQ ID NO: 1 include b) peptides containing amino acid sequences in which one or more amino acid residues are substituted, added, or deleted from the amino acid sequence described in SEQ ID NO: 1, or c) peptides containing amino acid sequences having 90% or more homology to the amino acid sequence described in SEQ ID NO: 1. Here, the number of amino acid residues to be substituted, added, or deleted is not particularly limited as long as it does not cause the above properties to be lost, but 4 or less is preferred, 3 or less is more preferred, 2 or less is even more preferred, and 1 is particularly preferred. Furthermore, the homology of the amino acid sequences is not particularly limited as long as it does not cause the above properties to be lost, but 90% or more is preferred, 95% or more is more preferred, 97% or more is even more preferred, and 99% or more is particularly preferred. The homology of the amino acid sequences can be calculated using the BLASTP program provided by NCBI (National Center of Biotechnology Information).

[0025] In the antibody of the present invention, the photosensitizer and quencher are bound to a peptide. The photosensitizer and quencher are usually bound to the side chains of amino acid residues constituting the peptide (for example, the amino group of a lysine residue). The peptide is also bound to the N-terminus of the antibody. The N-terminus may be the N-terminus of the light chain, but the N-terminus of the heavy chain is preferred. The photosensitizer is located closer to the antibody than the quencher, and a protease recognition sequence is located between the photosensitizer and the quencher. Therefore, in the presence of protease, the peptide is cleaved at the protease recognition sequence, and the quencher is released.

[0026] The antibody of the present invention can be used as a therapeutic agent for photoimmunotherapy. In photoimmunotherapy, an antibody conjugated with a photosensitizer is administered as a drug, and then laser light is irradiated onto the diseased area, such as cancer, to generate reactive oxygen species from the photosensitizer, thereby killing cancer cells and other organisms. In the present invention, "therapeutic agent for photoimmunotherapy" refers to a drug used in this photoimmunotherapy.

[0027] The photoimmunotherapy therapeutic agent of the present invention is mainly administered to humans, but may also be administered to animals other than humans. Examples of animals other than humans include mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, and monkeys.

[0028] The primary target of treatment is cancer, but other diseases may also be treated. Examples of cancers that can be treated include head and neck cancer, skin cancer, lung cancer, esophageal cancer, bladder cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, and pancreatic cancer.

[0029] The photoimmunotherapy therapeutic agent of the present invention can be formulated by mixing it with a pharmaceutically acceptable carrier or diluent according to known methods. The dosage form is not particularly limited and can be an injection, tablet, powder, granule, capsule, liquid, suppository, sustained-release, etc. The method of administration is also not particularly limited and can be administered orally or parenterally. Examples of parenteral administration methods include injection or infusion into the skin, peritoneal cavity, vein, artery, or cerebrospinal fluid. The dosage varies depending on the target patient, method of administration, etc., but for example, when administering the antibody of the present invention as an injection to an adult, the antibody of the present invention can be administered in 1 to several doses in a single treatment, with a dose of 2 to 8 mg / kg per dose.

[0030] After administering the photoimmunotherapy drug of the present invention, the diseased area is irradiated with light. The wavelength of the irradiated light can be appropriately determined depending on the type of photosensitizer used. [Examples]

[0031] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0032] [Materials and Methods] material The plasmid pVITRO1-Trastuzumab-IgG1 / κ (#61883) was obtained from Addgene (USA). Oligonucleotides were purchased from Eurofins Genomics. Restriction enzymes were purchased from New England Biolabs (USA). The In-Fusion HD cloning kit was purchased from Takara Bio. Trastuzumab (Traz) was purchased from Chugai Pharmaceutical Co., Ltd. 293fectin, used in FreeStyle293-F cells, Opti-MEM, and the FreeStyle 293 Expression System, was obtained from Thermo Fisher Scientific (USA). Protein G Mag Sepharose Xtra was purchased from Cytiva (USA). HER2 was obtained from Sino Biological Co. Ltd. (China). N(α)-Fmoc-protected amino acids and Fmoc-Gly-Wang resin were purchased from Novabiocam (USA). MB-NHS (ADMB2-21) and BHQ3-NHS (BHQ-3000S-5) were purchased from Funakoshi (Japan) and LGC Biosearch Technologies (USA), respectively. SOSG was purchased from Thermo Fisher Scientific (USA). MDA-MB-468 cells and SK-BR-3 cells were obtained from ATCC (USA). The WST-1 cell proliferation assay system was purchased from Takara Bio (Japan).

[0033] Construction of expression plasmids Using the plasmid pVITRO1-Trastuzumab-IgG1 / κ as a template, an expression plasmid of Traz was constructed by fusing the amino acid sequence GGGTG to the N-terminus of the heavy chain. The GGGTG-VH insert was amplified using the primers Traz-VH-back and Traz-VH-forward. The CH1 and Fc inserts were obtained by digesting the template with BsmBI and BspEI. For the light chain, the VL insert was amplified using the primers Traz-VL-back and Traz-VL-forward. The primers Traz-CL-back and Traz-CL-forward were used to amplify the CL insert. The template was digested with NotI and XbaI to obtain a vector. The four inserts were introduced into a single vector using the In-Fusion HD Cloning Kit to construct the plasmid pVITRO1-G3-Traz. Furthermore, to obtain higher expression levels, the heavy and light chains were introduced into the AgeI and EcoRV sites of pcDNA3.4, respectively, to construct pcDNA3.4-Tras-G3-HC and pcDNA3.4-Tras-G3-LC. [Table 1]

[0034] Antibody production and purification G3-Traz was prepared by diluting 30 μg of plasmid pVITRO1-G3-Traz (or 8 μg of pcDNA3.4-Tras-G3-HC and 17 μg of pcDNA3.4-Tras-G3-LC, respectively) produced in FreeStyle 293-F cells according to the FreeStyle 293 Expression System protocol, in 1 mL of Opti-MEM and gently mixing. Next, 60 μL of 293-fectin was mixed with 1 mL of Opti-MEM and incubated at 25°C for 5 minutes. The DNA and 293-infectin were gently mixed and incubated for a further 30 minutes to form the DNA-293-infectin complex. This complex was then divided into 1 × 10⁻⁶ units. 6The cell suspension was added dropwise to a 125 mL Erlenmeyer flask containing 30 mL of cells / mL. The cells were cultured on an orbital shaker rotating at 125 rpm at 37°C in 8% CO2 for 72 hours, after which the cells were harvested. The cell suspension was centrifuged at 10000 × g for 20 minutes at 4°C, and the supernatant was mixed with 200 μL of Protein G magnetic beads in a vertical tube rotator at 4°C for 2 hours. After centrifugation at 700 × g for 2 minutes at 4°C, the beads were transferred to a 1.5 mL tube and placed on a MagRack. The beads were washed three times with phosphate-buffered saline (PBS) at pH 7.4 and eluted with 100 μL of 100 mM glycine hydrochloride (pH 2.8). 4 microliters of 1 M Tris-HCl (pH 9.0) were added for neutralization. Finally, the eluate buffer was replaced with PBS using a G-25 Microspin column.

[0035] Synthesis of BQ-MB peptide The peptide CH3CONH-K(Boc)LPETGG-COOH was synthesized according to the Fmoc solid-phase peptide synthesis protocol using N(α)-Fmoc-protected amino acids immobilized on Fmoc-Gly-Wang resin. After the sequence was fully assembled on the resin, 25% acetic anhydride was added to the resin at 25°C for 5 minutes to acetylate the N-terminal primary amine group. The peptide was then deprotected by treatment with 95% trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% H2O at 25°C for 2 hours, purified by high-performance liquid chromatography (HPLC) (Chromaster, Hitachi, Japan), and identified by mass spectrometry (MALDI-TOF-MS) (UltrafleXtreme, Bruker, USA). The peptide CH3CONH-K(NH2)LPETGG-COOH and MB-NHS with a peptide / dye molar ratio of 10 were mixed in dimethylformamide and left in the dark at 25°C for 2 hours. HPLC purification and MS identification were performed to obtain CH3CONH-K(MB)LPETGG-COOH.

[0036] For the conjugation of the quencher and photosensitizer, peptides CH3CONH-K(Mtt)PLGLAGRSK(Boc)LPETGG-COOH, each containing a trypsin recognition sequence and an MMP2 / MMP9 recognition sequence, were assembled on a resin and acetylated using the same method. These peptides were treated with 1% trifluoroacetic acid and 5% triisopropylsilane in dichloromethane for 2 minutes to remove the Mtt protecting group. Subsequently, CH3CONH-K(NH2)PLGLAGRSK(Boc)LPETGG-COOH was incubated in 0.3 mM BHQ3-NHS and 6 mM N,N-diisopropylethylamine in dimethylformamide in the dark for 2 hours to deprotect it and obtain CH3CONH-K(BHQ3)PLGLAGRSK(NH2)LPETGG-COOH. This was further conjugated with MB using the same procedure as described above for CH3CONH-K(MB)LPETGG-COOH to obtain CH3CONH-K(BHQ3)PLGLAGRSK(MB)LPETGG-COOH.

[0037] Preparation of BQ-MB-Traz Three conjugate peptides, each containing an LPETGG sequence at the C-terminus, were ligated to G3-Traz using transpeptidase Sortase A, which cleaves the LPXTG motif and forms a covalent bond to the N-terminal glycine. To prepare BQ-MB-Traz, 5 μM G3-Traz, 100 μM BQ-MB-LPETGG, and 2 μM Sortase A were mixed overnight at 4°C in 50 mM Tris-HCl buffer (pH 8.0). Antibody purification and buffer exchange were performed as described above for G3-Traz production. MB-Traz and BQ-MB-Traz were prepared using the same procedure.

[0038] Fluorescence of BQ-MB-Traz upon antigen addition and 1 O 2 Generate The fluorescence intensity of solutions containing 30 nM BQ-MB-Traz and 60 nM HER2 mixed in PBST, as well as solutions without this mixture, was measured using a CLARIOstar in the dark at 25°C for 24 hours, with excitation wavelengths of 630 / 25 nm and emission wavelengths of 680 / 25 nm. Fluorescence intensity was plotted over time, and linear fitting was applied to evaluate the rate of change in MB fluorescence in the presence and absence of HER2. 1 To evaluate O2 generation, antibodies and antigens were prepared similarly, incubated for 0, 12, and 24 hours, and then mixed with 1 μM SOSG. A WISE-6100 LuminoGraph I was used with a wavelength of 630 nm and a light irradiation dose of 20 J / cm². 2 Irradiation continued until [the desired result was reached]. Fluorescence intensity was measured every 10 minutes using CLARIOstar at excitation wavelengths of 488 / 10 nm and emission wavelengths of 525 / 10 nm. From the fluorescence intensity-time plot... 1 The O2 generation rate was calculated and evaluated.

[0039] Preparation of randomly labeled Traz-MB 10 μM Traz and 150 μM MB-NHS were incubated in 0.1 M Na2HPO4, 150 mM NaCl, pH 8.5 in the dark at 25 °C for 3 hours. Purification and quantification of Traz-MB were performed in the same manner as for G3-Traz preparation described above.

[0040] cell culture HER2-negative MDA-MB-468 and HER2-positive SK-BR-3 breast cancer cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) high-glucose medium supplemented with 10% (v / v) fetal bovine serum, 100 IU / mL penicillin, and 100 μg / mL streptomycin in an incubator at 37°C and 5% CO2.

[0041] Cell viability assay for evaluating cytotoxicity Cells were seeded at a density of 5000 cells per well in 100 μL of medium into six 96-well clear flat-bottom polystyrene tissue culture-treated microplates (Corning, USA). The cells were allowed to adhere for 24 hours at 37°C under 5% CO₂. Subsequently, Traz, Traz-MB, or BQ-MB-Traz was added to the cells at final concentrations of 0, 15, 30, 60, 120, and 240 nM. Then, two types of experiments were performed: one in which 630 nm light irradiation was carried out immediately using an LED device (Revox, Switzerland), and another in which the cells were incubated for 24 hours, replaced with fresh DMEM, and then subjected to light irradiation. To estimate cell viability, 10 μL of Premix WST-1 reagent was added to each well, and the plates were incubated for 2 hours at 37°C in 5% CO₂. Absorbance was measured at 450 nm using a microplate reader SH-1000Lab with 655 nm as the reference wavelength.

[0042] [Results] Dequenching and 1 O 2 Generate A peptide (BQ-MB) conjugated with both a quencher (BHQ3) and a photosensitizer (methylene blue, MB) was linked to the N-terminus of the heavy chain of Traz, to prepare an antibody modified with the photosensitizer and the quencher (BQ-MB-Traz) (Figure 1A).

[0043] When the antigen (HER2) was added to BQ-MB-Traz, a gradual and steady increase in fluorescence over 24 hours was observed, which was not observed in the absence of HER2 (Figures 1B, C). After 24 hours of irradiation, BQ-MB-Traz in the presence of HER2 was approximately 5 times higher than BQ-MB-Traz in the absence of HER2 1 showed an O₂ generation rate, whereas almost no O₂ was detected without light irradiation 1 (Figure 1C). Taken together, these results indicate that when BQ-MB is linked to Traz, the linker between BHQ3 and MB interacts with the antibody, but after the antibody binds to the antigen, the interaction is reduced, the linker undergoes hydrolysis, leading to increased fluorescence and 1This suggests that it leads to O2 generation.

[0044] Evaluation of cytotoxicity (1) The cytotoxicity of BQ-MB-Traz against cancer cells was evaluated in an experimental system that mimicked the situation where antibodies do not bind to cells but circulate in the bloodstream (Figure 2A). HER2-negative breast cancer cell lines (MDA-MB-468) and HER2-positive breast cancer cell lines (SK-BR-3) were used as cancer cells. Cancer cells were treated with Traz, randomly labeled Traz with MB (Traz-MB), or BQ-MB-Traz, followed immediately by a dose of 80 J / cm³. 2 The cells were irradiated with optimized light, their absorbance was measured, and their viability was calculated (Figure 2A).

[0045] After irradiation, Traz treatment did not have a significant effect on cell viability, but Traz-MB caused significant cell death and IC 50 The levels were 67 nM in MDA-MB-468 cells and 50 nM in SK-BR-3 cells (Figure 2B, C). Notably, BQ-MB-Traz did not significantly reduce cell viability (Figure 2B, C). In either cell type, cell viability was not significantly affected by any antibody in the absence of irradiation. These results suggest that Traz-MB is effective near the cell, regardless of HER2 expression. 1 The generation of O2 induces cytotoxicity, and the suppression of BQ-MB-Traz photosensitivity effectively reduces cytotoxicity, suggesting that side effects in photodynamic therapy were minimized.

[0046] Evaluation of cytotoxicity (2) The cytotoxicity of BQ-MB-Traz against cancer cells was evaluated in an experimental system that mimicked antibody binding to antigen-presenting cancer cells (Figure 3A). In MDA-MB-468 cells, BQ-MB-Traz did not show a significant decrease in cell viability, and a protective effect against HER2-negative cell lines was confirmed, but Traz-MB showed an IC50 effect. 50This caused a slight decrease at 74 nM (Figure 3B). In SK-BR-3 cells, after irradiation, Traz-MB and BQ-MB-Traz showed equivalent IC50s of 14 nM and 20 nM, respectively. 50 This was shown (Figure 3C). Traz-MB is taken up by cells via HER2 binding and enters the cell. 1 It is thought that O2 was generated, resulting in cytotoxicity. In contrast, BQ-MB-Traz was thought to exhibit cytotoxicity either by binding to HER2 on the cell surface and being cleaved by exposure of the linker between BHQ3 and MB, or by being taken up into the cell and cleaved intracellularly. Taken together, these results suggest that BQ-MB-Traz may achieve effective therapeutic outcomes with minimal side effects in photodynamic therapy for HER2-positive breast cancer. [Industrial applicability]

[0047] Since the antibodies of the present invention are used as pharmaceuticals, the present invention is applicable to industries related to pharmaceuticals.

Claims

1. An antibody modified with a photosensitizer and a quencher, characterized in that: 1) the photosensitizer and quencher are bound to a peptide, which is bound to the N-terminus of the antibody; 2) the peptide contains a protease recognition sequence; 3) the photosensitizer is located closer to the antibody than the quencher, and the protease recognition sequence is located between the photosensitizer and the quencher; and 4) the peptide is encapsulated within the antibody when the antibody is not bound to an antigen, and is exposed from the antibody when the antibody is bound to an antigen.

2. The antibody according to claim 1, characterized in that the peptide comprises a trypsin recognition sequence and / or a matrix metalloproteinase recognition sequence.

3. The antibody according to claim 1, characterized in that the peptide is a) a peptide comprising the amino acid sequence described in SEQ ID NO: 1, b) a peptide comprising an amino acid sequence in which one or more amino acid residues are substituted, added, or deleted in the amino acid sequence described in SEQ ID NO: 1, or c) a peptide comprising an amino acid sequence having 90% or more homology to the amino acid sequence described in SEQ ID NO:

1.

4. The antibody according to claim 1, characterized in that the photosensitizer is a porphyrin-based photosensitizer, a texaphyllin-based photosensitizer, a chlorin-based photosensitizer, a bacteriochlorin-based photosensitizer, a phthalocyanine-based photosensitizer, or a dye-based photosensitizer.

5. The antibody according to claim 1, characterized in that the quencher is an azo quencher, a rhodamine quencher, a cyanine quencher, or DYQ quenchers.

6. The antibody according to claim 1, characterized in that the antibody targets a protein that is highly expressed in cancer cells.

7. A therapeutic agent for photoimmunotherapy, characterized by containing the antibody described in any one of claims 1 to 6.

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