Patent Application
20220016245
The present invention relates to a conjugate of a biotin-modified dimer and a phthalocyanine dye and the use thereof.
Avidin and biotin, or streptavidin and biotin have an extremely high affinity between them (Kd=10−15 to 10−14 M). This is one of the strongest interactions between two biomolecules. At present, the interaction between avidin/streptavidin and biotin has been widely applied in the field of biochemistry, molecular biology, or medicine. A drug delivery method and a pretargeting method, in which high binding ability between avidin/streptavidin and biotin is combined with an antibody molecule, have been devised. In connection with these studies, a mutant streptavidin with a reduced affinity for natural biotin and a biotin-modified dimer having a high affinity for the mutant streptavidin with a low affinity for natural biotin are reported in Patent Document 1.
On the other hand, photoimmunotherapy is a therapeutic method of using a photosensitizer and an irradiation light to destroy specific cells in a body. When a photosensitizer is exposed to a light with a specific wavelength, it generates cytotoxic reactive oxygen species capable of inducing apoptosis, necrosis, and/or autophagy to around cells. For example, Patent Document 2 discloses a method of killing cells, comprising: a step of allowing cells comprising a cell surface protein to come into contact with a therapeutically effective amount of one or more antibodies-IR700 molecules, wherein the antibodies specifically bind to the cell surface protein; a step of irradiating the cells with a light at a wavelength of 660 to 740 nm and at a dose of at least 1 Jcm−2; and a step of allowing the cells to come into contact with one or more therapeutic agents at approximately 0 to 8 hours after the irradiation, thereby killing the cells. Patent Document 3 discloses a method of inducing cytotoxicity to a subject affected with a disease or a pathology, comprising: (a) administering to a subject, a therapeutically effective drug comprising a phthalocyanine dye such as IRDye (registered trademark) 700DX conjugated with a probe specifically binding to the cell of the subject; and (b) irradiating the cell with an appropriate excitation light in an amount effective for inducing cell death.
Patent Document 1: International Publication WO2015/125820
Patent Document 2: Japanese Patent No. 6127045
Patent Document 3: JP Patent Publication (Kohyo) No. 2017-524659 A
It is an object of the present invention to provide a conjugate of a biotin-modified dimer and a phthalocyanine dye, which is used in photoimmunotherapy. It is another object of the present invention to provide a therapeutic kit, in which a combination of the above-described conjugate of a biotin-modified dimer and a phthalocyanine dye and a mutant streptavidin-molecular probe conjugate is used.
As a result of intensive studies directed towards achieving the above-described objects, the present inventor has found that the proliferation of cancer cells can be suppressed by photoimmunotherapy using a conjugate of a biotin-modified dimer and a phthalocyanine dye, thereby completing the present invention.
Specifically, according to the present invention, the following inventions are provided.
[1] A conjugate of a compound represented by the following formula (1) or a salt thereof and a phthalocyanine dye:
wherein
X1a, X1b, X2a and X2b each independently represent O or NH,
Y1 and Y2 each independently represent C or S,
Z1 and Z2 each independently represent O, S or NH,
V1 and V2 each independently represent 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 group comprising a functional group capable of binding to a phthalocyanine dye at the terminus, and
L4 represents a trivalent linking group.
[2] The conjugate according to the above [1], wherein the compound represented by the above formula (1) is represented by the following formula (2):
wherein each symbol is as defined in the above [1].
[3] The conjugate according to the above [1] or [2], wherein X1a, X1b, X2a and X2b represent NH; Y1 and Y2 represent C; Z1 and Z2 represent NH; and V1 and V2 represent S. [4] The conjugate according to any one of the above [1] to [3], wherein 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 containing 1 to 10 carbon atoms.
[5] The conjugate according to any one of the above [1] to [4], wherein L3 represents a group consisting of a combination of groups selected from —CONH—, —NHCO—, —COO—, —OCO—, —CO—, —O—, and an alkylene group containing 1 to 10 carbon atoms, and further comprising an amino group at the terminus.
[6] The conjugate according to any one of the above [1] to [5], wherein the phthalocyanine dye is represented by the following formula (21):
wherein L21 represents a divalent linking group; R21 represents a functional group capable of binding to the compound represented by the formula (1) or a salt thereof, and X and Y each independently represent a hydrophilic group, —OH, a hydrogen atom, or a substituent.
[7] The conjugate according to the above [6], wherein the hydrophilic group represented by X and/or Y is the following:
[8] The conjugate according to the above [6] or [7], wherein L21 represents a group consisting of a combination of groups selected from —CONH—, —NHCO—, —COO—, —OCO—, —CO—, —O—, and an alkylene group containing 1 to 10 carbon atoms.
[9] The conjugate according to any one of the above [6] to [8], wherein R21 represents an active ester group.
[10] A conjugate represented by any one of the following:
[11] A therapeutic agent comprising the conjugate according to any one of the above [1] to [10].
[12] A therapeutic kit, comprising: (1) the conjugate according to any one of the above [1] to [10]; and (b) a conjugate of a mutant streptavidin comprising the amino acid sequence as set forth in SEQ ID NO: 1 and a molecular probe.
[13] The therapeutic kit according to the above [12], wherein the molecular probe is an anti-EREG antibody, an anti-CEA antibody, or an anti-HER2 antibody.
[14] A fusion protein encoded by the nucleotide sequence as set forth in SEQ ID NO: 2, which can express a fusion protein of a mutant streptavidin comprising the amino acid sequence as set forth in SEQ ID NO: 1 and an scFv-type antibody that recognizes an EREG antigen.
The proliferation of cancer cells can be suppressed by photoimmunotherapy using the conjugate of a biotin-modified dimer and a phthalocyanine dye according to the present invention.
Hereinafter, the present invention will be described in more detail.
The present invention relates to a conjugate of a biotin-modified dimer and a phthalocyanine dye.
The biotin-modified dimer is a compound represented by the following formula (1) or a salt thereof, and is preferably a compound represented by the following formula (2) or a salt thereof. As such a biotin-modified dimer, the compound described in International Publication WO2015/125820 can be used.
wherein
X1a, X1b, X2a and X2b each independently represent O or NH,
Y1 and Y2 each independently represent C or S,
Z1 and Z2 each independently represent O, S or NH,
V1 and V2 each independently represent 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 group comprising a functional group capable of binding to a phthalocyanine dye at the terminus, and
L4 represents a trivalent linking group.
In the formula (1) and the formula (2), the portions represented by the following structures:
are preferably any one of the following portions, but are not limited thereto:
X1a, X1b, X2a and X2b preferably represent NH; Y1 and Y2 preferably represent C; Z1 and Z2 preferably represent NH; and V1 and V2 preferably represent S.
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 containing 1 to 10 carbon atoms.
Preferably, 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 containing 1 to 10 carbon atoms.
Preferably, L1 and L2 each independently represent a divalent linking group consisting of a combination of groups selected from —CONH—, —NHCO—, and an alkylene group containing 1 to 10 carbon atoms.
L4 represents a trivalent linking group, and is preferably the following:
(which is a benzene-derived trivalent linking group or a nitrogen atom).
L3 is preferably a group consisting of a combination of groups selected from —CONH—, —NHCO—, —COO—, —OCO—, —CO—, —O—, and an alkylene group containing 1 to 10 carbon atoms, and further comprising an amino group at the terminus.
The phthalocyanine dye is preferably a silicon phthalocyanine dye. Specific examples of the phthalocyanine dye, such as IRDye (registered trademark) 700DX, are described in, for example, U.S. Pat. No. 7,005,518.
As such a phthalocyanine dye, a dye represented by the following formula (21) can be used. As an example, a dye represented by the following formula (22) can be used.
In the above formulae, L21 represents a divalent linking group, and R21 represents a functional group capable of binding to the compound represented by the formula (1) or a salt thereof.
In the above formulae, X and Y each independently represent a hydrophilic group, —OH, a hydrogen atom, or a substituent. Examples of the substituent used herein may include, but are not particularly limited to, a halogen atom (a fluorine atom), a substituent containing a carbon atom (a hydrocarbo group, etc.), and a substituent containing a nitrogen atom (an amino group, etc.).
Specific examples of X and Y may include:
(i) a case where both X and Y are hydrophilic groups;
(ii) a case where either X or Y is a hydrophilic group, and the other is —OH or a hydrogen atom; and
(iii) a case where X and Y are —OH or hydrogen atoms.
The hydrophilic group(s) represented by X and/or Y are not particularly limited. One example is shown below.
As a phthalocyanine dye, a commercially available product such as IRDye (registered trademark) 700DX can be used. In the present invention, an NHS ester of IRDye (registered trademark) 700DX is used, and is allowed to react with a biotin-modified dimer having an amino group to produce a conjugate. Other variations of IRDye (registered trademark) 700DX are described in U.S. Pat. No. 7,005,518, and those can also be used.
R21 represents a functional group capable of binding to the compound represented by the formula (1) or a salt thereof. R21 is preferably a functional group that can react with a carboxyl group, amine or a thiol group on the biotin-modified dimer and can bind thereto. Preferred examples of R21 may include, but are not particularly limited to, activated ester, halogenated acyl, halogenated alkyl, appropriately substituted amine, anhydride, carboxylic acid, carbodiimide, hydroxyl, iodoacetamide, isocyanate, isothiocyanate, maleimide, NHS ester, phosphoramidite, sulfonic acid ester, thiol, and thiocyanate.
L21 represents a divalent linking group, and for example, it may include: any given combination of an ether, thioether, amine, ester, carbamate, urea, thiourea, oxy or amide bond, or a single, double, triple or aromatic carbon-carbon bond; or a phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bond; or an aromatic or heteroaromatic bond. L21 is preferably a group consisting of a combination of groups selected from —CONH—, —NHCO—, —COO—, —OCO—, —CO—, —O—, and an alkylene group containing 1 to 10 carbon atoms.
-L21-R21 may include a phosphoramidite group, NHS ester, active carboxylic acid, thiocyanate, isothiocyanate, maleimide, and iodoacetamide.
L21 contains a —(CH2)n— group, and in the formula, n is an integer of 1 to 10, and is preferably an integer of 1 to 4. As one example, -L21-R21 is —O—(CH2)3—OC(O)—NH—(CH2)5—C(O)O—N-succinimidyl.
According to the present invention, provided is a therapeutic kit, in which the conjugate of a biotin-modified dimer and a phthalocyanine dye of the present invention is combined with a mutant streptavidin-molecular probe conjugate.
As mutant streptavidins used herein, those described in International Publication WO2014/129446 and International Publication WO2015/125820 can be used. Particularly preferably, the mutant streptavidin 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 description of the present application) can be used.
Examples of the molecular probe used herein may include an antibody, a peptide, a nucleic acid, and an aptamer. Specifically, an antibody, a peptide, a nucleic acid, an aptamer, etc., which target the following antigens specifically expressed in cancer, can be used:
Epiregulin (EREG), ROBO 1, 2, 3, and 4, 1-40-β-amyloid, 4-1BB, SAC, 5T4, ACVR2B, adenocarcinoma antigen, α-fetoprotein, angiopoetin 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 (IL-2 receptor a chain), 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 β chain, fibronectin extra domain-B, folate receptor 1, Frizzled receptor, GD2, GD3 ganglioside, GMCSF receptor a chain, GPNMB, hepatitis B surface antigen, hepatitis B virus, HER1, HER2/neu, HER3, HGF, HIV-1, HLA-DRβ, HNGF, Hsp90, human β 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 αvβ, integrin γ induced protein, interferon receptor, interferon (α/β 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 α, 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, MUC1 tumor-specific glycosylation, TWEAK receptor, TYRP1 (glycoprotein 75), VEGF-A, VEGFR-1, VEGFR2, vimentin, and VWF.
Among the above-described antigens, epiregulin (EREG), CEA, and HER2 are preferable.
A fusion body of a molecular probe such as a tumor antigen-specific antibody molecule and a mutant streptavidin is prepared, and the prepared fusion body is then administered to a patient, so that the mutant streptavidin can be accumulated specifically in cancer cells. Subsequently, a conjugate of a biotin-modified dimer having an affinity for the above-described mutant streptavidin and a phthalocyanine dye is administered to the patient, so that the phthalocyanine dye can be accumulated exactly in the cancer cells.
Otherwise, in the present invention, a complex is prepared by binding a conjugate of a molecular probe such as a tumor antigen-specific antibody molecule and a mutant streptavidin with a conjugate of a biotin-modified dimer and a phthalocyanine dye, and the thus prepared complex can be administered to a patient.
Various types of molecules can be used as antibodies that are to be bound to the mutant streptavidin. Either a polyclonal antibody or a monoclonal antibody may be used. The subclass of the antibody is not particularly limited. Preferably, IgG, and particularly preferably, IgG1 is used. Furthermore, the term “antibody” includes all of modified antibodies and antibody fragments. Examples of such an antibody include, but are not limited to: a humanized antibody; a human type antibody; a human antibody; antibodies from various types of animals such as a mouse, a rabbit, a rat, a guinea pig and a monkey; a chimeric antibody between a human antibody and an antibody from a different type of animal; diabody; scFv; Fd; Fab; Fab; and F(ab)′2.
The conjugate of the mutant streptavidin and the antibody can be obtained by applying a method known to persons skilled in the art. For example, such a conjugate can be obtained by a chemical bond method (U.S. Pat. No. 5,608,060). Alternatively, DNA encoding the mutant streptavidin is ligated to DNA encoding an antibody, and using an expression vector or the like, the ligated DNA is then expressed in a host cell, so that such a conjugate can be obtained in the form of a fusion protein. The DNA encoding the mutant streptavidin may be ligated to the DNA encoding an antibody via DNA encoding a suitable peptide, called a linker. The mutant streptavidin-molecular probe conjugate is desirably produced, while keeping the specific binding force between an antibody and a target molecule.
The conjugate of a biotin-modified dimer and a phthalocyanine dye according to the present invention is administered to a subject, and the cells are then irradiated with an excitation light in an amount effective for suppression of cell proliferation or induction of cell death, so that the cell proliferation can be suppressed or the cell death can be induced, and thereby the subject can be treated.
Preferably a complex of the conjugate of a biotin-modified dimer and a phthalocyanine dye according to the present invention and a mutant streptavidin-molecular probe conjugate is administered to a subject, and the cells are then irradiated with an excitation light in an amount effective for suppression of cell proliferation or induction of cell death, so that the cell proliferation can be suppressed or the cell death can be induced, and thereby the subject can be treated.
The subject includes humans and non-human animals. Examples of the subject may include humans and experimental animals such as mice. The subject is preferably affected with a disease regarding which suppression of cell proliferation or induction of cell death is desired. For example, the subject is affected with cancer or solid tumor.
Examples of the “cancer” may include carcinoma, lymphoma, blastoma, sarcoma, and leukemia or malignant lymphoma. Specific examples of the cancer may include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer (“NSCLC”), pulmonary adenocarcinoma and pulmonary squamous cell carcinoma, peritoneal cancer, hepatocarcinoma, corpus ventriculi or stomach cancer, including digestive cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial membrane cancer or endometrial carcinoma, salivary gland carcinoma, kidney or renal region cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.
The solid tumor means a benign or malignant, abnormal cell mass that generally does not contain a capsule. Examples of the solid tumor may include glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal gland tumor, hemangioblastoma, acoustic neuroma, oligodendrocyte, meningioma, melanoma, neuroblastoma, and retinoblastoma.
In the photoimmunotherapy, the “conjugate of a biotin-modified dimer and a phthalocyanine dye” or the “complex of the conjugate of a biotin-modified dimer and a phthalocyanine dye and a mutant streptavidin-molecular probe conjugate” is administered to a subject, and thereafter, the subject is irradiated with a light, so that the subject can be treated.
Examples of the administration method to the subject may include, but are not limited to, a local route, an injection (a subcutaneous injection, an intramuscular injection, an intradermal injection, an intraperitoneal injection, an intratumoral injection, an intravenous injection, etc.), an oral route, an ocular route, a sublingual route, a rectal route, a percutaneous route, an intranasal route, a vaginal route, and an inhalation route.
The “conjugate of a biotin-modified dimer and a phthalocyanine dye” or the “complex of the conjugate of a biotin-modified dimer and a phthalocyanine dye and a mutant streptavidin-molecular probe conjugate” is preferably administered in a therapeutically effective amount. The therapeutically effective amount per 60 kg is at least 0.5 mg (mg/60 kg), at least 5 mg/60 kg, at least 10 mg/60 kg, at least 20 mg/60 kg, at least 30 mg/60 kg, or at least 50 mg/60 kg. For example, when it is intravenously administered, the applied dose is 1 mg/60 kg, 2 mg/60 kg, 5 mg/60 kg, 20 mg/60 kg, or 50 mg/60 kg, and it is, for example, 0.5 to 50 mg/60 kg. In another example, the therapeutically effective amount is at least 100 μg/kg, at least 500 μg/kg or at least 500 μg/kg, and it is, for example, at least 10 μg/kg. For example, when it is intratumorally or intraperitoneally administered, the dose is 100 μg/kg, 250 μg/kg, approximately 500 μg/kg, 750 μg/kg, or 1000 μg/kg, and it is, for example, 10 μg/kg to 1000 μg/kg. In one example, when it is administered in the form of a solution for local administration, the therapeutically effective amount is 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, 100 μg/ml or the like, or it is 20 μg/ml to 100 μg/ml, or it is at least 500 μg/ml, or at least 1 μg/ml.
The above-described dose can be administered once or divided doses over several administrations (2, 3, or 4 times, etc.), or as a single preparation.
The “conjugate of a biotin-modified dimer and a phthalocyanine dye” or the “complex of the conjugate of a biotin-modified dimer and a phthalocyanine dye and a mutant streptavidin-molecular probe conjugate” can be administered alone, or can also be administered in the presence of a pharmaceutically acceptable carrier, or can also be administered in the presence of other therapeutic agents (other anticancer agents, etc.).
The “conjugate of a biotin-modified dimer and a phthalocyanine dye” or the “complex of the conjugate of a biotin-modified dimer and a phthalocyanine dye and a mutant streptavidin-molecular probe conjugate” can bind to target cells or target tissues, such as circulating tumor cells or solid tumor cells. Thereafter, the target cells or tissues are irradiated with a light, so that the above-described conjugate or complex can absorb the light and can damage or destroy the target cells or tissues.
In the photoimmunotherapy, the wavelength of the irradiation light is preferably 660 to 740 nm, and the irradiation light has a wavelength of, for example, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, or 740 nm. Light irradiation may be carried out using a device equipped with a near infrared (NIR) light emitting diode.
The light irradiation amount is at least 1 J/cm2, for example, at least 4 J/cm2, at least 10 J/cm2, at least 15 J/cm2, at least 20 J/cm2, at least 50 J/cm2, or at least 100 J/cm2. It is, for example, 1 to 500 J/cm2. Light irradiation may be carried out several times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times).
The present invention will be more specifically described in the following examples. However, these examples are not intended to limit the scope of the present invention.
IR Dye 700 NHS Ester (3.0 mg, 1.5 μmop, disodium hydrogen phosphate buffer (pH 8.4, 144 μL) and dimethyl sulfoxide (120 μL) were added to Psyche J (1.8 mg, 1.6 μmop, light was then shielded with an aluminum foil, and the mixture was then stirred at mom temperature for 24 hours. Thereafter, the reaction solution diluted with water to 500 μL was purified by reverse phase HPLC (gradient: 0% for 5 min; 0-100% for 100 min acetonitrile in a 50 mM triethylammonium acetate aqueous solution (pH 6.5), retention time=47.2 min, YMC-Triart C18, flow rate=3.5 mL/min) to obtain a target compound 1 (dark blue green). Quantification was carried out from the reported molar absorption coefficient of IR700 at 694 nm in water (165,000 (M−1 cm−1)), and the yield (1.0 μmol, 72%) was calculated.
LRMS (ESI): m/z 1303 [M+2H]2+, 869 [M+3H]3+
A 0.1% formic acid aqueous solution (266 μL) and acetonitrile (133 μL) were added to the compound 1 (0.375 mg, 144 nmol), and the obtained mixture was stirred with a Vortex mixer and was then centrifuged. The resultant was left at rest under conditions of in a darkroom at 37° C. for 90 minutes. The resulting solution was purified by reverse phase HPLC (gradient: 0% for 5 min; 0-100% for 100 min acetonitrile in a 50 mM triethylammonium acetate aqueous solution (pH 6.5), retention time=51.6 min, YMC-Triart C18, flow rate=3.5 mL/min) to obtain a target compound 2 (dark blue green). Quantification was carried out in the same manner as described above, and the yield (48 nmol, 33%) was calculated.
LRMS (ESI): m/z 1054 [M-H2O+2H]2+, 703 [M-H2O+3H]3+
A 0.1% formic acid aqueous solution (266 μL) and acetonitrile (133 μL) were added to the compound 1 (0.375 mg, 144 nmol), and the obtained mixture was stirred with a Vortex mixer and was then centrifuged. The resultant was left at rest under conditions of in a darkroom at 37° C. for 28 hours. The resulting solution was purified by reverse phase HPLC (gradient: 0% for 5 min; 0-100% for 100 min acetonitrile in a 50 mM triethylammonium acetate aqueous solution (pH 6.5), retention time=56.2 min, YMC-Triart C18, flow rate=3.5 mL/min) to obtain a target compound 3 (dark blue green). Quantification was carried out in the same manner as described above, and the yield (15 nmol, 10%) was calculated.
LRMS (ESI): m/z 813 [M-H2O+2H]2+, 542 [M-H2O+3H]3+
<Preparation of scFv-Cupid Molecule Expression Vector>
The sequences of a gene ST01 (the nucleotide sequence and amino acid sequence thereof are as set forth in SEQ ID NO: 2 and SEQ ID NO: 3, respectively), which fuses an scFv-type antibody recognizing an EREG antigen with a Cupid molecule (the amino acid sequence thereof is as set forth in SEQ ID NO: 1 in the sequence listing) and expresses a fusion protein in Escherichia coli, and the gene sequences of a chaperone molecule skp, were obtained by artificial synthesis. For the expression of the protein in Escherichia coli, a pETDuet-1 vector was used. Specifically, the artificially synthesized chaperone skp gene sequences (the nucleotide sequence and amino acid sequence thereof are as set forth in SEQ ID NO: 4 and SEQ ID NO: 5, respectively) were amplified by PCR using the primers (skp_Fw: TACATATGGATAAAATTGCCATTGTTAAT (SEQ ID NO: 6), and skp_Rv: TITTATCCATATGTATATCTCCTTC (SEQ ID NO: 7)), and a band was separated by electrophoresis and was then purified. As such a pETDuet-1 vector, a vector linearized at the MCS2 NdeI site by a treatment with the restriction enzyme NdeI was produced. In accordance with the protocol and amount of In-Fusion HD Cloning Kit (Takara Bio, Inc.), skp was ligated to the linearized vector, and it was then confirmed by cloning and sequence analysis that a sequence of interest had been incorporated. The sequence of interest was incorporated into the MCS2 of the same vector to obtain an expression vector pETDuet_skp.
Thereafter, ST01 was incorporated into pETDuet_skp as follows. Using pETDuet_skp as a template, the vector was linearized using the primers (Linear1 Rv: GGTATATCTCCTTCTTAAAG (SEQ ID NO: 8), and Linear1 Fw: AATTCGAGCTCGGCGCGCCTG (SEQ ID NO: 9)). At the same time, the artificially synthesized ST01 gene was amplified by PCR using the primers (ST01 Fw: AGAAGGAGATATACCATGAAATATCTGCTGCCGAC (SEQ ID NO: 10), and ST01 Rv: CGCCGAGCTCGAATTTTAATGATGGTGATGATGATG (SEQ ID NO: 11)). The gel was cut out and was then purified to prepare an insert. The linearized vector and the insert were cloned in accordance with the protocol and amount of In-Fusion HD Cloning Kit (Takara Bio, Inc.), and thereafter, it was confirmed by sequence analysis that the incorporated gene was a correct one, which was then used as an ST01 expression vector.
<Expression and Purification of scFv-Cupid Protein>
A plasmid vector, which was confirmed by cloning and sequence analysis that a gene of interest had been incorporated therein, was introduced into competent cells BL21 (DE3) (ECOS Competent E. coli BL21 (DE3), Nippon Gene Co., Ltd.), so that the cells were transformed. A culture medium prepared by culturing the cells in 100 mL of 2×YT medium overnight was inoculated into 1 L of a culture solution, and the obtained mixture was then cultured at 37° C. When the OD value at 600 nm exceeded 2.0, IPTG was added to the culture to a final concentration of 0.5 mM, so that the expression of the protein was induced.
Regarding ST01, after induction of the expression with IPTG, it was cultured at 16° C. overnight, and thereafter, a culture supernatant was recovered.
Using Ni-NTA resin (cOmplete His-Tag Purification Resin, SIGMA-ALDRICH), the recovered culture supernatant was subjected to affinity purification with 6×His-Tag added to the C-terminus of the protein according to a batch method, so as to obtain a roughly purified product. Furthermore, using Protein L resin (Capto L, GE Healthcare), affinity purification with a x light chain was carried out. In this operation, as a binding/washing buffer, PBS was used, and as an elution buffer, a glycine-hydrochloric acid solution (10 mM, pH 2.0) was used. The purified product was concentrated with a centrifugal filtration filter (Vivaspin Turbo 15 100K MWCO, Sartorius). After completion of the concentration, a disposable column for buffer exchange (PD-10, GE Healthcare) was used to replace the buffer for the product with PBS.
DLD-1 cells (EREG-positive) (human colon adenocarcinoma-derived cells) were cultured in 10% FBS RPMI1640 (FUJIFILM Wako Pure Chemical Corporation). For a cytotoxicity test, the cells were seeded on a 96-well plate at a cell density of 3×103 to 5×103 cells per well. These cells were prepared the day before the cytotoxicity test.
Each ST01 and compounds 1, 2 and 3 dissolved in 100% DMSO were each mixed with each other at a molar ratio of 1:2 before dilution of the medium, and the obtained mixtures were then incubated at room temperature for 10 minutes. Thereafter, the reaction mixtures were diluted with a culture solution (10% FBS RPMI1640), so that the final concentration of scFv-Cupid could be 10 μg/mL (60 to 61 nM) and the final concentration of the Psyche compound could be 120 nM. Thereafter, the thus obtained mixtures were incubated at room temperature for 10 minutes to obtain test solutions. It is to be noted that a culture solution was used as a control without addition of scFv-Cupid, whereas DMSO was used as a control of compound.
The culture solution for the cells cultured the day before the cytotoxicity test, as mentioned above, was discarded, and 100 μL of the test solution was then added to each well, followed by performing culture for 4 hours. Thereafter, the resultant was irradiated with a light having a peak wavelength of 690 nm±10 nm. A measurement apparatus, in which a sensor with 400 to 1100 nm was connected with a photo power meter PM121D (both of which are manufactured by THORLABS), was used to measure an irradiation energy on the culture plate, and the irradiation time was then determined. The irradiation energy and the irradiation time were set to be 1.1 J/cm2 (2 min), 4.5 J/cm2 (8 min), and 18.4 J/cm2 (30 min). After completion of the irradiation at each irradiation time, culture was carried out in 5% CO2 at 37° C., and 48 hours later, cell proliferation was assayed using Cell Counting Kit-8 (DOJINDO LABORATORIES). Specifically, 10 μL of Cell Counting Kit-8 solution was added into each well, and it was then incubated in 5% CO2 at 37° C. for 1 to 2 hours. Thereafter, the absorbance at 450 nm was measured using an absorption spectrometer. A well containing only the medium was defined to be a background (BG), and a well containing only DMSO was defined to be 100% control (Cont.). The value of cell proliferation % under individual culture conditions was calculated according to the following expression: 100×(measurement value A−BG)/(Cont.−BG) (unit: %).
In
In
V2122 is a mutant streptavidin described in Example 3 of International Publication WO2015/125820 (SEQ ID NO: 4 shown in International Publication WO2015/125820). The amino acid sequence of V2122 (a sequence having a 6×His tag at the C-terminus) is as set forth in SEQ ID NO: 12 in the sequence listing.
scFv-V2122 is prepared by binding a single-chain antibody (scFv) against CEACAM5 with the above-described V2122. This scFv-type anti-CEACAM5 antibody is an scFv sequence described in a patent document U.S. Pat. No. 7,626,011B2. The amino acid sequence of the scFv-type anti-CEACAM5 antibody is as set forth in SEQ ID NO: 13 in the sequence listing. In addition, the amino acid sequence of CEA-V2122 prepared by binding the scFv-type anti-CEACAM5 antibody with V2122 via an amino acid linker (GGGGSGGGG) (SEQ ID NO: 22) is as set forth in SEQ ID NO: 14 in the sequence listing.
For the expression of a CEA-V2122 fusion protein, the DNA codon of a CEA-V2122 gene sequence, in which a pelB signal for secretion and expression in Escherichia coli had been incorporated into the N-terminus and a 6×His-Tag sequence had been incorporated into the C-terminus, was optimized for Escherichia coli, thereby synthesizing an artificial gene. This amino acid sequence is as set forth in SEQ ID NO: 15 in the sequence listing, and the DNA sequence is as set forth in SEQ ID NO: 16 in the sequence listing. Moreover, an outline of a domain structure is shown in
As a specific protein expression vector, a vector prepared by incorporating a chaperone skp gene into MCS2 of a pETDuet1 vector was used. Regarding the skp gene, the DNA codon was optimized for Escherichia coli based on the amino acid sequence as set forth in SEQ ID NO: 17 in the sequence listing, thereby synthesizing an artificial gene. The synthesized skp gene was amplified by PCR, using the primers (AAGGAGATATACATATGGATAAAATTGCCATTGTTAATAT (SEQ ID NO: 23), and TTGAGATCTGCCATATGTTATTTCACTTGTTTCAGAACG (SEQ ID NO: 24)), and the amplified gene was then cloned into MCS2 of the pETDue1 vector linearized with the restriction enzyme NdeI, using In-Fusion HD Cloning Kit, so as to obtain pETDuet_skp. Subsequently, the CEA-V2122 gene was incorporated into MCS1 of pETDuet_skp. Specifically, the artificially synthesized CEA-V2122 gene was amplified by PCR, using the primers (AGAAGGAGATATACCATGAAATATCTGCTGCCGAC (SEQ ID NO: 25), and CGCCGAGCTCGAATTTTAATGATGGTGATGATGATG (SEQ ID NO: 26)). Moreover, pETDuet_skp was linearized by PCR, using the primers (GGTATATCTCCTTCTTAAAGTTAAAC (SEQ ID NO: 27), and AATTCGAGCTCGGCGCGCCTGCAG (SEQ ID NO: 28)). The CEA-V2122 amplified by PCR and the linearized pETDuet_skp were subjected to cloning using In-Fusion HD Cloning Kit. The cloned vector was confirmed by sequencing, in terms of the gene sequence incorporated therein, and thereafter, it was referred to as pETDuet_CEA-V2122_skp.
For the expression of the protein, pETDuet_CEA-V2122_skp was transformed into BL21(DE3) (Nippon Gene Co., Ltd.), which was then pre-cultured in 2×YT medium (SIGMA-ALDRICH) at 37° C. overnight. The medium used in the pre-culture was added to a new medium for 100-fold dilution, and culture was then carried out at 37° C., until OD (600 nm) became 0.5 to 2.0. Subsequently, IPTG was added to the culture to a final concentration of 0.5 mM, and the obtained mixture was then cultured at 37° C. for 4 hours. Thereafter, a culture supernatant was recovered and was then preserved at 4° C.
The CEA-V2122 protein was roughly purified according to a batch method utilizing 6×His-Tag added to the C-terminus. Specifically, cOmplete His-Tag Purification Resin equilibrated with buffer A (50 mM Tris-HCl, 0.2 M NaCl, 1 mM EDTA, and 5 mM Imidazole; pH 8.0) was added to the culture supernatant preserved at 4° C. The obtained mixture was stirred for 2 hours to overnight at 4° C., so that the protein was allowed to bind to the resin. Subsequently, the resin was recovered into a column, and a 20 column volume of washing operation was performed with buffer A. Thereafter, a roughly purified product of CEA-V2122 was recovered by elution with buffer B (50 mM Tris-HCl, 0.2 M NaCl, 1 mM EDTA, and 400 mM Imidazole; pH 8.0).
Subsequently, the roughly purified product was purified using a Protein L column. Specifically, 1 mL of Capto L (GE Healthcare Life Sciences) was filled into a PD-10 column, and was then equilibrated with 10 column volume of PBS, and the aforementioned roughly purified product was then applied thereto. Thereafter, the resultant was washed with 10 column volume of PBS, was then eluted with 10 mM glycine hydrochloride (pH 2.0), and was then subjected to centrifugal concentration using Vivaspin Turbo 15 (MWCO 100,000). Moreover, using PD-10 (GE Healthcare Life Science), the buffer was replaced with PBS, and centrifugal concentration was further carried out using Vivaspin Turbo 4 (MWCO 100,000) to obtain a finally purified product. After completion of SDS-PAGE electrophoresis, the purity of tetramer CEA-V2122 was assayed by CBB staining. The results are shown in
From
The affinity of CEA-V2122 for the antigen CEACAM5 was evaluated using a surface plasmon resonance (SPR) measuring device, Biacore T200 (GE Healthcare Life Sciences). Specifically, Recombinant Human CEACAM-5/CD66e Protein, CF (R & D SYSTEMS) was immobilized on Sensor Chip CMS (GE Healthcare Life Sciences) using an amine-coupling kit (GE Healthcare Life Sciences). The final amount of the ligand immobilized was 279 RU. Moreover, with regard to the purified CEA-V2122, two-fold serial dilutions from 1E-08 M to 6.25E-10 M were prepared as analytes. Regarding interaction analysis, data were obtained by single-cycle kinetics analysis. Using Biacore T200 Evaluation Software, version 2.0, the obtained data were subjected to curve fitting in a bivalent analysis mode, and the following values were obtained: ka1=3.208E+5, and kd1=3.461E−7. Moreover, since evaluation can be carried out at KD=kd1/ka1 in the bivalent analysis, the evaluation value KD=kd1/ka1=3.461E−7/3.208E+5=1.078E−12 was obtained. These results are shown in
From the KD value in the sensorgram shown in
Furthermore, the interaction between CEA-V2122 and a modified biotin was also analyzed using Biacore T200. The modified biotin was specifically the title compound 14 described in Example 1 of International Publication WO2018/07239. In addition, the analysis method was specifically as follows. That is, an amine-coupling kit was used, a target value was set to be 5000 RU, and the purified CEA-V2122 was immobilized on Sensor Chip CMS. With regard to the concentrations of the analytes, 5 types of two-fold serial dilutions from 1E-08 M to 6.25E-10 M were used. Regarding interaction analysis, data were obtained by single-cycle kinetics analysis. Using Biacore T200 Evaluation Software, version 2.0, the obtained data were subjected to curve fitting in a bivalent analysis mode, and the following values were obtained: ka1=3.792E+4, and kd1=4.424E−6. Moreover, since evaluation can be carried out at KD=kd1/ka1 in the bivalent analysis, the evaluation value KD=kd1/ka1=3.792E+4/4.424E−6=1.167E−10 was obtained. These results are shown in
From the KD value in the sensorgram shown in
In order to stain a CEACAM5 expression-positive cancer cell line, FITC labeling was carried out on the cell line, using 100 μg of a purified CEA-V2122 protein. Specifically, labeling was carried out using Fluorescein Labeling Kit—NH2 (DOJINDO LABORATORIES) in accordance with the protocol and amount described in the operating manual included with the kit, and the obtained product was defined to be CEA-V2122-FITC. Specific staining of the CEACAM5 expression-positive cancer cell line is as follows. That is, CEACAM5-positive human stomach cancer-derived MKN-45 cells and CEACAM5-negative human colon cancer-derived DLD1 cells were each seeded on a CELLSTAR, μClear, 96-well plate (Greiner) to a cell density of 2.0×104 cells/well, and thereafter, the cells were cultured overnight. Subsequently, a culture solution containing 20 nM CEA-V2122-FITC and 1 μM Hoechist was added to the 96-well plate to a concentration of 100 μL/well, and the obtained mixture was then reacted at 4° C. for 30 minutes. Thereafter, each image was taken using In Cell Analyzer 6000 (GE Healthcare Life Sciences). The results are shown in
From the results shown in
A cytotoxicity test was carried out using the photoactivatable compound-labeled modified biotins, namely, Compound 1, Compound 2, and Compound 3. These compounds are described in Japanese Patent Application No. 2018-149295. Compound 1, Compound 2, and Compound 3 are shown in
Such complex serial dilution solutions were each added in an amount of 50 μL/well to the cells which were cultured overnight, so that the final concentrations became 10 μg/mL, 2.5 μg/mL, 0.625 μg/mL, 0.156 μg/mL, and 0.039 μg/mL One hour and two hours after addition of the complex, the cells were irradiated with a light, using LED emitting a light having a wavelength of 690±10 nm, so that the irradiation energy became 100 J/cm2. Thereafter, the cells were cultured for 48 hours, and thereafter, a comparison was made in terms of the number of surviving cells, using Cell Counting Kit-8 (DOJINDO LABORATORIES). Each condition was set to be n=3. The protocol and amount were determined in accordance with the instruction manuals included with the kit, and after addition of the reagent, the mixture was incubated for 1.5 hours at 37° C., in a CO2 incubator. Thereafter, the absorbance at 450 nm was measured, and the mean value was then calculated, followed by background collection. The control was set to be 100%, and the ratio of cell proliferation to the control under each condition was calculated. The results are shown in
As shown in
MKN45 cells were subcutaneously transplanted into nude mice to produce xenograft mouse models. That is, 4-week-old female nude mice were purchased, and the mice were then acclimated for 1 week. Thereafter, the MKN45 cells were transplanted in a cell count of 2×105 cells per mouse into the subcutis thereof. Approximately 10 days after completion of the transplantation, 100 μg of a complex of CEA-V2122 and a photoactivatable compound-labeled modified biotin (Compound 1), as described in Example 4, was administered to the mice via the caudal vein thereof. Using an LED light source emitting a light having a wavelength of 690 nm (USHIO OPTO SEMICONDUCTORS, INC., LD690D-66-60-550.), T-Cube LED Driver (THORLABS, NC0713145), and T-CUBE 15V POWER SUPPLY (THORLABS, TPS001), 6 hours after the administration, the mice were irradiated with the light having a wavelength of 690 nm, so that the irradiation energy became 230 J/cm2. Twenty-four hours after administration of the complex, the mice were irradiated again with the light having a wavelength of 690 nm, so that the irradiation energy became 230 J/cm2. A graph showing a change in the tumor volume is shown in
Regarding hematoxylin and eosin staining, the sliced pathological specimen was immersed in a xylene solution (Wako Pure Chemical Industries, Ltd., 241-00091) at room temperature for 10 minutes to perform deparaffinization, and thereafter, hematoxylin staining (Sakura Finetek Japan Co., Ltd., #8650) and eosin staining (Sakura Finetek Japan Co., Ltd., #8660) were carried out. The results are shown in
Moreover, immunostaining on CEACAM5 was carried out as follows. The sliced pathological specimen was immersed in a xylene solution (Wako Pure Chemical Industries, Ltd., 241-00091) at room temperature for 10 minutes to perform deparaffinization, and thereafter, the antigen was activated by performing an autoclave treatment (121° C., 5 minutes) using a citrate buffer (pH=6.0). Thereafter, the resulting specimen was immersed in a 0.3% hydrogen peroxide (Wako Pure Chemical Industries, Ltd., 081-04215)/methanol (Wako Pure Chemical Industries, Ltd., 137-01823) solution at room temperature for 10 minutes to remove endogenous peroxidase. Non-specific reactions were blocked with a 2% BSA (Sigma Aldrich, A1470)/phosphate buffered saline solution. An anti-CEACAM5 antibody (R & D SYSTEMS, MAB41281, concentration: 1/100) was reacted with the specimen at 4° C. overnight, and thereafter, immunostaining signals were visualized using Histostar (trademark) (MBL, #8460) and DAB Substrate Solution (MBL, #8469). Finally, nuclear staining was carried out using hematoxylin (Sakura Finetek Japan Co., Ltd., #8650). The results are shown in
From
V2122 is a mutant streptavidin described in Example 3 of International Publication WO2015/125820 (SEQ ID NO: 4 shown in International Publication WO2015/125820). The amino acid sequence of V2122 is as set forth in SEQ ID NO: 12 in the sequence listing.
scFv-V2122 is prepared by binding a single-chain antibody (scFv) against HER2 (ERBB2) with the above-described V2122. This scFv-type anti-HER2 antibody is an scFv sequence described in Zhang H, et al., Therapeutic potential of an anti-HER2 single chain antibody-DM1 conjugates for the treatment of HER2-positive cancer. Signal Transduct Target Ther. 2017 May 19; 2: 17015. doi: 10.1038/sigtrans. 2017.15. The amino acid sequence of the scFv-type anti-HER2 antibody is as set forth in SEQ ID NO: 18 in the sequence listing. In addition, the structural drawing of HER2-V2122 prepared by binding the scFv-type anti-HER2 antibody with V2122 via an amino acid linker (GGGGGSGGGGG) (SEQ ID NO: 29) is shown in
For the expression of a HER2-V2122 fusion protein, the DNA codon of a HER2-V2122 gene sequence, in which a pelB signal for secretion and expression in Escherichia coli had been incorporated into the N-terminus and a 6×His-Tag sequence had been incorporated into the C-terminus, was optimized for Escherichia coli, thereby synthesizing an artificial gene. This amino acid sequence is as set forth in SEQ ID NO: 20 in the sequence listing, and the DNA sequence is as set forth in SEQ ID NO: 21 in the sequence listing.
As a specific protein expression vector, a vector prepared by incorporating a chaperone skp gene into MCS2 of a pETDuet1 vector was used. Regarding the skp gene, the DNA codon was optimized for Escherichia coli based on the amino acid sequence as set forth in SEQ ID NO: 6 in the sequence listing, thereby synthesizing an artificial gene. The synthesized skp gene was amplified by PCR, using the primers (AAGGAGATATACATATGGATAAAATTGCCATTGTTAATAT (SEQ ID NO: 23), and TTGAGATCTGCCATATGTTATTTCACTTGTTTCAGAACG (SEQ ID NO: 24)), and the amplified gene was then cloned into MCS2 of the pETDue1 vector linearized with the restriction enzyme NdeI, using In-Fusion HD Cloning Kit, so as to obtain pETDuet_skp. Subsequently, the HER2-V2122 gene was incorporated into MCS1 of pETDuet_skp. Specifically, the artificially synthesized HER2-V2122 gene was amplified by PCR, using the primers (AGAAGGAGATATACCATGAAATATCTGCTGCCGAC (SEQ ID NO: 25), and CGCCGAGCTCGAATTTTAATGATGGTGATGATGATG (SEQ ID NO: 26)). Moreover, pETDuet_skp was linearized by PCR, using the primers (GGTATATCTCCTTCTTAAAGTTAAAC (SEQ ID NO: 27), and AATTCGAGCTCGGCGCGCCTGCAG (SEQ ID NO: 28)). The HER2-V2122 amplified by PCR and the linearized pETDuet_skp were subjected to cloning using In-Fusion HD Cloning Kit. The cloned vector was confirmed by sequencing, in terms of the gene sequence incorporated therein, and thereafter, it was referred to as pETDuet HER2-V2122_4 (p.
For the expression of the protein, pETDuet HER2-V2122_skp was transformed into BL21(DE3) (Nippon Gene Co., Ltd.), which was then pre-cultured in 2×YT medium (SIGMA-ALDRICH) at 37° C. overnight. The medium used in the pre-culture was added to a new medium for 100-fold dilution, and culture was then carried out at 37° C., until OD (600 nm) became 0.5 to 2.0. Subsequently, IPTG was added to the culture to a final concentration of 0.5 mM, and the obtained mixture was then cultured at 37° C. for 4 hours. Thereafter, a culture supernatant was recovered and was then preserved at 4° C.
The HER2-V2122 protein was roughly purified according to a batch method utilizing a 6×His-Tag added to the C-terminus. Specifically, cOmplete His-Tag Purification Resin equilibrated with buffer A (50 mM Tris-HCl, 0.2 M NaCl, 1 mM EDTA, and 5 mM Imidazole; pH 8.0) was added to the culture supernatant preserved at 4° C. The obtained mixture was stirred for 2 hours to overnight at 4° C., so that the protein was allowed to bind to the resin. Subsequently, the resin was recovered into a column, and a 20 column volume of washing operation was performed with buffer A. Thereafter, a roughly purified product of HER2-V2122 was recovered by elution with buffer B (50 mM Tris-HCl, 0.2 M NaCl, 1 mM EDTA, and 400 mM Imidazole; pH 8.0).
Subsequently, the roughly purified product was purified using a Protein L column. Specifically, 1 mL of Capto L (GE Healthcare) was filled into a PD-10 column and was then equilibrated with 10 column volume of PBS, and the aforementioned roughly purified product was then applied thereto. Thereafter, the resultant was washed with 10 column volume of PBS, was then eluted with 10 mM glycine hydrochloride (pH 2.0), and was then subjected to centrifugal concentration using Vivaspin Turbo 15 (MWCO 100,000). Moreover, using PD-10 (GE Healthcare), the buffer was replaced with PBS, and centrifugal concentration was further carried out using Vivaspin Turbo 4 (MWCO 100,000) to obtain a finally purified product. The purified product was subjected to CBB staining, and the purity of tetramer HER2-V2122 was assayed. The results are shown in
From
The affinity of HER2-V2122 for the antigen CEACAM5 was evaluated using a surface plasmon resonance (SPR) measuring device, Biacore T200 (GE Healthcare Life Sciences). Specifically, Recombinant Human ErbB2/Fc Chimera, Carrier Free (R & D SYSTEMS, 1129-ER-050) was immobilized on Sensor Chip CMS (GE Healthcare Life Sciences) using an amine-coupling kit (GE Healthcare Life Sciences). The final amount of the ligand immobilized was 279 RU. Moreover, with regard to the purified HER2-V2122, two-fold serial dilutions from 1E-08 M to 6.25E-10 M were prepared as analytes. Regarding interaction analysis, data were obtained by single-cycle kinetics analysis. Using Biacore T200 Evaluation Software, version 2.0, the obtained data were subjected to curve fitting in a bivalent analysis mode, and the following values were obtained: ka1=3.857E+5, and kd1=1.710E−6. Moreover, since evaluation can be carried out at KD=kd1/ka1 in the bivalent analysis, the evaluation value KD=kd1/ka1=1.710E−6/3.857E+5=4.433E−12 was obtained. These results are shown in
From the sensorgram and the calculated KD value shown in
Furthermore, the interaction between HER2-V2122 and a modified biotin (Production Example 1, the compound represented by Formula 17) was also analyzed using Biacore T200. Specifically, an amine-coupling kit was used, a target value was set to be 5000 RU, and the purified CEA-V2122 was immobilized on Sensor Chip CMS. With regard to the concentrations of the analytes, 5 types of two-fold serial dilutions from 1E-08 M to 6.25E-10 M were used. Regarding interaction analysis, data were obtained by single-cycle kinetics analysis. Using Biacore T200 Evaluation Software, version 2.0, the obtained data were subjected to curve fitting in a bivalent analysis mode, and the following values were obtained: ka1=1.4E+4, and kd1=1.0E−3. Moreover, since evaluation can be carried out at KD=kd1/ka1 in the bivalent analysis, the evaluation value KD=kd1/ka1=kd1=1.0E−3/1.4E+4=7.4E−8 was obtained. These results are shown in
A cytotoxicity test was carried out using the photoactivatable compound-labeled modified biotins, namely, Compound 1, Compound 2, and Compound 3. These compounds are described in Japanese Patent Application No. 2018-149295. Compound 1, Compound 2, and Compound 3 are shown in
Such complex serial dilution solutions were each added in an amount of 50 μL/well to the cells cultured overnight, so that the final concentrations became 10 μg/mL, 2.5 μg/mL, 0.625 μg/mL, 0.156 μg/mL, and 0.039 μg/mL. One hour and two hours after addition of the complex, the cells were irradiated with a light, using LED emitting a light having a wavelength of 690±10 nm, so that the irradiation energy became 100 J/cm2. Thereafter, the cells were cultured for 48 hours, and thereafter, a comparison was made in terms of the number of surviving cells, using Cell Counting Kit-8 (DOJINDO LABORATORIES). Each condition was set to be n=3. The dosage and administration were determined in accordance with the instruction manuals included with the kit, and addition of the reagent, the mixture was incubated for 1.5 hours at 37° C., in a CO2 incubator. Thereafter, the absorbance at 450 nm was measured, and the mean value was then calculated, followed by background collection. The control was set to be 100%, and the ratio of cell proliferation to the control under each condition was calculated. The results are shown in
As shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-149295 | Aug 2018 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2019/031336 | 8/8/2019 | WO | 00 |
