WO2023008515A1 - 抗原結合分子とストレプトアビジン変異体との融合タンパク質 - Google Patents
抗原結合分子とストレプトアビジン変異体との融合タンパク質 Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Definitions
- the present invention relates to fusion proteins of antigen-binding molecules and streptavidin mutants, and uses thereof.
- Patent Document 1 describes a streptavidin mutant whose immunogenicity is reduced by introducing mutations into amino acids. Patent Document 1 also describes a therapeutic or diagnostic kit containing the streptavidin mutant and a diagnostic or therapeutic substance labeled with biotin or a derivative thereof.
- the problem to be solved in the present invention is to provide a fusion protein between a molecule that recognizes cancer cells and a streptavidin mutant for use in the treatment or diagnosis of cancer.
- a further object of the present invention is to provide a means for treating cancer or a means for diagnosing cancer using the fusion protein described above.
- the present inventor selected a molecule with a molecular weight smaller than that of an antibody as a molecule that recognizes cancer cells, and prepared a fusion protein between the above molecule and a streptavidin mutant. Then, the present inventors have found that photoimmunotherapy using the fusion protein and a conjugate of biotin and a phthalocyanine dye can suppress the growth of cancer cells, and have completed the present invention.
- ⁇ 3> The fusion protein of ⁇ 1> or ⁇ 2>, wherein the antigen-binding molecule binds to an antigen expressed on cancer cells.
- ⁇ 4> The fusion protein of any one of ⁇ 1> to ⁇ 3>, wherein the antigen-binding molecule is a molecule that binds to Her2.
- ⁇ 5> The fusion protein of any one of ⁇ 1> to ⁇ 4>, wherein the antigen-binding molecule has the amino acid sequence of SEQ ID NO:2.
- ⁇ 6> The fusion protein according to any one of ⁇ 1> to ⁇ 5>, wherein the linker sequence consists of glycine and serine residues and has 5 to 25 amino acid residues.
- the linker sequence is an amino acid sequence represented by [(Gly) m -Ser] n (wherein m represents an integer of 1 to 10 and n represents an integer of 1 to 5) ⁇ 1 > to ⁇ 6>.
- a cancer therapeutic agent or cancer diagnostic agent comprising the fusion protein according to any one of ⁇ 1> to ⁇ 8>.
- fusion protein according to any one of ⁇ 1> to ⁇ 8>, and (2) a conjugate of biotin and a diagnostic substance or a therapeutic substance, a kit for treating or diagnosing cancer .
- the diagnostic or therapeutic substance is a phthalocyanine dye.
- ⁇ 14> The method according to ⁇ 13>, wherein the fusion protein is expressed in a bacterial inclusion body and recovered.
- the growth of cancer cells can be suppressed.
- FIG. 1 shows the results of unwinding studies of MFL2-G5Sx3-His and MFL2-G5Sx3-del5.
- Figure 2 shows the results of the Biacore assay (MFL2-Hisvs.HER2.
- Figure 3 shows the results of the Biacore assay (MFL2-Hisvs.biotin-SiPc).
- Figure 4 shows the results of the MFL2-G5Sx3-His-SiPc cytotoxicity assay.
- FIG. 5 shows the results of expression/purification and performance evaluation of MFL2-G5Sx1-PSAAS, MFL2-G5Sx2-PSAAS, and MFL2-G5Sx3-PSAAS.
- the present invention will be described in more detail below.
- the fusion protein of the present invention has the core streptavidin amino acid sequence shown in SEQ ID NO: 17 (provided that part or all of the amino acid sequence consisting of C-terminal Pro-Ser-Ala-Ala-Ser is deleted).
- N-terminal side and/or C-terminal side of a streptavidin mutant that contains an amino acid sequence having the following mutations (1) to (6) in (good) and has reduced immunogenicity compared to wild-type streptavidin is a fusion protein in which an antigen-binding molecule with a molecular weight of 20,000 or less is bound via a linker sequence.
- the antigen-binding molecules When an antigen-binding molecule having a molecular weight of 20,000 or less is bound to both the N-terminal side and the C-terminal side of the streptavidin mutant via a linker sequence, the antigen-binding molecules may be the same or different. good.
- the fusion protein of the present invention is a fusion protein having, from the N-terminal side to the C-terminal side, an antigen-binding molecule with a molecular weight of 20,000 or less, a linker sequence, and the amino acid sequence of the streptavidin mutant in this order. be.
- the streptavidin mutant in the present invention is a streptavidin mutant whose immunogenicity is reduced by introducing mutations into amino acids, described in International Publication WO2010/95455. That is, the streptavidin mutant of the present invention has the core streptavidin amino acid sequence shown in SEQ ID NO: 17 (provided that part or all of the amino acid sequence consisting of C-terminal Pro-Ser-Ala-Ala-Ser is deleted).
- streptavidin variant comprising an amino acid sequence with the following mutations: (1) A mutation in which tyrosine at the tenth amino acid residue is replaced with serine or threonine: (2) A mutation in which tyrosine at the 71st amino acid residue is replaced with alanine or serine: (3) A mutation in which arginine at the 72nd amino acid residue is substituted with lysine: (4) A mutation in which glutamic acid at the 89th amino acid residue is substituted with aspartic acid: (5) A mutation in which arginine at the 91st amino acid residue is substituted with lysine: (6) A mutation in which glutamic acid at the 104th amino acid residue is substituted with glutamine or asparagine:
- immunogenicity is reduced compared to wild-type streptavidin, which means reduced immunogenicity when a streptavidin mutant is administered to a mammal such as a human.
- Decreased immunogenicity can be confirmed, for example, by the following method. That is, the mutant streptavidin of the present invention was analyzed for reactivity to anti-streptavidin antiserum obtained by immunizing cynomolgus monkeys with wild-type streptavidin, and the reactivity to the anti-streptavidin antiserum was determined to be the same as that of wild-type streptavidin.
- the streptavidin mutant of the present invention has an immunogenicity of preferably 80% or less, more preferably 60% or less, and more preferably 60% or less, compared to wild-type streptavidin. It is preferably 20% or less, more preferably 15% or less, still more preferably 10% or less, particularly preferably 5% or less.
- the fusion protein of the present invention is a fusion protein between an antigen-binding molecule with a molecular weight of 20,000 or less and a streptavidin mutant.
- the fusion protein of the present invention forms a tetramer due to the affinity between the streptavidin mutant amino acid sequences.
- the molecular weight of the tetramer of the fusion protein of the present invention is about 96 kDa.
- the fusion protein of the present invention is administered to a living body to treat cancer, it should have good tumor uptake, good clearance rate, and good It is important to simultaneously achieve good tumor penetration.
- the molecular weight of the tetramer described above in the present invention (approximately 96 kDa) is assumed to be the molecular weight that can simultaneously achieve the above three parameters.
- the molecular weight of the antigen-binding molecule may be 20,000 or less, and the molecular weight of the antigen-binding molecule is generally 4,000 or more and 20,000 or less, preferably 4,000 or more and 10,000 or less. , more preferably 4,000 or more and 8,000 or less.
- the antigen-binding molecule in the present invention is a molecule with a concept different from that of an antibody. While the molecular weight of IgG antibodies is usually about 150,000, antigen-binding molecules in the present invention are molecules with much smaller molecular weights than IgG antibodies.
- An antigen-binding molecule in the present invention may be a molecule that mimics an antibody.
- a molecule that binds to the desired antigen can be appropriately selected by screening from proteins with a molecular weight of about 6,000 that are prepared by partially modifying the IgG-binding domain of Protein A (VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK) (SEQ ID NO: 18). can be done.
- the antigen in the antigen-binding molecule is not particularly limited, but is preferably an antigen expressed in cancer cells.
- Antigens that are specifically expressed in cancer include the following antigens.
- Epiregulin EREG
- ROBO1,2,3,4, 1-40- ⁇ -amyloid 4-1BB,5AC, 5T4, ACVR2B, adenocarcinoma antigen, ⁇ -fetoprotein, angiopoietin 2, anthrax toxin, AOC3 ( VAP-1), B-lymphoma cells, B7-H3, BAFF, ⁇ -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 ⁇ chain), CD28, CD3, CD30 (TNFRSF8), CD33, CD37, CD
- coli Shiga toxin type 1 E. coli Shiga toxin type 2, EGFL7, EGFR, endotoxin, EpCAM, episialin, ERBB3, Escherichia coli, respiration 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-DR ⁇ , HNGF, Hsp90, human ⁇ amyloid , human scatterfactor receptor kinase, human TNF, ICAM-1(CD54), IFN- ⁇ , IFN- ⁇ , IgE, IgEFc region, IGF-1 receptor, IGF-I, IgG4,IGHE, IL- 1 ⁇ , IL-12, IL-13, IL
- HER2 is particularly preferred.
- An example of an antigen-binding molecule is that it can bind to HER2.
- a protein having the amino acid sequence set forth in SEQ ID NO:2 can be mentioned.
- the linker sequence is not particularly limited as long as the effects of the present invention can be achieved, but the number of amino acids is preferably 5 to 25 amino acids, more preferably 10 to 25 amino acids, and even more preferably 15 to 20 amino acids.
- a specific example of the linker sequence is a sequence consisting of glycine and serine residues.
- an amino acid sequence represented by [(Gly) m -Ser] n (wherein m represents an integer of 1 to 10 and n represents an integer of 1 to 5) can be used. can.
- linker sequences include Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser. Not limited.
- a specific example of the fusion protein of the present invention is a fusion protein having the amino acid sequence set forth in SEQ ID NO: 3 or 9.
- the present invention further provides a nucleic acid (eg, DNA) encoding the fusion protein of the present invention described above.
- a specific example of the nucleic acid of the present invention is a nucleic acid encoding a fusion protein having the amino acid sequence set forth in SEQ ID NO:3 or 9.
- An example of the nucleic acid of the present invention is a nucleic acid having the base sequence of SEQ ID NO: 4 or 10.
- a nucleic acid (eg, DNA) encoding the fusion protein of the present invention can be used by incorporating it into a vector.
- the fusion protein of the present invention can be expressed by incorporating a nucleic acid encoding the fusion protein of the present invention into an expression vector and transforming the expression vector into a host. . That is, the present invention provides a method for producing the fusion protein of the present invention, comprising the step of expressing a nucleic acid encoding the fusion protein of the present invention in a host.
- the fusion protein is preferably expressed and recovered in bacterial inclusion bodies.
- the vector When E. coli is used as a host, the vector has an origin of replication (ori) and a gene for selecting a transformed host (for example, a drug such as ampicillin, tetracycline, kanamycin or chloramphenicol). It is preferable to have a drug resistance gene against In the case of an expression vector, it preferably has a promoter such as the lacZ promoter or T7 promoter that allows efficient expression of the streptavidin mutant of the present invention in the host.
- a promoter such as the lacZ promoter or T7 promoter that allows efficient expression of the streptavidin mutant of the present invention in the host.
- vectors examples include M13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system” (Qiagen), pEGFP, or pET (In this case, it is preferable to use BL21 expressing T7 RNA polymerase as the host).
- a tag for improving solubility such as a sequence encoding glutathione-S-transferase, thioredoxin, or maltose binding protein, may be added. It also encodes tags designed to facilitate purification, such as polyhistidine tags, Myc epitopes, hemagglutinin (HA) epitopes, T7 epitopes, Xpress tags, FLAG peptide tags, and other known tag sequences. A sequence may be added.
- expression vectors derived from mammals e.g., pcDNA3 (manufactured by Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322), pEF, pCDM8), insect cell-derived Expression vectors (e.g., "Bac-to-BAC baculovairus expression system” (Gibco BRL), pBacPAK8), plant-derived expression vectors (e.g., pMH1, pMH2), animal virus-derived expression vectors (e.g., pHSV, pMV, pAdexLcw ), a retrovirus-derived expression vector (e.g., pZIPneo), a yeast-derived expression vector (e.g., "Pichia Expression Kit” (manufactured by Invitrogen), pNV11, SP-Q01), a Bacillus subtilis-derived expression vector (e.g.,
- a promoter necessary for intracellular expression such as the SV40 promoter (Muligan et al., Nature (1979) 277, 108), It is essential to have MMLV-LTR promoter, EF1 ⁇ promoter (Mizushima et al., Nucleic Acids Res. (1990) 18,5322), CMV promoter, and the like. It is more preferable to have a drug resistance gene that can be identified by a drug (neomycin, G418, etc.). Vectors with such properties include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, and the like.
- the host cell into which the vector is introduced is not particularly limited, and may be either prokaryotic or eukaryotic.
- E. coli and various animal cells can be used.
- animal cells for example, animal cells, plant cells, and fungal cells can be used as hosts.
- animal cells mammalian cells such as CHO cells, COS cells, 3T3 cells, HeLa cells, Vero cells, or insect cells such as Sf9, Sf21 and Tn5 can be used.
- animal cells CHO cells are particularly preferred for the purpose of large-scale expression.
- a vector can be introduced into a host cell by, for example, calcium phosphate method, DEAE dextran method, method using cationic ribosome DOTAP (manufactured by Boehringer Mannheim), electroporation method, lipofection and the like.
- Nicotiana tabacum-derived cells are known as a protein production system, and callus can be cultured from these cells.
- Known fungal cells include yeast such as Saccharomyces genus such as Saccharomyces cerevisiae, and filamentous fungi such as Aspergillus genus such as Aspergillus niger.
- Escherichia coli such as JM109, DH5 ⁇ , HB101, etc., and Bacillus subtilis are known.
- the fusion protein of the present invention can be obtained by transforming these cells with the nucleic acid of the present invention and culturing the transformed cells in vitro.
- Culture can be performed according to a known method.
- DMEM, MEM, RPMI1640, and IMDM can be used as animal cell culture media.
- a serum replacement fluid such as fetal calf serum (FCS) can be used in combination, or serum-free culture can be performed.
- FCS fetal calf serum
- the pH during culture is preferably about 6-8. Cultivation is usually carried out at about 30-40° C. for about 15-200 hours, and medium replacement, aeration and agitation are added as necessary.
- growth factors may be added to promote cell growth.
- the fusion protein of the present invention is useful as a cancer therapeutic agent or cancer diagnostic agent.
- a cancer treatment or diagnostic kit comprising (1) the fusion protein of the present invention and (2) biotin and a diagnostic agent or a conjugate of a therapeutic agent.
- a molecule that binds to an antigen present on cancer cells is used as the antigen-binding molecule
- administration of the fusion protein of the present invention to a patient enables specific accumulation of streptavidin mutants in cancer cells. can be done.
- administering a conjugate of biotin and a diagnostic or therapeutic substance to a patient it becomes possible to accurately accumulate the diagnostic or therapeutic substance in cancer cells.
- the "fusion protein of the present invention” and the “conjugate of biotin and a diagnostic or therapeutic substance” can be combined to prepare a complex, and this complex can be administered to a patient.
- a conjugate of biotin and a diagnostic or therapeutic agent can be prepared by conjugating a diagnostic or therapeutic agent to biotin.
- Diagnostic or therapeutic substances include fluorescent dyes, chemiluminescent agents, radioisotopes, sensitizers composed of metal compounds and the like, neutron capture agents composed of metal compounds and the like, phthalocyanine dyes, low-molecular-weight compounds, micro- or nanobubbles, Proteins and the like can be mentioned. Phthalocyanine dyes can preferably be used.
- phthalocyanine dye specifically, a compound represented by the following formula (1) or a salt thereof can be used.
- X represents a substituent having a hydrophilic group at its end.
- hydrophilic group a sulfonic acid group, a phosphoric acid group, an ammonium group and the like are preferable.
- X is preferably a substituent having a terminal sulfonic acid group.
- X is A group represented by can be mentioned.
- Me represents a methyl group.
- L 3 , L 4 , L 5 and L 6 each independently represent a divalent linking group.
- L3 is preferably (Wherein, m represents an integer of 1 to 5.) is.
- L 4 is preferably -[(CH 2 ) p -O)] q - (Wherein, p and q each independently represents an integer of 1 to 5.) is.
- L 5 is preferably -CONH-, -NHCO-, -COO- or -OCO-, more preferably -CONH-.
- L 6 is preferably -(CH 2 ) r -, -(CH 2 ) r -O-, or -(CH 2 ) r -Si(R 1 )(R 2 )-O- (where r represents an integer of 1 to 5.
- R 1 and R 2 each independently represent an alkyl group having 1 to 4 carbon atoms.) is.
- R 1 and R 2 are particularly preferably methyl radicals.
- Y represents a group that can bind to an antigen-binding molecule.
- Y is preferably an active ester of a carboxyl group, more preferably a succinimidyl ester of a carboxyl group.
- R 3 is an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, —SR 11 , —SOR 12 , an aryl group having 6 to 10 carbon atoms, —N(R 13 )(R 14 ), or represents —NO 2 , or two adjacent R 3 may together form an aryl group having 6 to 10 carbon atoms.
- R 11 , R 12 , R 13 and R 14 each independently represent an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms.
- the aryl group may be substituted with an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms.
- the alkyl group and alkoxy group may be substituted with a halogen atom.
- each may be the same or different.
- s represents an integer of 0-4. Preferably s is zero.
- the halogen atom may be fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.
- the phthalocyanine dye compound can be synthesized according to the methods described in Production Examples 1 to 5 of the Examples.
- Photoimmunotherapy is a treatment that uses photosensitizers and irradiated light to destroy specific cells in the body.
- Photosensitizers produce cytotoxic reactive oxygen species that can induce apoptosis, necrosis, and/or autophagy in nearby cells when exposed to specific wavelengths of light.
- 6,127,045 discloses a method of killing cells comprising contacting cells containing cell surface proteins with a therapeutically effective amount of one or more antibody-IR700 molecules, wherein the antibodies are specifically binding to a cell surface protein; irradiating said cells at a wavelength of 660-740 nm and a dose of at least 1 Jcm ⁇ 2 ; and approximately 0-8 hours after irradiating said cells, with one or more therapeutic agents, thereby killing the cell.
- 2017-524659 discloses a method for inducing cytotoxicity in a subject suffering from a disease or condition, comprising: (a) IRDye (registered trademark) conjugated to a probe that specifically binds to the cells of the subject; ) administering to said subject a therapeutically effective agent comprising a phthalocyanine dye such as 700DX; and (b) irradiating said cells with a suitable excitation light in an amount effective to induce cell death.
- IRDye registered trademark
- a therapeutically effective agent comprising a phthalocyanine dye such as 700DX
- irradiating said cells with a suitable excitation light in an amount effective to induce cell death.
- administering the fusion protein of the present invention and a conjugate of a biotin-modified dimer and a phthalocyanine dye to a subject, and irradiating the cells with excitation light in an amount effective to inhibit cell growth or induce cell death; can inhibit cell proliferation or induce cell death to treat a subject.
- the fusion protein of the present invention by administering the fusion protein of the present invention and a conjugate of biotin and a phthalocyanine dye to a subject and irradiating the cells with excitation light in an amount effective to inhibit cell proliferation or induce cell death. , can inhibit cell proliferation or induce cell death to treat a subject.
- Subjects include humans and non-human mammals, including humans and laboratory animals such as mice.
- the subject is preferably a subject suffering from a disease for which suppression of cell proliferation or induction of cell death is desired, and examples thereof include subjects having cancer or solid tumors.
- Cancer includes carcinoma, lymphoma, blastoma, sarcoma, and leukemia or malignant lymphoma.
- Specific examples of cancers 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 cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectum cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal carcinoma, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.
- squamous cell carcinoma e.g. epithelial squamous cell carcinoma
- a solid tumor is an abnormal mass of cells, benign or malignant, that usually does not contain a cyst.
- Solid tumors include glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineocytoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, Neuroblastoma, and retinoblastoma.
- Administration methods to subjects include topical routes, injections (subcutaneous injections, intramuscular injections, intradermal injections, intraperitoneal injections, intratumoral injections, intravenous injections, etc.), oral routes, ocular routes, sublingual routes, and rectal routes. Routes include, but are not limited to, transdermal, intranasal, vaginal, and inhalation routes.
- the biotin-modified dimer-phthalocyanine dye conjugate and the fusion protein of the invention are each preferably administered in therapeutically effective amounts.
- a therapeutically effective amount is at least 0.5 milligrams per 60 kilograms (mg/60kg), at least 5mg/60kg, at least 10mg/60kg, at least 20mg/60kg, at least 30mg/60kg, for each of the above conjugates and fusion proteins; At least 50 mg/60 kg.
- the therapeutically effective amount is at least 10 ⁇ g/kg, such as at least 100 ⁇ g/kg, at least 500 ⁇ g/kg or at least 500 ⁇ g/kg, for example 100 ⁇ g/kg, 250 ⁇ g/kg when administered intratumorally or intraperitoneally. kg, such as doses of about 500 ⁇ g/kg, 750 ⁇ g/kg, or 1000 ⁇ g/kg, eg, 10 ⁇ g/kg to 1000 ⁇ g/kg.
- the therapeutically effective amount is 10 ⁇ g/ml, 20 ⁇ g/ml, 30 ⁇ g/ml, 40 ⁇ g/ml, 50 ⁇ g/ml, 60 ⁇ g/ml, 70 ⁇ g/ml, 80 ⁇ g/ml, 90 ⁇ g/ml administered in a topical solution. , or at least 1 ⁇ g/ml, such as at least 500 ⁇ g/ml, such as between 20 ⁇ g/ml and 100 ⁇ g/ml, such as 100 ⁇ g/ml.
- the doses described above can be administered in one or more divided doses (such as 2, 3, or 4 doses) or in a single formulation.
- Conjugates of biotin and phthalocyanine dyes and fusion proteins of the invention can each be administered alone or in the presence of a pharmaceutically acceptable carrier, other therapeutic agents (such as other It can also be administered in the presence of an anticancer drug, etc.).
- Conjugates of biotin and phthalocyanine dyes and fusion proteins of the invention can bind to target cells or tissues, such as circulating tumor cells or cells of solid tumors. Upon subsequent irradiation with light, the conjugate or complex can absorb the light and damage or destroy the target cell or tissue.
- the wavelength of irradiation light in photoimmunotherapy 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.
- Light irradiation may be performed by using a device with near-infrared (NIR) light emitting diodes.
- NIR near-infrared
- the dose of light is at least 1 J/cm 2 , such as 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 . , for example, from 1 to 500 J/cm 2 .
- Light irradiation may be performed multiple times (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times).
- a LISA314 molecule (SEQ ID NO: 1) and a molecule that recognizes the HER2 antigen (Lofblom, J. FEBSLett. 584(12):2670-80(2010)) (SEQ ID NO: 2) are fused, and both the biotin molecule and Her2/ErbB2 A protein (hereinafter referred to as MFL2-G5Sx3-His) having a binding property to E. coli was synthesized in E. coli (SEQ ID NO: 3).
- the gene sequence of MFL2-G5Sx3-His (SEQ ID NO: 4) was artificially synthesized by Eurofins Genomics.
- an expression vector was constructed according to a standard method. Specifically, a primer set (Rv: catggtatatctccttctttaaagttaac (SEQ ID NO: 5), Fw: cgcagcttaattaacctaggctgctgccac (SEQ ID NO: 6)) was used to linearize the vector by PCR reaction.
- the sequence prepared by artificial gene synthesis was amplified by PCR using a primer set (Fw: aggagatataccatgGTGGACAACAAATTCAACAAAGAG (SEQ ID NO: 7), Rv: gttaattaagctgcgTTAATGATGGTGGTGATGATGCGATG (SEQ ID NO: 8)). Both PCR products were subjected to agarose gel electrophoresis to cut out bands and purify them. The ligation reaction of the purified vector and the insert was performed using the In-Fusion HD Cloning Kit (TaKaRa Bio) according to the usage volume of the manual.
- the MFL2-G5Sx3-His expression vector was modified to express MFL2-G5Sx3-del5 having the amino acid sequence shown in SEQ ID NO: 9, which lacks the C-terminal amino acid sequence PSAASHHHHHH of the amino acid sequence shown in SEQ ID NO: 3.
- a deletion mutant was produced (SEQ ID NO: 10). Specifically, a primer set (Fw; AAGTCAAATAACGCAGCTTAATTAACCTAG (SEQ ID NO: 11), Rw; TGCGTTATTTGACTTTGGTAAAGGTGTCATG (SEQ ID NO: 12)) was used, and the expression vector was used as a template for PCR reaction. Thereafter, the reaction solution was treated with DpnI at 37° C.
- Competent cells BL21 (DE3) ( ECOS Competent E.coli BL21 (DE3), Nippon Gene) and transformed. 100 mL of 2xYT medium overnight culture was phagocytosed into 1 liter of culture medium and cultured at 37°C. IPTG was added so as to obtain the desired amount, culture was performed at 37°C for 4 hours, and then the cells were collected by centrifugation (7500 xg, 20 min at 4°C).
- IB insoluble fraction
- B-PER ThermoSCIENTIFIC
- IB insoluble fraction
- B-PER ThermoSCIENTIFIC
- the recovered IB is resuspended in 10-fold diluted B-PER buffer (no Benzonase added), centrifuged, discarded the supernatant, and the IB washed 3 times.After 3 washes, recovered by centrifugation.
- the obtained IB was resuspended in ultrapure water (MilliQ), dispensed into 1.5 mL tubes in 1 mL portions, and stored frozen at -80°C.
- the denaturation and unwinding of IB are shown below.
- Add denature buffer 0.1 M Tris-HCl, pH 8.5, 6 M guanidine hydrochloride, 10 mM EDTA
- IB dissolve by pipetting, incubate overnight at 4°C while stirring with a rotator, and centrifuge (15,000 x g, 20 min at 4°C) and the supernatant was recovered.
- Mix the refolding buffer (0.05 M sodium phosphate, 0.4 M arginine hydrochloride, pH 6.8) prepared with denature buffer so that the protein concentration (OD280nm) of the recovered supernatant is 30-50 mg/mL.
- MFL2-G5Sx3-His was analyzed for binding activity to HER2 (ErbB2) using SPR (BiacoreT200, Cytiva).
- Her2 antigen RecombinantHumanErbB2/Her2 FcChimera Protein, R&D SYSTEMS
- Sensor ChipCM5 ChipCM5
- AdI AminCoupling Kit, Cytiva
- MFL2-G5Sx3-His and photosensitizer Psyche> Her2-positive cells (KPL-4) were seeded in a 96-well plate at a cell number of 1 ⁇ 10 4 cells/well and a culture solution of 50 ⁇ L/well, and cultured overnight.
- Purified MFL2-G5Sx3-His and biotin-SiPc produced in Production Example 1 were mixed at a molar ratio of 1:4 (hereinafter referred to as MFL2-G5Sx3-His complex), and 10 ⁇ g/ Eight 10-fold dilution series (including zero concentration) were prepared from mL.
- the complex was added to the cells, and 24 hours later, light was irradiated from the bottom of the culture plate with an LED that emits light of 690 nm at 100 J/cm 2 . Then, after culturing for 24 hours, the medium containing the MFL2-G5Sx3-His complex was removed, the cells were washed once with PBS, fresh medium was added, and viable cells were counted using Cell Counting Kit-8 (Dojindo). A number of comparisons were made.
- a gene sequence (SEQ ID NOS: 14-16) encoding an amino acid sequence having each linker length and C-terminus described in SEQ ID NOS: 11-13 was produced by artificial gene synthesis.
- the pET45b vector was linearized by PCR using a primer set (Rv: catggtatatctccttcttaaagttaac (SEQ ID NO: 5), Fw: cgcagcttaattaacctaggctgctgccac (SEQ ID NO: 6)).
- the sequence prepared by artificial gene synthesis was amplified by PCR reaction using a primer set (Fw: aggagatataccatgGTGGACAACAAATTCAACAAAGAG (SEQ ID NO: 7), Rv: gttaattaagctgcgTTAATGATGGTGGTGATGATGCGATG (SEQ ID NO: 8)). Bands from both PCR reaction products were excised and purified by agarose gel electrophoresis, and the ligation reaction of the vector and insert was performed using the In-Fusion HD Cloning Kit (TaKaRa Bio) according to the usage volume of the instructions.
- pET45-MFL2-G4Sx1-PSAAS, pET45-MFL2-G4Sx2-PSAAS, and pET45-MFL2-G4Sx3-PSAAS were introduced into competent cells BL21 (DE3) (ECOS Competent E.coli BL21 (DE3), Nippon Gene) for transformation. did 100 mL of 2xYT medium overnight culture was phagocytosed into 1 liter of culture medium and cultured at 37°C. IPTG was added so as to obtain the desired amount, and culture was performed at 37° C. for 4 hours.
- IB insoluble fraction
- B-PER ThermoSCIENTIFIC
- IB insoluble fraction
- B-PER ThermoSCIENTIFIC
- the recovered IB is resuspended in 10-fold diluted B-PER buffer (no Benzonase added), centrifuged, discarded the supernatant, and the IB washed 3 times.After 3 washes, recovered by centrifugation.
- the obtained IB was resuspended in ultrapure water (MilliQ), dispensed into 1.5 mL tubes in 1 mL portions, and stored frozen at -80°C.
- degeneration of IB is shown below.
- denature buffer 0.1 M Tris-HCl, pH 8.5, 6 M guanidine hydrochloride, 10 mM EDTA
- IB 0.1 M Tris-HCl, pH 8.5, 6 M guanidine hydrochloride, 10 mM EDTA
- the protein concentration (OD280nm) of the recovered supernatant was adjusted to 30-50 mg/mL using a denature buffer.
- the refolding buffer is made by adding 0.4 M arginine hydrochloride to McIlvain buffer and adjusting the pH to pH 4.0, pH 4.5, pH 5.0, pH 6.5, pH 7.5, pH 8.0, etc. Prepared in NaOH. To 1 mL of refolding buffer at each pH, 25 ⁇ L of denaturing solution supernatant was added to unwind by dilution. The samples were incubated at 4°C, and the formation of tetramers was confirmed by SDS-PAGE electrophoresis.
- Fig. 5B shows the confirmation results of tetramer formation 1 hour after unwinding and 24 hours after unwinding for MFL2-G4Sx2-PSAAS and MFL2-G4Sx3-PSAAS at each pH. From this result, it was confirmed that the tetramer formation was promoted from the monomer at pH5.0-pH6.5. After 72 hours, however, the disappearance of the tetramer band was confirmed at all pHs.
- SEQ ID NO: 3 Amino acid sequence of MFL2-G5Sx3-His MVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANLLAEAKKLNDAQAPKSGGGGGSGGGGGSGGGGGSAEAGITGTWSNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH*
- SEQ ID NO: 9 Amino acid sequence of MFL2-G5Sx3-del5 MVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANLLAEAKKLNDAQAPKSGGGGGSGGGGGSGGGGGSAEAGITGTWSNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVK
- SEQ ID NO: 11 Amino acid sequence of MFL2-G4Sx1-PSAAS MVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANLLAEAKKLNDAQAPKGGGGSAEAGITGTWSNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAAS*
- SEQ ID NO: 12 amino acid sequence of MFL2-G4Sx2-PSAAS MVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANLLAEAKKLNDAQAPKGGGGSGGGGSAEAGITGTWSNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAAS*
- SEQ ID NO: 13 Amino acid sequence of MFL2-G4Sx3-PSAAS MVDNKFNKEMRNAYWEIALLPNLNNQKRAFIRSIYDDPSQSANLLAEAKKLNDAQAPKGGGGSGGGGSGGGGSAEAGITGTWSNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAAS*
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Abstract
Description
<1> 配列番号17に記載のコアストレプトアビジンのアミノ酸配列(但し、C末端のPro-Ser-Ala-Ala-Serからなるアミノ酸配列の一部又は全部は欠失していてもよい)において下記(1)~(6)の変異を有するアミノ酸配列を含み、野生型ストレプトアビジンと比較して免疫原性が低下しているストレプトアビジン変異体のN末端側及び/又はC末端側に、リンカー配列を介して、分子量20,000以下の抗原結合分子が結合している、融合タンパク質。
(1)10番目のアミノ酸残基のチロシンがセリン又はトレオニンに置換している変異:
(2)71番目のアミノ酸残基のチロシンがアラニン又はセリンに置換している変異:
(3)72番目のアミノ酸残基のアルギニンがリジンに置換している変異:
(4)89番目のアミノ酸残基のグルタミン酸がアスパラギン酸に置換している変異:
(5)91番目のアミノ酸残基のアルギニンがリジンに置換している変異:
(6)104番目のアミノ酸残基のグルタミン酸がグルタミン又はアスパラギンに置換している変異:
<2> N末端側からC末端側に、分子量20,000以下の抗原結合分子と、リンカー配列と、前記ストレプトアビジン変異体のアミノ酸配列とをこの順に有する、<1>に記載の融合タンパク質。
<3> 抗原結合分子が、がん細胞に発現している抗原に結合する分子である、<1>又は<2>に記載の融合タンパク質。
<4> 抗原結合分子が、Her2に結合する分子である、<1>から<3>の何れか一に記載の融合タンパク質。
<5> 抗原結合分子が、配列番号2に記載のアミン酸配列を有する、<1>から<4>の何れか一に記載の融合タンパク質。
<6> リンカー配列が、グリシン残基及びセリン残基からなり、アミノ酸残基の数が5から25個である、<1>から<5>の何れか一に記載の融合タンパク質。
<7> リンカー配列が、[(Gly)m-Ser]n(式中、mは1~10の整数を示し、nは1~5の整数を示す)で示されるアミノ酸配列である、<1>から<6>の何れか一に記載の融合タンパク質。
<8> 配列番号3又は9に記載のアミノ酸配列を有する、<1>から<7>の何れか一に記載の融合タンパク質。
<9> 配列番号3又は9に記載のアミノ酸配列を有する融合タンパク質をコードする核酸。
<10> <1>から<8>の何れか一に記載の融合タンパク質を含む、がん治療剤またはがん診断剤。
<11> (1)<1>から<8>の何れか一に記載の融合タンパク質、及び(2)ビオチンと診断用物質又は治療用物質とのコンジュゲートを含む、がんの治療または診断キット。
<12> 診断用物質又は治療用物質が、フタロシアニン染料である、<11>に記載のキット。
<13> <1>から<8>の何れか一に記載の融合タンパク質をコードする核酸を宿主で発現させる工程を含む、<1>から<8>の何れか一に記載の融合タンパク質の製造方法。
<14> 前記融合タンパク質を、細菌の封入体において発現させて回収する、<13>に記載の方法。
<抗原結合分子とストレプトアビジン変異体との融合タンパク質>
本発明の融合タンパク質は、配列番号17に記載のコアストレプトアビジンのアミノ酸配列(但し、C末端のPro-Ser-Ala-Ala-Serからなるアミノ酸配列の一部又は全部は欠失していてもよい)において下記(1)~(6)の変異を有するアミノ酸配列を含み、野生型ストレプトアビジンと比較して免疫原性が低下しているストレプトアビジン変異体のN末端側及び/又はC末端側に、リンカー配列を介して、分子量20,000以下の抗原結合分子が結合している、融合タンパク質である。
(1)10番目のアミノ酸残基のチロシンがセリン又はトレオニンに置換している変異:
(2)71番目のアミノ酸残基のチロシンがアラニン又はセリンに置換している変異:
(3)72番目のアミノ酸残基のアルギニンがリジンに置換している変異:
(4)89番目のアミノ酸残基のグルタミン酸がアスパラギン酸に置換している変異:
(5)91番目のアミノ酸残基のアルギニンがリジンに置換している変異:
(6)104番目のアミノ酸残基のグルタミン酸がグルタミン又はアスパラギンに置換している変異:
(1)10番目のアミノ酸残基のチロシンがセリン又はトレオニンに置換している変異:
(2)71番目のアミノ酸残基のチロシンがアラニン又はセリンに置換している変異:
(3)72番目のアミノ酸残基のアルギニンがリジンに置換している変異:
(4)89番目のアミノ酸残基のグルタミン酸がアスパラギン酸に置換している変異:
(5)91番目のアミノ酸残基のアルギニンがリジンに置換している変異:
(6)104番目のアミノ酸残基のグルタミン酸がグルタミン又はアスパラギンに置換している変異:
リンカー配列の具体例としては、グリシン残基及びセリン残基からなる配列を挙げることができる。リンカー配列としては、例えば、[(Gly)m-Ser]n(式中、mは1~10の整数を示し、nは1~5の整数を示す)で示されるアミノ酸配列を使用することができる。リンカー配列の具体例としては、Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Serを挙げることができるが特に限定されない。
本発明の融合タンパク質は、がん治療剤またはがん診断剤として有用である。
本発明によれば、(1)本発明の融合タンパク質、及び(2)ビオチンと、診断用物質又は治療用物質とのコンジュゲートを含む、がんの治療または診断キットが提供される。
ビオチンに、診断用物質又は治療用物質を結合させることにより、ビオチンと診断用物質又は治療用物質とのコンジュゲートを調製することができる。診断用物質又は治療用物質としては、蛍光色素、化学発光剤、放射性同位元素、金属化合物等からなる増感剤、金属化合物等からなる中性子捕捉剤、フタロシアニン染料、低分子化合物、マイクロあるいはナノバブル、タンパク質などを挙げることができる。好ましくは、フタロシアニン染料を使用することができる。
Xは、スルホン酸基を末端に有する置換基であることが好ましい。
(式中、p及びqはそれぞれ独立に1~5の整数を示す。)
である。
である。R1及びR2は特に好ましくはメチル基である。
ここで、アリール基は、炭素数1から6のアルキル基または炭素数1から6のアルコキシ基で置換されていてもよい。
複数個のR3が存在する場合、それぞれ同一でも異なっていてもよい。
ハロゲン原子としては、フッ素、塩素、臭素、ヨウ素のいずれでもよいが、好ましくはフッ素、塩素、又は臭素である。
光免疫療法とは、体内で特定の細胞を破壊するために光増感剤及び照射光を使用する治療法である。光増感剤は、特異的な波長の光に晒されるとき、付近の細胞のアポトーシス、ネクローシス、及び/又は自食作用を誘発できる細胞傷害性の活性酸素種を生じる。例えば、特許6127045号公報には、細胞を死滅させる方法であって、細胞表面タンパク質を含む細胞を、治療有効量の1または複数の抗体-IR700分子と接触させるステップであって、該抗体が該細胞表面タンパク質に特異的に結合するステップと;該細胞に、660~740nmの波長で、かつ少なくとも1Jcm-2の線量で照射するステップと;該細胞に照射した約0~8時間後に、該細胞を1または複数の治療剤と接触させ、これにより、該細胞を死滅させるステップとを含む方法が記載されている。特表2017-524659号公報には、疾患又は病態を患っている対象において細胞毒性を誘発する方法であって、(a)対象の細胞に特異的に結合するプローブにコンジュゲートしたIRDye(登録商標)700DXなどのフタロシアニン染料を包含する治療的に有効な薬剤を該対象に投与し;そして、(b)細胞死を誘発するのに有効な量で適切な励起光を前記細胞に照射することを含む、方法が記載されている。
治療有効量は、上記のコンジュゲートと融合タンパク質のそれぞれについて、60キログラム当たり少なくとも0.5ミリグラム(mg/60kg)、少なくとも5mg/60kg、少なくとも10mg/60kg、少なくとも20mg/60kg、少なくとも30mg/60kg、少なくとも50mg/60kgである。例えば、静脈内投与される場合、1mg/60kg、2mg/60kg、5mg/60kg、20mg/60kg、または50mg/60kgの用量など、例えば、0.5~50mg/60kgである。別の例では、治療有効量は、少なくとも100μg/kg、少なくとも500μg/kgまたは少なくとも500μg/kgなど、少なくとも10μg/kg、例えば、腫瘍内投与または腹腔内投与される場合、100μg/kg、250μg/kg、約500μg/kg、750μg/kg、または1000μg/kgの用量など、例えば、10μg/kg~1000μg/kgである。一例では、治療有効量は、局所用溶液で投与される10μg/ml、20μg/ml、30μg/ml、40μg/ml、50μg/ml、60μg/ml、70μg/ml、80μg/ml、90μg/ml、または100μg/mlなど、20μg/ml~100μg/mlの間など、少なくとも500μg/mlなど、少なくとも1μg/mlである。
LRMS (ESI): m/z 941.45 [M-2H]2-
LISA314分子(配列番号1)とHER2抗原を認識する分子(Lofblom, J. FEBSLett. 584(12):2670-80(2010))(配列番号2)を融合し、ビオチン分子およびHer2/ErbB2の両方に結合性を持つタンパク質(以下、MFL2-G5Sx3-Hisと示す。)を大腸菌にて合成を行った(配列番号3)。MFL2-G5Sx3-Hisの遺伝子配列(配列番号4)はEurofinsGenomics社で人工遺伝子合成を行った。発現ベクターとしてpET45b(+)を使用し定法に従い発現ベクターの作製を行った。具体的にはプライマーセット(Rv: catggtatatctccttcttaaagttaaac(配列番号5),Fw:cgcagcttaattaacctaggctgctgccac(配列番号6))を用いPCR反応でベクターの直鎖化を行った。人工遺伝子合成にて調製された配列は、プライマーセット(Fw: aggagatataccatgGTGGACAACAAATTCAACAAAGAG(配列番号7), Rv: gttaattaagctgcgTTAATGATGGTGGTGATGATGCGATG(配列番号8))を用いPCR反応で増幅を行った。PCR反応物はともにアガロースゲル電気泳動にてバンドを切り出して精製をおこなった。In-Fusion HD Cloning Kit(TaKaRa Bio)を用い精製されたベクターとインサートのライゲーション反応を説明書の用法容量に従いクローニングを実施した。
MFL2-G5Sx3-HisについてSPR(BiacoreT200,Cytiva)を用いてHER2(ErbB2)との結合活性について解析を行った。Sensor ChipCM5(Cytiva)にアミンカップリング法 (AmineCoupling Kit, Cytiva) でHer2抗原 (RecombinantHumanErbB2/Her2 FcChimera Protein, R&D SYSTEMS)の固定化を行った。次に、FLの希釈系列(1E-08 Mから6.25E-10Mまで2倍希釈系列)を用いて結合活性の測定を実施した。実施結果を図2に示す。結果より安定した結合を示すことが確認された。解析の結果、ka = 8.509E+6、kd = 0.006598、KD = 7.753E-10となった。
96ウェルプレートに細胞数が 1 x 104cells/well、培養液50μL/wellとなるようにHer2陽性細胞(KPL-4)を播種し、一晩培養を行った。精製したMFL2-G5Sx3-Hisとビオチン-SiPc(製造例1において製造したもの)をモル比が1:4になるように混合し(以下、MFL2-G5Sx3-His複合体と示す。)、10μg/mLから10倍希釈系列8系列(ゼロ濃度度を含む)を作製した。希釈系列調整後、複合体を細胞に添加し24時間後に培養プレート底面から100 J/cm2 となるように690 nm の光を出すLEDで光照射を行った。その後、24時間培養し、MFL2-G5Sx3-His複合体含有培地を除去したのち、PBSで細胞を1回洗浄し、新しい培地を添加し Cell Counting Kit-8 (同仁化学社) を用いて生細胞数の比較を行なった。用法用量は取扱説明書に従い試薬添加1時間半37℃、CO2インキュベーターでインキュベーションしたのち、吸光度450nmを測定し、平均値を計算しバックグラウンド補正後、コントロールを100%として各条件のコントロールに対する細胞増殖の割合を計算した。実施結果を図4に示す。FLと光増感剤ビオチン-SiPc化合物の複合体は、光照射依存的かつ濃度依存的な細胞傷害性を有することが確認された。
次にMFL2-G5Sx3-del5におけるHER2抗原を認識する分子とCupid-del5をつなぐアミノ酸リンカーの長さがちがい、C末端に5アミノ酸PSAASを付加したタンパク質発現ベクターの構築を行なった。具体的には図5Aに示すように、グリシン4つとセリン1つを組み合わせたペプチドを基本単位とし、1単位、2単位、3単位の長さのリンカーを有し、C末端がPSAASとなる発現ベクターを作製した。作製方法は、以下の通り。
pET45-MFL2-G4Sx1-PSAAS、pET45-MFL2-G4Sx2-PSAAS、pET45-MFL2-G4Sx3-PSAASをコンピテントセルBL21 (DE3)(ECOS Competent E.coli BL21 (DE3), ニッポンジーン社)に導入し形質転換を行った。100 mL の2xYT培地で一晩培養された培養液を1リットルの培養液に食菌し37℃にて培養を行い、600 nm のOD値が0.5-0.8になった時点で最終濃度0.5 mM になるようにIPTGを添加し、37℃にて4時間の培養を行ったのち菌体を遠心(7500 x g, 20 min at 4℃)にて回収した。
AEAGITGTWSNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH*
VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANLLAEAKKLNDAQAPK
MVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANLLAEAKKLNDAQAPKSGGGGGSGGGGGSGGGGGSAEAGITGTWSNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAASHHHHHH*
ATGGTGGACAACAAATTCAACAAAGAGATGCGCAATGCGTACTGGGAAATTGCCCTGTTACCGAACCTGAACAACCAGCAGAAACGTGCCTTCATTCGCAGCATCTATGACGATCCAAGCCAATCTGCGAATCTGCTTGCAGAAGCGAAGAAACTGAATGATGCTCAAGCTCCGAAAAGCGGAGGCGGCGGCGGGTCTGGTGGTGGCGGTGGCAGTGGCGGAGGTGGCGGTTCAGCGGAAGCCGGTATTACCGGGACCTGGTCCAATCAACTCGGCTCCACGTTTATCGTGACAGCAGGCGCGGATGGCGCGCTGACAGGGACGTACGAGTCCGCGGTAGGCAATGCGGAAAGCCGCTATGTGTTGACCGGTCGTTACGATTCGGCTCCGGCTACGGATGGTTCCGGTACAGCCTTAGGGTGGACCGTTGCGTGGAAGAACAACTCGAAGAATGCCCACAGTGCTACCACTTGGTCAGGCCAGTATGTTGGCGGGGCGGATGCGAAAATCAACACTCAGTGGCTTCTGACCAGCGGAACCACGAATGCCAATGCGTGGAAATCCACGCTGGTCGGTCATGACACCTTTACCAAAGTCAAACCGAGTGCAGCATCGCATCATCACCACCATCATTAA
MVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANLLAEAKKLNDAQAPKSGGGGGSGGGGGSGGGGGSAEAGITGTWSNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVK
ATGGTGGACAACAAATTCAACAAAGAGATGCGCAATGCGTACTGGGAAATTGCCCTGTTACCGAACCTGAACAACCAGCAGAAACGTGCCTTCATTCGCAGCATCTATGACGATCCAAGCCAATCTGCGAATCTGCTTGCAGAAGCGAAGAAACTGAATGATGCTCAAGCTCCGAAAAGCGGAGGCGGCGGCGGGTCTGGTGGTGGCGGTGGCAGTGGCGGAGGTGGCGGTTCAGCGGAAGCCGGTATTACCGGGACCTGGTCCAATCAACTCGGCTCCACGTTTATCGTGACAGCAGGCGCGGATGGCGCGCTGACAGGGACGTACGAGTCCGCGGTAGGCAATGCGGAAAGCCGCTATGTGTTGACCGGTCGTTACGATTCGGCTCCGGCTACGGATGGTTCCGGTACAGCCTTAGGGTGGACCGTTGCGTGGAAGAACAACTCGAAGAATGCCCACAGTGCTACCACTTGGTCAGGCCAGTATGTTGGCGGGGCGGATGCGAAAATCAACACTCAGTGGCTTCTGACCAGCGGAACCACGAATGCCAATGCGTGGAAATCCACGCTGGTCGGTCATGACACCTTTACCAAAGTCAAA
MVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANLLAEAKKLNDAQAPKGGGGSAEAGITGTWSNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAAS*
MVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANLLAEAKKLNDAQAPKGGGGSGGGGSAEAGITGTWSNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAAS*
MVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSIYDDPSQSANLLAEAKKLNDAQAPKGGGGSGGGGSGGGGSAEAGITGTWSNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNSKNAHSATTWSGQYVGGADAKINTQWLLTSGTTNANAWKSTLVGHDTFTKVKPSAAS*
ATGGTGGACAACAAATTCAACAAAGAGATGCGCAATGCGTACTGGGAAATTGCCCTGTTACCGAACCTGAACAACCAGCAGAAACGTGCCTTCATTCGCAGCATCTATGACGATCCAAGCCAATCTGCGAATCTGCTTGCAGAAGCGAAGAAACTGAATGATGCTCAAGCTCCGAAAGGAGGCGGAGGGTCTGCGGAAGCCGGTATTACCGGGACCTGGTCCAATCAACTCGGCTCCACGTTTATCGTGACAGCAGGCGCGGATGGCGCGCTGACAGGGACGTACGAGTCCGCGGTAGGCAATGCGGAAAGCCGCTATGTGTTGACCGGTCGTTACGATTCGGCTCCGGCTACGGATGGTTCCGGTACAGCCTTAGGGTGGACCGTTGCGTGGAAGAACAACTCGAAGAATGCCCACAGTGCTACCACTTGGTCAGGCCAGTATGTTGGCGGGGCGGATGCGAAAATCAACACTCAGTGGCTTCTGACCAGCGGAACCACGAATGCCAATGCGTGGAAATCCACGCTGGTCGGTCATGACACCTTTACCAAAGTCAAACCGAGTGCAGCATCGTAA
ATGGTGGACAACAAATTCAACAAAGAGATGCGCAATGCGTACTGGGAAATTGCCCTGTTACCGAACCTGAACAACCAGCAGAAACGTGCCTTCATTCGCAGCATCTATGACGATCCAAGCCAATCTGCGAATCTGCTTGCAGAAGCGAAGAAACTGAATGATGCTCAAGCTCCGAAAGGAGGCGGAGGGTCTGGAGGTGGCGGTTCAGCGGAAGCCGGTATTACCGGGACCTGGTCCAATCAACTCGGCTCCACGTTTATCGTGACAGCAGGCGCGGATGGCGCGCTGACAGGGACGTACGAGTCCGCGGTAGGCAATGCGGAAAGCCGCTATGTGTTGACCGGTCGTTACGATTCGGCTCCGGCTACGGATGGTTCCGGTACAGCCTTAGGGTGGACCGTTGCGTGGAAGAACAACTCGAAGAATGCCCACAGTGCTACCACTTGGTCAGGCCAGTATGTTGGCGGGGCGGATGCGAAAATCAACACTCAGTGGCTTCTGACCAGCGGAACCACGAATGCCAATGCGTGGAAATCCACGCTGGTCGGTCATGACACCTTTACCAAAGTCAAACCGAGTGCAGCATCGTAA
ATGGTGGACAACAAATTCAACAAAGAGATGCGCAATGCGTACTGGGAAATTGCCCTGTTACCGAACCTGAACAACCAGCAGAAACGTGCCTTCATTCGCAGCATCTATGACGATCCAAGCCAATCTGCGAATCTGCTTGCAGAAGCGAAGAAACTGAATGATGCTCAAGCTCCGAAAGGAGGCGGAGGGTCTGGAGGTGGCGGTTCAGGTGGCGGTGGCAGTGCGGAAGCCGGTATTACCGGGACCTGGTCCAATCAACTCGGCTCCACGTTTATCGTGACAGCAGGCGCGGATGGCGCGCTGACAGGGACGTACGAGTCCGCGGTAGGCAATGCGGAAAGCCGCTATGTGTTGACCGGTCGTTACGATTCGGCTCCGGCTACGGATGGTTCCGGTACAGCCTTAGGGTGGACCGTTGCGTGGAAGAACAACTCGAAGAATGCCCACAGTGCTACCACTTGGTCAGGCCAGTATGTTGGCGGGGCGGATGCGAAAATCAACACTCAGTGGCTTCTGACCAGCGGAACCACGAATGCCAATGCGTGGAAATCCACGCTGGTCGGTCATGACACCTTTACCAAAGTCAAACCGAGTGCAGCATCGTAA
Ala Glu Ala Gly Ile Thr Gly Thr TrpTyr Asn Gln Leu Gly Ser Thr
Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly ThrTyr Glu
Ser Ala Val Gly Asn Ala GluSer Arg Tyr Val Leu Thr GlyArg Tyr
Asp Ser Ala Pro Ala Thr Asp GlySer Gly Thr Ala Leu Gly Trp Thr
Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp
Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp
Leu Leu Thr Ser Gly Thr ThrGlu Ala Asn Ala Trp Lys SerThr Leu
Val Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser
Claims (14)
- 配列番号17に記載のコアストレプトアビジンのアミノ酸配列(但し、C末端のPro-Ser-Ala-Ala-Serからなるアミノ酸配列の一部又は全部は欠失していてもよい)において下記(1)~(6)の変異を有するアミノ酸配列を含み、野生型ストレプトアビジンと比較して免疫原性が低下しているストレプトアビジン変異体のN末端側及び/又はC末端側に、リンカー配列を介して、分子量20,000以下の抗原結合分子が結合している、融合タンパク質。
(1)10番目のアミノ酸残基のチロシンがセリン又はトレオニンに置換している変異:
(2)71番目のアミノ酸残基のチロシンがアラニン又はセリンに置換している変異:
(3)72番目のアミノ酸残基のアルギニンがリジンに置換している変異:
(4)89番目のアミノ酸残基のグルタミン酸がアスパラギン酸に置換している変異:
(5)91番目のアミノ酸残基のアルギニンがリジンに置換している変異:
(6)104番目のアミノ酸残基のグルタミン酸がグルタミン又はアスパラギンに置換している変異: - N末端側からC末端側に、分子量20,000以下の抗原結合分子と、リンカー配列と、前記ストレプトアビジン変異体のアミノ酸配列とをこの順に有する、請求項1に記載の融合タンパク質。
- 抗原結合分子が、がん細胞に発現している抗原に結合する分子である、請求項1又は2に記載の融合タンパク質。
- 抗原結合分子が、Her2に結合する分子である、請求項1から3の何れか一項に記載の融合タンパク質。
- 抗原結合分子が、配列番号2に記載のアミン酸配列を有する、請求項1から4の何れか一項に記載の融合タンパク質。
- リンカー配列が、グリシン残基及びセリン残基からなり、アミノ酸残基の数が5から25個である、請求項1から5の何れか一項に記載の融合タンパク質。
- リンカー配列が、[(Gly)m-Ser]n(式中、mは1~10の整数を示し、nは1~5の整数を示す)で示されるアミノ酸配列である、請求項1から6の何れか一項に記載の融合タンパク質。
- 配列番号3又は9に記載のアミノ酸配列を有する、請求項1から7の何れか一項に記載の融合タンパク質。
- 配列番号3又は9に記載のアミノ酸配列を有する融合タンパク質をコードする核酸。
- 請求項1から8の何れか1項に記載の融合タンパク質を含む、がん治療剤またはがん診断剤。
- (1)請求項1から8の何れか1項に記載の融合タンパク質、及び(2)ビオチンと診断用物質又は治療用物質とのコンジュゲートを含む、がんの治療または診断キット。
- 診断用物質又は治療用物質が、フタロシアニン染料である、請求項11に記載のキット。
- 請求項1から8の何れか一項に記載の融合タンパク質をコードする核酸を宿主で発現させる工程を含む、請求項1から8の何れか一項に記載の融合タンパク質の製造方法。
- 前記融合タンパク質を、細菌の封入体において発現させて回収する、請求項13に記載の方法。
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WO2010095455A1 (ja) | 2009-02-20 | 2010-08-26 | 国立大学法人 東京大学 | 低免疫原性ストレプトアビジンおよびその利用 |
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