WO2016140344A1 - 結合体及びその使用 - Google Patents
結合体及びその使用 Download PDFInfo
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- WO2016140344A1 WO2016140344A1 PCT/JP2016/056797 JP2016056797W WO2016140344A1 WO 2016140344 A1 WO2016140344 A1 WO 2016140344A1 JP 2016056797 W JP2016056797 W JP 2016056797W WO 2016140344 A1 WO2016140344 A1 WO 2016140344A1
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- Prior art keywords
- conjugate
- compound
- target substance
- hydrogen atom
- alkyl group
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Images
Classifications
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- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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Definitions
- the present invention relates to a conjugate and its use.
- This application claims priority based on Japanese Patent Application No. 2015-043459 filed in Japan on March 5, 2015, the contents of which are incorporated herein by reference.
- Antibody pharmaceuticals are purified using a protein A binding resin that binds to the Fc region during the manufacturing process (see, for example, Patent Document 1). Protein A binding resins are expensive and have limited restrictions on reuse after washing. Therefore, in spite of various improvements, the purification cost of the antibody has not decreased so much, which is one of the causes of the expensive antibody drug.
- antibody drugs are highly humanized, there is almost no difference from a large amount of antibodies present in the body, and it is impossible to selectively adsorb and remove the administered antibody drugs using a plasma purification device or the like.
- An object of the present invention is to provide a technique capable of easily purifying a biologically active protein such as an antibody at low cost.
- the present invention also includes conjugates, vectors, pharmaceutical compositions, pharmaceuticals, conjugate purification methods, conjugate capture solid phases, target substance removal methods, target substance removal devices, conjugate detection label compounds, and conjugate detection. It is an object to provide a method, a compound detection conjugate, and a compound detection method.
- the present invention includes the following aspects.
- a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence comprising one or several amino acids deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 1, and —OH and —OR 1
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- the functional molecule is a target substance capturing molecule.
- the target substance-capturing molecule is an antibody, antibody fragment, aptamer or receptor against the target substance.
- TNFR tumor necrosis factor receptor
- a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence comprising one or several amino acids deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 1, and —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- Drug and —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group or —PO 3 H 2 . And a conjugate according to any one of (1) to (5) bound to the compound. (8) A pharmaceutical comprising the conjugate according to any one of (1) to (5) as an active ingredient. (9) —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group or —PO 3 H 2 , wherein the conjugate according to any one of (1) to (5) is immobilized on a solid phase.
- a method for purifying a conjugate comprising the step of contacting with a compound having the formula: (10)
- a method of removing the conjugate comprising the step of contacting with a compound having the formula: (12) A plasma containing a complex of the target substance and the conjugate according to any one of (2) to (5), -OH and -OR 1 [R 1 is a hydrogen atom, alkyl Represents the group or —PO 3 H 2 .
- a method for removing the target substance comprising the step of contacting the compound with (13)
- a method for removing a target substance comprising the step of bringing plasma containing a target substance into contact with the conjugate according to any one of (2) to (5) immobilized on a solid phase.
- the conjugate removing apparatus further comprising a solid phase on which a compound having the formula:
- a target substance removing apparatus comprising a solid phase on which a compound having (16) A removal means for removing the target substance from plasma containing the target substance is provided, and the removal means comprises a solid phase on which the conjugate according to any one of (2) to (5) is fixed.
- a target substance removing device 17.
- a labeled compound for detecting a conjugate (18) In the conjugate according to any one of (1) to (5), —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- a method of detecting a labeled compound in the complex and a step of detecting a labeled compound in the complex.
- a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence comprising one or several amino acids deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 1, and —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 , characterized in that it is a conjugate of a protein having binding activity to a compound having . ] The compound detection conjugate
- a compound detection conjugate according to (19) is bound to a compound having the following formula: and a step of detecting the labeling substance in the complex.
- OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 . ] The detection method of the compound which has this.
- conjugates it is possible to provide a technique capable of easily purifying a biologically active protein such as an antibody at low cost.
- conjugates, vectors, pharmaceutical compositions, pharmaceuticals, conjugate purification methods, conjugate capture solid phases, target substance removal methods, target substance removal devices, conjugate detection label compounds, conjugate detection methods, compounds A detection conjugate and a method for detecting a compound can be provided.
- FIG. 10 is a graph showing the results of Experimental Example 2. 10 is a graph showing the results of Experimental Example 2. 10 is a graph showing the results of Experimental Example 2. 10 is a graph showing the results of Experimental Example 2.
- 10 is a graph showing the results of Experimental Example 3.
- 10 is a graph showing the results of Experimental Example 4.
- 10 is a photograph showing the results of Experimental Example 5. It is a general formula showing the structures of 1-monoacylglycerol, 2-monoacylglycerol and lysophosphatidic acid, and R represents an alkyl group.
- 10 is a graph showing the results of Experimental Example 7.
- 10 is a photograph showing the results of Experimental Example 7.
- 10 is a graph showing the results of Experimental Example 8.
- 10 is a graph showing the results of Experimental Example 9.
- 10 is a photograph showing the results of Experimental Example 9.
- 10 is a photograph showing the results of Experimental Example 10.
- 10 is a photograph showing the results of Experimental Example 10.
- 14 is a graph showing the results of Experimental Example 11.
- 14 is a graph showing the results of Experimental Example 11. It is a graph which shows the result of Experimental example 12.
- 14 is a graph showing the results of Experimental Example 13. It is a graph which shows the result of Experimental example 14.
- inlectin which is a kind of lectin (the amino acid sequence of human inlectin-1 is shown in SEQ ID NO: 1) to a compound having a specific structure typified by a diol structure described later. As a result, the present invention has been completed.
- the present invention comprises a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 1.
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- R 1 is a hydrogen atom
- the above compound is a compound having two —OH. Examples of such compounds include diols, details of which will be described later.
- one or several is, for example, 1 to 60, for example 1 to 55, for example 1 to 50, for example 1 to 40, for example 1 to 30, for example 1 to 20, for example 1 to 10.
- the above protein may be referred to as “interlectin or a mutant thereof”.
- R 1 is an alkyl group
- the alkyl group has, for example, 1 to 6 carbon atoms, for example 1 to 3.
- mouse inlectin-1 is —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 . It was found to have a binding activity to a compound having Human inlectin-1 and mouse inlectin-1 differ in 51 amino acid residues. Therefore, the inlectin in the conjugate of this embodiment or a variant thereof may have at least 51 mutations in the amino acid sequence shown in SEQ ID NO: 1.
- the amino acid sequence of mouse inlectin-1 is shown in SEQ ID NO: 7, and the base sequence is shown in SEQ ID NO: 8.
- Wild-type human inlectin-1 forms a trimer, but a mutant (C31, 48S) in which the 31st and 48th cysteine residues are substituted with serine residues, respectively, is a 3 It is produced as a monomer without forming a monomer (“Tsuji S., et al., Differential structure and activity between human and mouse intelectin-1: Human intelectin-1 is a disulfide-linked trimer, whereas mouse homologue is a monomer, Glycobiology 17 (10), 1045-1051, 2007. ”
- the inventors have found that the C31,48S mutant of human inlectin- 1 is —OH and —OR 1 [R 1 is a hydrogen atom, an alkyl group, as in human inlectin- 1 . Or represents -PO 3 H 2 . It was found to have a binding activity to a compound having
- inlectin or a variant thereof may be produced as a monomer, for example, may be produced as a multimer such as a trimer.
- the above compound is preferably a compound represented by the following formula (1) from the viewpoint of high binding activity of intelectin or a mutant thereof.
- R 1 represents a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or —PO 3 H 2 .
- Y 1 represents a single bond or a methylene group.
- R 2 to R 5 each independently represents a hydrogen atom or an organic group which may be substituted. When any of R 2 to R 5 is an organic group, the organic group may form a branch or a ring, and may have an ester bond, an ether bond, or the like.
- Substituents include alkyl groups, hydroxyl groups, acyl groups, phosphate groups, phosphate ester groups, carboxyl groups, amino groups, nitro groups, thiol groups, sulfo groups, carbonyl groups, acetamide groups, benzoyl groups, alkylene groups, alkynyls. Group, aryl group, silicon compound, halogen atom and the like.
- inlectin or a mutant thereof exhibits high adsorption activity (binding activity) for the compound represented by the above formula (1).
- R 1 to R 4 of the compound represented by the above formula (1) are preferably hydrogen atoms.
- R 1 to R 4 of the compound represented by the above formula (1) are hydrogen atoms, the structure of R 5 tends not to affect the binding activity of inlectin or a mutant thereof.
- R 1 is a hydrogen atom
- either R 2 or R 3 is an alkyl group
- either R 4 or R 5 is an alkyl group.
- a meso form is preferable to a cis form or a trans form from the viewpoint of higher binding activity of intelectin or a mutant thereof.
- Specific examples of the compound represented by the formula (1) include, for example, L-ribose, D-ribose, 2-deoxygalactose, 2-acetamido-2-deoxy-4-O-beta-D-galactopyranosyl.
- a plurality of the compounds represented by the above formula (1) may be linked to form a polymer.
- the polymer represented by following formula (2), polyvinyl alcohol, etc. are mentioned, for example.
- Y 1 represents the same meaning as Y 1 in formula (1)
- Y 2 to Y 4 each independently represents a divalent linking group
- m, n, and p each independently represents 0.
- q represents an integer of 2 or more.
- the divalent linking group include —CH 2 —, —O—, —S—, —NH—, —CO—, —CONH—, —CH (COOH) —.
- the upper limit of q is about 5000, for example.
- Examples of the compound represented by the above formula (2) include polyethylene glycol having a diol structure introduced into the side chain, ⁇ -polyglutamic acid having a diol structure introduced into the side chain, and the like.
- the functional molecule in the conjugate of the present embodiment is not particularly limited as long as it has a desired function, and may be a therapeutic drug, a diagnostic drug, or the like.
- the pharmaceutical product may be, for example, a high molecular compound such as a protein or a low molecular compound.
- More specific functional molecules include, for example, target substance capturing molecules; anticancer agents, cytotoxic agents, therapeutic radioisotopes (eg, P 32 , Y 90 , I 131 , I 125 , Sm 153 , Re 186 , Re 188 , At 211 , Bi 212 , Pb 212 , Lu, etc.) pharmaceutical composition containing a compound, etc .; computer tomography (CT), magnetic resonance imaging (MRI), etc. Contrast agent used for the above; a probe for positron emission tomography (PET) and the like.
- CT computer tomography
- MRI magnetic resonance imaging
- PET positron emission tomography
- the functional molecule may be a high molecular compound such as a protein or nucleic acid used as a reagent, or may be a low molecular compound.
- target substance capture molecule examples include an antibody against the target substance, an antibody fragment, an aptamer, and a receptor.
- the antibody or antibody fragment is preferably humanized.
- antibody fragments include Fv, Fab, scFv and the like.
- the antibody or antibody fragment may be derived from an antibody drug.
- Antibody drugs are highly humanized, so there is almost no difference from antibodies present in the body and cannot be removed once administered to a patient.
- the conjugate of the present embodiment can be removed from the patient's body by using a target substance removing device, which will be described later, as necessary.
- An aptamer is a substance having a specific binding ability with a target substance.
- examples of aptamers include nucleic acid aptamers and peptide aptamers.
- a nucleic acid aptamer having a specific binding ability to a target substance can be selected by, for example, a systematic evolution of ligand by exponential enrichment (SELEX) method.
- Peptide aptamers having specific binding ability to the target substance can be selected by, for example, the two-hybrid method using yeast.
- the conjugate of this embodiment may be a fusion protein with the target substance capturing molecule.
- the conjugate of this embodiment can be obtained by cross-linking inlectin or a mutant thereof and the target substance capturing molecule using a chemical linker or the like. Obtainable.
- the target substance may be, for example, a cancer antigen, for example, a cell membrane antigen, for example, a cytokine, for example, an arbitrary protein or the like that is a research object in basic research or the like.
- the cancer antigen is not particularly limited, and examples thereof include HER2, MAGE, CEA, WT1, and KL-6.
- Examples of cell membrane antigens include CD3, CTLA-4, and PD-1.
- cytokines include inflammatory cytokines, and specific examples include TNF- ⁇ , TNF- ⁇ , LT- ⁇ , interleukin (IL) -1 ⁇ , IL-6, and the like.
- the target substance capturing molecule is a receptor
- examples of the receptor include cytokine receptors, receptors that bind to cell membrane antigens, and the like.
- cytokine receptors include tumor necrosis factor receptor (TNFR), IL-1 receptor, IL-6 receptor, TRAIL receptor and the like.
- TNFR includes TNFR1, TNFR2, and the like, but TNFR2 is preferable because of its higher affinity for TNF.
- the conjugate of the present embodiment is a cytokine receptor
- the conjugate can be used, for example, for removal of cytokines in blood. Details will be described later.
- inlectin or a mutant thereof can be produced as a monomer, for example, as a multimer such as a trimer.
- multimers have higher affinity for the target substance than monomers, so if you want to increase the affinity for the target substance, you can produce inlectin or its variant as a multimer. Also good.
- inlectin or its variant for example as a purification tag etc., you may produce as a monomer.
- Human inlectin is a plasma protein and does not induce a physiological response due to the high concentration of body fluid. Therefore, it is considered that the conjugate of this embodiment is unlikely to exhibit harmful side effects even when administered to humans or non-human animals as a pharmaceutical product.
- the present invention provides a vector comprising a nucleic acid encoding a fusion protein of an intellactin or a variant thereof and an antibody, antibody fragment, peptide aptamer or receptor for a target substance.
- the nucleic acid encoding inlectin or a variant thereof include, for example, a nucleic acid comprising the base sequence described in SEQ ID NO: 2, or 80% or more, such as 85% or more, for example 90%, with the base sequence described in SEQ ID NO: 2. As described above, for example, they have 95% or more identity, and —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- a nucleic acid comprising a base sequence encoding a protein having a binding activity to a compound having The base sequence shown in SEQ ID NO: 2 is the base sequence of human inlectin-1.
- the vector of this embodiment is preferably an expression vector.
- the expression vector is not particularly limited.
- plasmids derived from E. coli such as pBR322, pBR325, pUC12, and pUC13
- plasmids derived from Bacillus subtilis such as pUB110, pTP5, and pC194
- plasmids derived from yeast such as pSH19 and pSH15
- Bacteriophages viruses such as adenoviruses, adeno-associated viruses, lentiviruses, vaccinia viruses, baculoviruses; and modified vectors thereof.
- the promoter for expression of the conjugate is not particularly limited, and for example, EF1 ⁇ promoter, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-tk promoter, etc. should be used. Can do.
- the above expression vector may further have an enhancer, a splicing signal, a poly A addition signal, a selection marker, a replication origin, and the like.
- the present invention relates to a drug and —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group or —PO 3 H 2 . And a conjugate containing the compound bound to the compound, and a pharmaceutical composition comprising the above-mentioned conjugate.
- the pharmaceutical composition of this embodiment may be a drug delivery system.
- the compound is preferably a compound represented by the formula (1) or (2).
- FIG. 1 is a schematic diagram illustrating the structure of the pharmaceutical composition of the present embodiment.
- the pharmaceutical composition 100 includes a carrier 130 containing a drug 110 and a compound 120, and a conjugate 140 bound to the compound 120.
- the compound 120 is preferably exposed on the surface of the carrier 130.
- the conjugate 140 is a conjugate of the above-mentioned inlectin or its variant 141 and, for example, an antibody 142 against a cancer antigen.
- the carrier 130 is not particularly limited as long as it is used in a drug delivery system, and examples thereof include micelles and liposomes.
- the micelle is not particularly limited as long as it is used in a drug delivery system.
- a micelle formed from a block copolymer having a hydrophilic region and a hydrophobic region can be used.
- the liposome is not particularly limited as long as it is used in a drug delivery system, and for example, a liposome formed from phospholipid can be used.
- the micelles and liposomes are formed after adding the compound 120 to these micelle and liposome materials.
- the compound 120 include monoacylglycerols such as 1-monoolein.
- the drug 110 examples include anticancer agents such as paclitaxel, docetaxel, vincristine, vinorelbine, vinblastine, vindesine, eribulin mesylate, oxaliplatin, cisplatin, carboplatin and the like.
- anticancer agents such as paclitaxel, docetaxel, vincristine, vinorelbine, vinblastine, vindesine, eribulin mesylate, oxaliplatin, cisplatin, carboplatin and the like.
- the drug 110 can be encapsulated in the micelle or liposome by forming micelles or liposomes in the presence of the drug 110.
- the conjugate 140 is easily bound when it is brought into contact with the compound 120. Therefore, the pharmaceutical composition 100 can be easily produced by mixing the carrier 130 containing the compound 120 and the conjugate 140.
- the pharmaceutical composition 100 of this embodiment can deliver the drug 110 specifically to tissues or cells that express cancer antigens. For this reason, cytotoxic activity can be exerted specifically for cancer cells, and side effects can be reduced.
- the pharmaceutical composition of this embodiment not only specifically delivers an anticancer agent targeting the above-described cancer cells, but also specifically delivers an antibacterial agent or antiviral agent targeting, for example, bacteria or virus-infected cells; Targeting cells according to the purpose, specifically delivering metabolic regulators such as enzymes, siRNAs, peptide aptamers, nucleic acid aptamers, nucleic acids (genes); targeting the cells according to the purpose, radioisotopes, fluorescent dyes It can also be used for specifically delivering a labeling substance such as a fluorescent protein.
- metabolic regulators such as enzymes, siRNAs, peptide aptamers, nucleic acid aptamers, nucleic acids (genes)
- targeting the cells according to the purpose radioisotopes, fluorescent dyes
- a labeling substance such as a fluorescent protein.
- the present invention relates to an anticancer agent, antibacterial agent, antiviral agent, metabolic regulator or labeling substance, and —OH and —OR 1
- R 1 represents a hydrogen atom, an alkyl group or —PO 3 H 2
- a pharmaceutical composition comprising a carrier comprising a compound having the following formula: and a conjugate of an antibody, antibody fragment or aptamer to cancer antigen or a desired antigen and interlectin or a variant thereof bound to the compound.
- a method of treating or diagnosing cancer or an infectious disease comprising the step of administering is provided.
- the present invention provides an anticancer agent, antibacterial agent, antiviral agent, metabolic regulator or labeling substance for the treatment of cancer or infectious diseases, and —OH and —OR 1 [where R 1 is a hydrogen atom, alkyl Represents the group or —PO 3 H 2 . And a conjugate of an antibody, antibody fragment or aptamer against a cancer antigen or a desired antigen and intelectin or a variant thereof bound to the compound.
- the present invention relates to an antibody, an antibody fragment or an aptamer against a cancer antigen or a desired antigen for producing a pharmaceutical composition for treating cancer or a pharmaceutical composition for treating infectious disease, and an intectin or a variant thereof.
- the present invention provides a medicament containing the above-mentioned conjugate as an active ingredient.
- the medicament of the present embodiment may be, for example, an antibody medicament, for example, a hypercytokinemia therapeutic agent (anti-cytokine drug).
- the conjugate include a conjugate of an antibody against the target substance, an antibody fragment or a peptide aptamer and intelectin or a variant thereof; a conjugate of a cytokine receptor and intelectin or a variant thereof.
- the target substance, cytokine and cytokine receptor are as described above.
- the medicine of this embodiment can also be applied to existing antibody medicines. Thereby, the antibody drug can be easily purified at low cost.
- the target diseases are systemic inflammatory response syndrome (SIRS) such as sepsis, which is hypercytokinemia, rheumatism, cancer cachexia, psoriasis, etc. Is mentioned.
- SIRRS systemic inflammatory response syndrome
- the anti-cytokine drug of this embodiment to a patient with severe SIRS, the inflammatory cytokine in the blood can be adsorbed and the symptoms can be alleviated.
- the anti-cytokine drug of this embodiment to patients with rheumatism, cancer cachexia, psoriasis and the like, inflammatory cytokines in the body can be adsorbed and the symptoms can be alleviated.
- the present invention provides a method for treating cancer, comprising the step of administering to a mammal a medicament comprising an antibody, antibody fragment or aptamer against a cancer antigen, and a conjugate of intelectin or a variant thereof. To do.
- the present invention provides a conjugate of an antibody, antibody fragment, or aptamer against a cancer antigen and inlectin or a variant thereof for the treatment of cancer.
- the present invention provides the use of a conjugate of an antibody, antibody fragment or aptamer against cancer antigen and inlectin or a variant thereof for producing a medicament for treating cancer.
- the present invention comprises the step of administering to a mammal a medicament comprising an antibody against a cytokine, an antibody fragment or an aptamer, or a conjugate of a cytokine receptor and intelectin or a variant thereof.
- a method for treating a disease caused by the disease is provided.
- the present invention provides a conjugate of an antibody against an cytokine, an antibody fragment or an aptamer, or a cytokine receptor, and an inlectin or a variant thereof, for the treatment of a disease caused by high cytokine production.
- the present invention provides a conjugate of an antibody against an cytokine, an antibody fragment or an aptamer, or a cytokine receptor and inlectin or a variant thereof for producing a medicament for treating a disease caused by high cytokine production.
- a conjugate of an antibody against an cytokine, an antibody fragment or an aptamer, or a cytokine receptor and inlectin or a variant thereof for producing a medicament for treating a disease caused by high cytokine production.
- the present invention provides a genetic recombinant that expresses the conjugate described above.
- genetic recombinants include cultured cells such as bacteria, yeast, insect cells and animal cells in which the above-described conjugate expression vector has been introduced; insect organisms such as silkworms in which the above-described conjugate expression vector has been introduced. Examples thereof include animals such as goats, sheep, cows, and chickens that have been genetically modified to express the above-mentioned conjugates in milk and eggs.
- the above-described conjugate can be produced from the gene recombinant of this embodiment.
- the produced conjugate can be easily purified by the method described later.
- the present invention provides —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 , wherein the above-described conjugate is immobilized on a solid phase.
- a method for purifying a conjugate comprising the step of contacting with a compound having the formula: The compound is preferably a compound represented by the formula (1) or (2).
- the solid phase (solid phase for capturing the conjugate) on which the above compound is immobilized will be described later.
- purification can be called “separation”, “recovery”, “adsorption”, “binding”, and the like.
- a conjugate expressed in the above-described culture of a genetic recombinant, an insect in vivo, milk, or egg can be easily purified at low cost.
- the conjugate contained in the culture, insect in vivo, milk, egg and —OH and —OR 1 [R 1 is a hydrogen atom, an alkyl group or It represents a -PO 3 H 2. ] Is contacted with the solid phase on which the compound having Then, the conjugate can be adsorbed to the solid phase by adsorbing intelectin or a mutant region thereof to the compound.
- conjugate adsorbed on the solid phase is washed with a buffer solution or the like.
- a buffer solution or the like for example, —OH and —OR 1
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 in a free state on the conjugate adsorbed on the solid phase.
- the conjugate can be dissociated from the solid phase and recovered. Examples of the above-mentioned compound in the free state include glycerol, propylene glycol, ribose and the like.
- the inventors have intellectin or a derivative thereof and —OH and —OR 1
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- EDTA ethylenediaminetetraacetic acid
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- the solid phase on which the compound having the formula is fixed is very inexpensive as compared with, for example, a protein A-binding resin and has resistance to denaturing washing treatment. Therefore, when the conjugate is, for example, a conjugate of intelectin or a variant thereof and an antibody that is an antibody drug, the purification method of the present embodiment can significantly reduce the manufacturing cost of the antibody drug. Can be provided at low cost.
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 typified by diols.
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 typified by diols.
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 typified by diols.
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 typified by diols.
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 typified by diols.
- the present invention is a solid phase for capturing the above-described conjugate, and —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- a solid phase for capturing a conjugate characterized in that a compound having the formula:
- the compound is preferably a compound represented by the formula (1) or (2).
- the solid phase include beads, membranes, hollow fiber membranes, plastic substrates, well plates, slide glasses, sensor chips of measuring instruments such as surface plasmon resonance measuring devices, and the like.
- a solid phase in the form of beads, membranes, etc. may be packed in a column.
- —OH and —OR 1 represented by diols
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- a diepoxide such as ethylene glycol glycidyl ether or 1,4-butanediol diglycidyl ether used as a cross-linking agent is subjected to an alkali hydrolysis or acid hydrolysis treatment, whereby a hydroxyl group, an amino group,
- the compound can be easily immobilized on a solid phase having a thiol group.
- 3-amino-1,2-propanediol is bonded onto a solid phase having a carboxyl group via 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC).
- EDC 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride
- the compound can be immobilized.
- the compound can be immobilized on a solid phase having an amino group by binding glyceric acid via EDC.
- protein A binding resin is limited in re-use because protein A deteriorates due to washing and sterilization.
- purifying an antibody using a protein A binding resin it is necessary to denature and elute the antibody with an acidic buffer, neutralize the eluted antibody, and further purify it with an ion exchange column. There are many steps required for purification.
- —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 . ],
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- the compound-capturing solid phase of this embodiment can use almost all cleaning and sterilization methods that the solid phase can withstand. It is.
- the binding rate with the binding agent (intellectin) is fast, the purification purity is high, and the recovery efficiency is as high as 95% or more.
- eluents such as glycerol, propylene glycol, and ribose have low toxicity and protein denaturation action, for example, the conjugate eluted from the conjugate-capturing solid phase of this embodiment can be adsorbed to the ion exchange column as it is. The conjugate can be easily purified.
- the present invention relates to plasma containing a complex of a target substance and the above-described conjugate with —OH and —OR 1
- R 1 is a hydrogen atom, an alkyl group, or —PO 3 , which is immobilized on a solid phase. representing the H 2.
- a method for removing a target substance, comprising the step of contacting the compound with The compound is preferably a compound represented by the formula (1) or (2).
- the target substance removal method of this embodiment can also be referred to as a method for removing a target substance from plasma or a method for removing a conjugate from plasma.
- the target substance removal method of this embodiment is a kind of apheresis, which is a method of collecting a desired blood component from whole blood.
- conjugates include antibody pharmaceuticals, which are conjugates of intellectin or a variant thereof and an antibody or antibody fragment, conjugates of antibodies or antibody fragments or peptide aptamers to the target substance, intelectin or a variant thereof, intelectin or Examples include a conjugate of the mutant and a cytokine receptor.
- the target substance include toxins, pathogenic substances, inflammatory cytokines, viruses, bacteria, prions and the like.
- plasma separated from a patient's blood by a membrane plasma separator or the like can be used.
- FIG. 2 is a schematic diagram for explaining the first embodiment of the target substance removing method.
- plasma 230 is preliminarily administered with a conjugate 230 of antibodies, antibody fragments, peptide aptamers, cytokine receptors, and the like against intelectin or a variant thereof and a target substance.
- a composite 240 is formed.
- the target substance removal method of this embodiment includes a step of bringing the above-described plasma 220 into contact with the above-described conjugate-capturing solid phase 250.
- the solid phase 250 is packed in a column 255.
- the solid phase 250 includes —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 . Embedded image is fixed.
- the inlectin of the conjugate 230 constituting the complex 240 or a mutant portion thereof binds to the compound 251.
- the complex 240 is adsorbed on the solid phase 250 and the target substance 210 is removed from the plasma 220.
- the target substance 210 is removed from the plasma 220 ′ discharged from the column 255.
- the target substance 210 in the plasma 220 can be specifically removed by the method of the present embodiment.
- the plasma 220 'from which the target substance 210 has been removed can be returned to the patient. Therefore, it can be said that the conjugate 230 is a target substance recovery tool for apheresis.
- the present invention provides a method for removing a target substance, comprising the step of bringing plasma containing the target substance into contact with the above-mentioned conjugate immobilized on a solid phase.
- the target substance removal method of the present embodiment is a kind of apheresis, which is a method of collecting a desired blood component from whole blood.
- FIG. 3 is a schematic diagram for explaining a second embodiment of the target substance removing method.
- the column 255 is packed with the solid phase 250 to which the conjugate 230 is bound.
- the solid phase 250 includes —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 . ] Is fixed, and the conjugate 230 is bound to the solid phase 250 by binding of the intelctin of the conjugate 230 or a mutant portion thereof to the compound 251.
- the conjugate 230 binds to the target substance 210.
- the target substance 210 is adsorbed on the solid phase 250, and the target substance 210 is removed from the plasma 220.
- the target substance 210 is removed from the plasma 220 ′ discharged from the column 255.
- the target substance 210 in the plasma 220 can be specifically removed by the method of the present embodiment.
- the plasma 220 'from which the target substance 210 has been removed can be returned to the patient. Therefore, also in this embodiment, it can be said that the conjugate 230 is a target substance recovery tool for apheresis.
- SIRS systemic inflammatory response syndrome
- the conjugate 230 if a conjugate that binds to a cytokine is used as the conjugate 230, the cytokine in the plasma can be removed. Thereby, it is considered possible to prevent multi-organ failure due to serious SIRS and to greatly improve the lifesaving rate.
- the method of the present embodiment can also be applied to rheumatism, cancer cachexia, psoriasis and the like, which are caused by increased cytokines in the body.
- the present invention relates to plasma containing the above conjugate, —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 , immobilized on a solid phase.
- a method for removing the conjugate comprising the step of contacting with a compound having the formula: According to the removal method of the present embodiment, for example, the above-mentioned conjugate administered into the blood of the patient can be removed from the patient's body.
- the above conjugate having a contrast agent as a functional molecule is administered to a patient and examined by computed tomography (CT), magnetic resonance imaging (MRI), etc., blood For example, removing the contrast agent in the medium. Thereby, the side effect by a contrast agent can be reduced.
- CT computed tomography
- MRI magnetic resonance imaging
- the present invention comprises a removing means for removing the complex from plasma containing a complex of a target substance and the above-mentioned conjugate, wherein the removing means comprises —OH and —OR 1 [R 1 is Represents a hydrogen atom, an alkyl group or —PO 3 H 2 .
- a target substance removing apparatus comprising a solid phase to which a compound having The compound is preferably a compound represented by the formula (1) or (2).
- FIG. 2 is a schematic diagram for explaining the target substance removing apparatus 300 of the present embodiment.
- the target substance removal apparatus 300 is an apparatus suitable for carrying out the first embodiment of the target substance removal method described above.
- the target substance removing apparatus 300 includes the above-described conjugate capturing solid phase (removing means) 250.
- the target substance removing apparatus 300 is further separated by a blood circuit 260 for circulating the patient's blood, a pump 270a for transferring the blood in the blood circuit 260, a plasma separating apparatus 280 for separating plasma from the blood, and a plasma separating apparatus 280.
- a plasma circuit 290 for circulating the plasma 220, a pump 270b for transferring the plasma in the plasma circuit 290, and the like may be provided.
- a patient 230 to which the target substance removing apparatus 300 is applied has been administered in advance with a conjugate 230 of an antibody against intelectin or a variant thereof and the target substance 210, an antibody fragment, a peptide aptamer, a cytokine receptor, etc.
- the target substance 210 forms a complex 240 and a complex 240.
- Blood collected from the patient circulates in the blood circuit 260 by the pump 270a. Eventually, the blood is separated into plasma 220 and blood cell components in plasma separator 280. The separated plasma 220 is circulated in the plasma circuit 290 by the pump 270b. Eventually, the plasma 220 comes into contact with the removal means 250.
- the removing means 250 is packed in the column 255.
- the removal means 250 includes —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 . Embedded image is fixed.
- the removing means 250 is brought into contact with the plasma 220, the inlectin of the conjugate 230 constituting the complex 240 or a mutant portion thereof is bound to the compound 251.
- the complex 240 is adsorbed by the removing means 250 and the target substance 210 is removed from the plasma 220.
- the target substance 210 is removed from the plasma 220 ′ discharged from the column 255.
- the plasma 220 ′ from which the target substance 210 has been removed is returned to the blood circuit 260.
- the target substance removal device of the present embodiment can specifically remove the target substance 210 in the plasma 220.
- the plasma 220 'from which the target substance 210 has been removed can be returned to the patient.
- the present invention comprises a removing means for removing the target substance from plasma containing the target substance, and the removing means comprises a solid phase on which the above-mentioned conjugate is immobilized.
- a removal device is provided.
- conjugate the same ones that can be used in the first embodiment of the target substance removing method described above can be used.
- FIG. 3 is a schematic diagram for explaining the target substance removing device 400 of the present embodiment.
- the target substance removal apparatus 400 is an apparatus suitable for carrying out the second embodiment of the target substance removal method described above.
- the target substance removing apparatus 400 includes the above-described conjugate (removing means) 230 coupled to the above-described conjugate-capturing solid phase 250.
- the target substance removing apparatus 400 is further separated by a blood circuit 260 for circulating the patient's blood, a pump 270a for transferring the blood in the blood circuit 260, a plasma separating apparatus 280 for separating plasma from the blood, and a plasma separating apparatus 280.
- a plasma circuit 290 for circulating the plasma 220, a pump 270b for transferring the plasma in the plasma circuit 290, and the like may be provided.
- Blood collected from the patient circulates in the blood circuit 260 by the pump 270a. Eventually, the blood is separated into plasma 220 and blood cell components in plasma separator 280. The separated plasma 220 is circulated in the plasma circuit 290 by the pump 270b. Eventually, the plasma 220 comes into contact with the removing means 230.
- the removing means 230 is packed in the column 255.
- the removing means 230 is —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group or —PO 3 H 2, which is fixed to the solid phase 250. ] Is bound to compound 251 having
- the conjugate 230 binds to the target substance 210.
- the target substance 210 is adsorbed on the solid phase 250, and the target substance 210 is removed from the plasma 220.
- the target substance 210 is removed from the plasma 220 ′ discharged from the column 255.
- the plasma 220 ′ from which the target substance 210 has been removed is returned to the blood circuit 260.
- the target substance removal device of the present embodiment can specifically remove the target substance 210 in the plasma 220.
- the plasma 220 'from which the target substance 210 has been removed can be returned to the patient.
- a conjugate 230 that binds to cytokines it can be used as a cytokine removing device that removes cytokines in plasma.
- the present invention comprises a removing means for removing the conjugate from plasma containing the conjugate, wherein the removing means comprises —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group or — Represents PO 3 H 2 .
- the conjugate removing apparatus is provided with a solid phase on which a compound having the formula: According to the removing device of the present embodiment, for example, the above-mentioned conjugate administered in the blood of the patient can be removed from the patient's body.
- the present invention is a labeled compound for detecting the above-mentioned conjugate, and —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- a labeled compound for detecting a conjugate comprising: From the viewpoint of high binding activity to inlectin or a mutant thereof, the conjugate detection labeling compound of the present embodiment includes a compound represented by the above formula (2), a radioisotope, a fluorescent substance, an enzyme, and a fluorescent protein. It is preferably a conjugate with a labeling substance such as
- More specific labeling substances include alkaline phosphatase, horseradish peroxidase, GFP, biotin, FITC, Cy2, Cy3, Cy5, rhodamine, Alexa Fluor dye, and the like.
- the labeled compound for detecting a conjugate of the present embodiment specifically binds to the above-described intelectin or a mutant portion thereof. For this reason, for example, a conjugate having an intectin or a mutant part thereof and an antibody, antibody fragment or peptide aptamer part against the target substance is used instead of the primary antibody, and the conjugate detection labeling compound of the present embodiment is used. By using it instead of the secondary antibody, the presence of the target substance recognized by the conjugate can be detected.
- FIG. 4 is a schematic diagram illustrating the conjugate-detecting labeled compound of the present embodiment.
- the conjugate detection labeling compound 400 is —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- a labeling substance 420 is horseradish peroxidase.
- conjugate detection labeling compound 400 can specifically bind to, for example, the intelectin or its mutant part of the fusion protein (conjugate) 430 of intelectin or its mutant and antibody, Can be used for detection.
- conjugate 430 an antibody is bound, but an aptamer, a receptor, or the like may be bound instead of the antibody.
- the present invention provides —OH and —OR 1 wherein R 1 represents a hydrogen atom, an alkyl group or —PO 3 H 2 in the above-described conjugate.
- the method comprises: a step of binding a labeling compound having a compound to form a complex; and a step of detecting the labeling compound in the complex.
- the labeling compound is preferably a compound represented by the above formula (2).
- a conjugate having an intelectin or a mutant portion thereof and an antibody, antibody fragment or peptide aptamer portion against a target substance is used instead of the primary antibody, and the conjugate detection described above
- a labeled compound can be used in place of the secondary antibody to detect the presence of the target substance recognized by the conjugate.
- the detection of the conjugate detection labeling compound can be performed by using a fluorescence microscope or the like when the labeling substance in the conjugate detection labeling compound is a fluorescent substance or a fluorescent protein. Further, when the labeling substance is an enzyme, it can be carried out by reacting an appropriate substrate and detecting color development, luminescence, fluorescence and the like.
- the present invention relates to —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group or —PO 3, representing the H 2. And a conjugate for detecting a compound.
- FIG. 5 is a schematic diagram illustrating the conjugate for detecting a compound of the present embodiment.
- the compound detection conjugate 500 is a conjugate of intelectin or a variant 510 thereof and a labeling substance 520.
- the labeling substance 520 the same substance as the labeling substance in the conjugate detection labeling compound described above can be used.
- the compound detection conjugate 500 is —OH and —OR 1
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 at the portion of intelectin or a variant 510 thereof.
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- the compound 531 labeled with the compound 531 is used as the primary antibody, and the compound detection conjugate 500 is used in place of the secondary antibody to detect the presence of the target substance recognized by the antibody 530. Can do.
- the labeling substance 520 is a fluorescent substance
- the compound 531 is polyethylene glycol having a diol structure introduced in the side chain.
- antibody 530 is shown, but aptamers, receptors, and the like can be used instead of antibodies.
- the present invention relates to —OH and —OR 1 [R 1 represents a hydrogen atom, an alkyl group or —PO 3 H 2 .
- the compound detection conjugate is bound to a compound having the above structure to form a complex, and the labeling substance in the complex is detected.
- the compound is preferably a compound represented by the above formula (2).
- the detection method of the present embodiment for example, by using the antibody 530 shown in FIG. 5 as the primary antibody and using the compound detection conjugate 500 instead of the secondary antibody, the target substance recognized by the antibody 530 can be detected. The presence can be detected.
- R 1 represents a hydrogen atom, an alkyl group, or —PO 3 H 2 .
- the detection of the labeling substance can be performed in the same manner as in the compound detection method described above.
- Example 1 (Binding of inlectin and diol group) Beads having a diol group on the surface and beads not having a diol group on the surface were prepared.
- beads having a diol group on the surface beads obtained by binding 3-amino-1-propanediol to the surface of polystyrene beads via amino groups (diol beads), and 1,4-bis (2,3-epoxy) (Propyl) butane-introduced Sepharose beads (epoxy-activated Sepharose 6B (GE Healthcare Bioscience)) were used that had been hydrolyzed with an alkali (diol Sepharose beads) and had no diol groups on the surface.
- polystyrene beads and beads obtained by binding 3-amino-1-propanol to polystyrene beads (hydroxy beads) were used.
- FIG. 6A is a photograph showing the results of SDS-PAGE
- FIG. 6B is a view showing the structure of each bead.
- inlectin selectively binds to diol beads and diol sepharose beads.
- Epoxy groups of Sepharose beads (epoxy-activated Sepharose 6B (GE Healthcare Bioscience)) into which 1,4-bis (2,3-epoxypropyl) butane has been introduced as diol Sepharose beads having diol groups on the surface. Similar results were obtained when experiments were carried out using acid hydrolyzed water.
- glycerol 1,2-propanediol, 1,3-propanediol, 1-propanol, ethylene glycol, ethanolamine, ethanol, 1 , 2-butanediol, 1,3-butanediol, 1,4-butanediol, D-2,3-butanediol, L-2,3-butanediol, meso-2,3-butanediol, 3-amino -1,2-propanediol, 3-amino-1-propanol, galactose, glucose, mannose and ribose.
- the concentration of inlectin measured using a sample to which the above compound was not added was set to 100%, and the concentration of inlectin measured using a medium containing no inlectin was set to 0%.
- the inhibition rate of the bond between diol groups was calculated.
- FIGS. 7A to 7C are graphs showing the experimental results. Inlectin showed a high affinity for compounds having a 1,2-diol group. It also showed a low affinity for compounds having a 2,3-diol structure.
- Inlectin was used at a final concentration of 0.5 ⁇ g / mL. Further, 10 mM HEPES (pH 7.0) containing 1 mM CaCl 2 , 150 mM NaCl, and 0.03% Tween 20 was used as a measurement buffer. The binding rate of inlectin in each sample was measured with the binding amount of inlectin measured using the sample to which the above compound was not added being 100%.
- FIG. 8 is a graph showing the experimental results. Inlectin showed a high affinity for compounds having a 1,2-diol group. It also showed a low affinity for compounds having a 2,3-diol structure. Moreover, it showed almost no affinity for compounds having no diol group.
- FIG. 9 is a graph showing the measurement results.
- the association rate constant K a 2.03 ⁇ 10 ⁇ 5 M ⁇ 1 s ⁇ 1
- the dissociation rate constant K d 1.79 ⁇ 10 ⁇ 3 s ⁇ 1
- the dissociation constant K D 8.84 ⁇ 10 -9 M was calculated.
- the precipitate was collected by centrifugation, washed once with a buffer solution, and dissolved with a sample buffer. Subsequently, the sample was separated by SDS-PAGE and then stained with Coomassie Brilliant Blue.
- FIG. 10A is a photograph showing the result of SDS-PAGE. As a result, it was revealed that inlectin was bound to 1-monopalmitin and 2-monopalmitin. It was also revealed that inlectin binds only slightly to 1-palmitoyl lysophosphatidic acid.
- FIG. 10B shows 1-monoacylglycerol represented by 1-monopalmitin, 2-monoacylglycerol represented by 2-monopalmitin, and lysophosphatidic acid represented by 1-palmitoyl lysophosphatidic acid. It is the general formula which shows a structure.
- R represents an alkyl group.
- the above expression plasmid and an expression plasmid incorporating a base sequence fragment encoding the L chain of monoclonal antibody MH166 were introduced into a rabbit kidney cell line RK-13 for co-expression.
- the nucleotide sequence encoding the mature protein of mutant recombinant human inlectin-1 (S165A) (including one glycine residue of the spacer) is shown in SEQ ID NO: 3.
- the base sequence encoding the Fab region of the H chain of MH166 is shown in SEQ ID NO: 4, and the base sequence encoding the L chain of MH166 is shown in SEQ ID NO: 5.
- the culture supernatant of the above cell line was collected and added to diol sepharose beads, the beads were washed, and 20 mM Tris buffer (pH 7. 5 containing 10% 1,2-propanediol and 0.05% Tween 20). Eluted in 4). The eluted fraction was added to a Resource Q column (1 mL, GE Healthcare Bioscience) and eluted with a NaCl concentration gradient of 0 to 1M.
- the culture supernatant of the above cell line was collected and added to diol sepharose beads, the beads were washed, and 20 mM Tris buffer (pH 7. 5 containing 10% 1,2-propanediol and 0.05% Tween 20). Eluted in 4). The eluted fraction was added to a Resource Q column (1 mL, GE Healthcare Bioscience) and eluted with a NaCl concentration gradient of 0 to 1M.
- FIG. 11A is a graph showing the elution profile of the conjugate by anion exchange chromatography.
- FIG. 11B is a photograph showing the results of SDS-PAGE. As a result, it was shown that the conjugate of TNFR2 and inlectin can be highly purified by diol sepharose beads and anion exchange chromatography.
- IL-6 protein is adsorbed on an ELISA plate, the conjugate prepared in Experimental Example 6 is reacted as a primary antibody, and a horseradish peroxidase-labeled anti-intellectin-1 monoclonal antibody (2D2) is used as a secondary antibody.
- 2D2 horseradish peroxidase-labeled anti-intellectin-1 monoclonal antibody
- FIG. 12 is a graph showing the experimental results. As a result, it was revealed that the conjugate prepared in Experimental Example 6 binds to IL-6 protein.
- FIGS. 13A and 13B are graphs showing the results of the first and second experiments, respectively. As a result, it was shown that the conjugate produced in Experimental Example 7 can adsorb and remove TNF- ⁇ in the solution.
- Example 10 (Binding of mouse intellectin-1 and human inlectin-1 monomer to diol group) Beads having a diol group on the surface and beads not having a diol group on the surface were prepared. As beads, the diol beads, diol sepharose beads and hydroxy beads used in Experimental Example 1 were used.
- Wild type mouse inlectin-1 and mutant type human inlectin-1 were used as intelctin.
- Wild type mouse inlectin-1 is produced as a monomer.
- Mouse inlectin-1 is different from human inlectin-1 in 51 amino acid residues at the amino acid level.
- wild type human inlectin-1 forms a trimer.
- mutant (C31, 48S) in which the 31st and 48th cysteine residues of human inlectin-1 are respectively substituted with serine residues does not form a trimer via a disulfide bond, Produced as.
- SEQ ID NO: 9 shows the amino acid sequence of the C31,48S (C31S, C48S) mutant of human inlectin-1.
- SEQ ID NO: 10 shows the base sequence of the C31,48S mutant of human inlectin-1.
- each bead was added to 5 mL of a medium containing recombinant mouse inlectin-1 or 1 mL of a medium containing C31,48S mutant of human inlectin-1 and stirred at 25 ° C. for 18 hours.
- FIG. 14A is a photograph showing the results of SDS-PAGE of mouse inlectin-1.
- FIG. 14B is a photograph showing the results of SDS-PAGE of the C31,48S mutant of human inlectin-1.
- TNF- ⁇ Recombinant human TNF- ⁇ (Life Technology) was used as TNF- ⁇ .
- TNF- ⁇ was used as an analyte, and the conjugate or etanercept prepared in Experimental Example 7 was immobilized on a sensor chip via an amino group and used as a ligand.
- As a buffer for affinity measurement 10 mM HEPES (pH 7.0) containing 1 mM CaCl 2 , 150 mM NaCl, and 0.03% Tween 20 was used.
- FIG. 15A is a graph showing the results of measuring the affinity between the conjugate prepared in Experimental Example 7 and TNF- ⁇ .
- FIG. 15B is a graph showing the results of measuring the affinity between etanercept and TNF- ⁇ .
- FIG. 16 is a graph showing experimental results. As a result, it was revealed that the conjugate prepared in Experimental Example 7 and etanercept had almost the same cytotoxicity inhibiting activity.
- FIG. 17 is a graph showing experimental results. As a result, all mice in the control group died within 12 hours. However, no mice in the test group were debilitated or died until 4 days, and the observation was terminated after 4 days.
- FIG. 18 is a graph showing the experimental results.
- the intelctin did not bind to the column, and the peak of intelectin was detected in the vicinity of 11 mL of the eluate (indicated by an arrow in the figure).
- the serum sample to which EDTA was not added inlectin was not detected in any fraction, and it was revealed that inlectin was adsorbed on the column.
- conjugates it is possible to provide a technique capable of easily purifying a biologically active protein such as an antibody at low cost.
- conjugates, vectors, pharmaceutical compositions, pharmaceuticals, conjugate purification methods, conjugate capture solid phases, target substance removal methods, target substance removal devices, conjugate detection label compounds, conjugate detection methods, compounds A detection conjugate and a method for detecting a compound can be provided.
- DESCRIPTION OF SYMBOLS 100 ... Pharmaceutical composition, 110 ... Drug, 120,251 ... Compound, 130 ... Carrier, 140,230,430 ... Conjugate (removal means), 141 ... Interlectin or its variant, 142 ... Antibody, 300,400 ... Target substance removing device 210 ... Target substance 220, 220 '... Plasma, 240 ... Complex, 250 ... Solid phase (removing means), 255 ... Column, 260 ... Blood circuit, 270a, 270b ... Pump, 280 ... Plasma separation Device, 290 ... Plasma circuit.
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Abstract
Description
(1)配列番号1に記載のアミノ酸配列からなるタンパク質、又は配列番号1に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり且つ-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物への結合活性を有するタンパク質と、機能性分子との結合体。
(2)前記機能性分子が標的物質捕捉分子である、(1)に記載の結合体。
(3)前記標的物質捕捉分子が、該標的物質に対する抗体、抗体断片、アプタマー又は受容体である、(2)に記載の結合体。
(4)前記標的物質捕捉分子が腫瘍壊死因子受容体(TNFR)である、(2)又は(3)に記載の結合体。
(5)前記標的物質がサイトカインである、(2)~(4)のいずれかに記載の結合体。
(6)配列番号1に記載のアミノ酸配列からなるタンパク質、又は配列番号1に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり且つ-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物への結合活性を有するタンパク質と、標的物質に対する抗体、抗体断片、ペプチドアプタマー又は受容体との融合タンパク質をコードする核酸を含むことを特徴とするベクター。
(7)薬物と、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物を含む担体と、前記化合物に結合した(1)~(5)のいずれかに記載の結合体と、を備えたことを特徴とする医薬組成物。
(8)(1)~(5)のいずれかに記載の結合体を有効成分として含有することを特徴とする医薬。
(9)(1)~(5)のいずれかに記載の結合体を、固相に固定された-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物に接触させる工程を有することを特徴とする結合体の精製方法。
(10)(1)~(5)のいずれかに記載の結合体を捕捉するための固相であって、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物が固定されたことを特徴とする結合体捕捉用固相。
(11)(1)~(5)のいずれかに記載の結合体を含む血漿を、固相に固定された-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物に接触させる工程を有することを特徴とする前記結合体の除去方法。
(12)標的物質と(2)~(5)のいずれかに記載の結合体との複合体を含む血漿を、固相に固定された-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物に接触させる工程を有することを特徴とする標的物質除去方法。
(13)標的物質を含む血漿を、固相に固定された(2)~(5)のいずれかに記載の結合体に接触させる工程を有することを特徴とする標的物質除去方法。
(14)(1)~(5)のいずれかに記載の結合体を含む血漿から前記結合体を除去する除去手段を備え、前記除去手段が、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物が固定された固相を備えることを特徴とする前記結合体の除去装置。
(15)標的物質と(2)~(5)のいずれかに記載の結合体との複合体を含む血漿から前記複合体を除去する除去手段を備え、前記除去手段が、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物が固定された固相を備えることを特徴とする標的物質除去装置。
(16)標的物質を含む血漿から前記標的物質を除去する除去手段を備え、前記除去手段が、(2)~(5)のいずれかに記載の結合体が固定された固相を備えることを特徴とする標的物質除去装置。
(17)(1)~(5)のいずれかに記載の結合体を検出するための標識化合物であって、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有することを特徴とする結合体検出用標識化合物。
(18)(1)~(5)のいずれかに記載の結合体に-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する標識化合物を結合させ複合体を形成させる工程と、該複合体中の標識化合物を検出する工程と、を有することを特徴とする結合体の検出方法。
(19)配列番号1に記載のアミノ酸配列からなるタンパク質、又は配列番号1に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり且つ-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物への結合活性を有するタンパク質と、標識物質との結合体であることを特徴とする、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物検出用結合体。
(20)-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物に(19)に記載の化合物検出用結合体を結合させ複合体を形成させる工程と、該複合体中の前記標識物質を検出する工程と、を有することを特徴とする、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物の検出方法。
発明者らは、レクチンの1種であるインテレクチン(ヒトインテレクチン-1のアミノ酸配列を配列番号1に示す。)が、後述するジオール構造に代表される特定構造を有する化合物に特異的に結合することを見出し、本発明を完成させた。
本実施形態の結合体における機能性分子は、所望の機能を有する分子であれば特に制限されず、治療用医薬品、診断用医薬品等であってもよい。上記の医薬品は、例えば、タンパク質等の高分子化合物であってもよく、低分子化合物であってもよい。より具体的な機能性分子としては、例えば、標的物質捕捉分子;抗がん剤、細胞傷害剤、治療用放射線同位体(例えば、P32、Y90、I131、I125、Sm153、Re186、Re188、At211、Bi212、Pb212、Lu等の放射性同位体)等を結合させた化合物を含有する医薬組成物;コンピュータ断層撮影法(CT)、磁気共鳴画像法(MRI)等に用いる造影剤;陽電子放射断層撮影法(PET)用プローブ等が挙げられる。後述するように、本実施形態の結合体によれば、例えば機能性分子を生体に投与した後、必要に応じて回収することができる。機能性分子である薬剤を回収することを可能にすることにより、副作用の軽減を図ることや、抗体等のタンパク質製剤等における自己抗体の産生のタイミングを遅延させる等の効果が期待できる。結合体は医療用途だけではなく研究ツール等の研究試薬分野に応用することもできる。その場合にも、機能性分子は試薬に利用されるタンパク質、核酸等の高分子化合物であってもよく、低分子化合物であってもよい。
本実施形態の結合体における標的物質捕捉分子としては、該標的物質に対する抗体、抗体断片、アプタマー、受容体等が挙げられる。
1実施形態において、本発明は、インテレクチン又はその変異体と、標的物質に対する抗体、抗体断片、ペプチドアプタマー又は受容体との融合タンパク質をコードする核酸を含むことを特徴とするベクターを提供する。インテレクチン又はその変異体をコードする核酸としては、例えば、配列番号2に記載の塩基配列からなる核酸、又は、配列番号2に記載の塩基配列と80%以上、例えば85%以上、例えば90%以上、例えば95%以上の同一性を有し且つ-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物への結合活性を有するタンパク質をコードする塩基配列からなる核酸が挙げられる。なお、配列番号2に示す塩基配列は、ヒトインテレクチン-1の塩基配列である。
1実施形態において、本発明は、薬物と、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物を含む担体と、前記化合物に結合した、上述した結合体と、を備えたことを特徴とする医薬組成物を提供する。本実施形態の医薬組成物は、ドラッグデリバリーシステムであってもよい。上記化合物は、上記式(1)又は(2)で示される化合物であることが好ましい。
本実施形態の医薬組成物は、上述した癌細胞を標的として抗癌剤を特異的に送達するだけでなく、例えば、細菌やウイルス感染細胞を標的として抗菌剤や抗ウイルス剤を特異的に送達する;目的に応じた細胞を標的として、酵素やsiRNA、ペプチドアプタマー、核酸アプタマー、核酸(遺伝子)等の代謝調節物質を特異的に送達する;目的に応じた細胞を標的として、放射性同位体、蛍光色素、蛍光タンパク質等の標識物質を特異的に送達する等に利用することもできる。
1実施形態において、本発明は、上述した結合体を有効成分として含有する、医薬を提供する。
1実施形態において、本発明は、上述した結合体を発現する遺伝子組換え体を提供する。遺伝子組換え体としては、上述した結合体の発現ベクターが導入された、細菌、酵母、昆虫細胞、動物細胞等の培養細胞;上述した結合体の発現ベクターが導入された、カイコ等の昆虫生体;例えば乳中、卵中に上述した結合体を発現するように遺伝子改変された、ヤギ、ヒツジ、ウシ、ニワトリ等の動物等が挙げられる。
1実施形態において、本発明は、上述した結合体を、固相に固定された-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物に接触させる工程を有することを特徴とする結合体の精製方法を提供する。上記化合物は、上記式(1)又は(2)で示される化合物であることが好ましい。上記化合物が固定された固相(結合体捕捉用固相)については後述する。
1実施形態において、本発明は、上述した結合体を捕捉するための固相であって、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物が固定されたことを特徴とする結合体捕捉用固相を提供する。上記化合物は、上記式(1)又は(2)で示される化合物であることが好ましい。固相としては、ビーズ、膜、中空糸膜、プラスチック基板、ウェルプレート、スライドガラス、表面プラズモン共鳴測定装置等の測定機器のセンサーチップ等が挙げられる。例えば、ビーズ、膜等の形態である固相は、カラムに充填されていてもよい。
(第1実施形態)
1実施形態において、本発明は、標的物質と上述した結合体との複合体を含む血漿を、固相に固定された-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物に接触させる工程を有することを特徴とする標的物質除去方法を提供する。上記化合物は、上記式(1)又は(2)で示される化合物であることが好ましい。本実施形態の標的物質除去方法は、血漿から標的物質を除去する方法、血漿から結合体を除去する方法ということもできる。また、本実施形態の標的物質除去方法は、全血から所望の血液成分を採取する方法である、アフェレーシスの一種であるともいえる。
1実施形態において、本発明は、標的物質を含む血漿を、固相に固定された、上述した結合体に接触させる工程を有することを特徴とする標的物質除去方法を提供する。本実施形態の標的物質除去方法は、全血から所望の血液成分を採取する方法である、アフェレーシスの一種であるともいえる。
1実施形態において、本発明は、上記の結合体を含む血漿を、固相に固定された-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物に接触させる工程を有することを特徴とする前記結合体の除去方法を提供する。本実施形態の除去方法によれば、例えば患者の血液中に投与された上記の結合体を、患者の体内から除去することができる。
(第1実施形態)
1実施形態において、本発明は、標的物質と上述した結合体との複合体を含む血漿から前記複合体を除去する除去手段を備え、前記除去手段が、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物が固定された固相を備えることを特徴とする標的物質除去装置を提供する。上記化合物は、上記式(1)又は(2)で示される化合物であることが好ましい。
1実施形態において、本発明は、標的物質を含む血漿から前記標的物質を除去する除去手段を備え、前記除去手段が、上述した結合体が固定された固相を備えることを特徴とする標的物質除去装置を提供する。
1実施形態において、本発明は、上記の結合体を含む血漿から前記結合体を除去する除去手段を備え、前記除去手段が、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物が固定された固相を備えることを特徴とする前記結合体の除去装置を提供する。本実施形態の除去装置によれば、例えば患者の血液中に投与された上記の結合体を、患者の体内から除去することができる。
1実施形態において、本発明は、上述した結合体を検出するための標識化合物であって、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有することを特徴とする結合体検出用標識化合物を提供する。インテレクチン又はその変異体との結合活性が高い観点から、本実施形態の結合体検出用標識化合物は、上記式(2)で表される化合物と、放射性同位体、蛍光物質、酵素、蛍光タンパク質等の標識物質との結合体であることが好ましい。
1実施形態において、本発明は、上述した結合体に-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する標識化合物を結合させ複合体を形成させる工程と、該複合体中の標識化合物を検出する工程と、を有することを特徴とする結合体の検出方法を提供する。上記の標識化合物は、上記式(2)で表される化合物であることが好ましい。
1実施形態において、本発明は、インテレクチン又はその変異体と、標識物質との結合体であることを特徴とする、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物検出用結合体を提供する。
1実施形態において、本発明は、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物に上述した化合物検出用結合体を結合させ複合体を形成させる工程と、該複合体中の前記標識物質を検出する工程と、を有することを特徴とする、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物の検出方法を提供する。上記の化合物は、上記式(2)で表される化合物であることが好ましい。
(インテレクチンとジオール基との結合)
表面にジオール基を有するビーズ及び表面にジオール基を有しないビーズを準備した。表面にジオール基を有するビーズとしては、ポリスチレンビーズの表面に3-アミノ-1-プロパンジオールをアミノ基を介して結合させたビーズ(ジオールビーズ)、及び1,4-ビス(2,3-エポキシプロピル)ブタンが導入されたセファロースビーズ(epoxy-activated Sepharose 6B(GEヘルスケアバイオサイエンス社)のエポキシ基をアルカリ加水分解したもの(ジオールセファロースビーズ)を使用した。また、表面にジオール基を有しないビーズとしては、ポリスチレンビーズ、及びポリスチレンビーズに3-アミノ-1-プロパノールを結合させたビーズ(ヒドロキシビーズ)を使用した。
(各種化合物によるインテレクチンとジオール基との結合の阻害1)
インテレクチンを含む培地35μLに7.5μLのジオールセファロースビーズ及び段階希釈された様々な種類の化合物を含む緩衝液(1mM CaCl2、150mM NaCl、0.1%Tween20を含む20mM Tris緩衝液(pH7.0))65μLを加え、4℃で36時間撹拌した。インテレクチンとしては、リコンビナントヒトインテレクチン-1を使用した。また、上記化合物はジオール基を含む又は含まない化合物であり、具体的には、グリセロール、1,2-プロパンジオール、1,3-プロパンジオール、1-プロパノール、エチレングリコール、エタノールアミン、エタノール、1,2-ブタンジオール、1,3-ブタンジオール、1,4-ブタンジオール、D-2,3-ブタンジオール、L-2,3-ブタンジオール、メソ-2,3-ブタンジオール、3-アミノ-1,2-プロパンジオール、3-アミノ-1-プロパノール、ガラクトース、グルコース、マンノース、リボースであった。
(各種化合物によるインテレクチンとジオール基との結合の阻害2)
ビアコア(GEヘルスケアバイオサイエンス社)を用いて、段階希釈された様々な種類の化合物の存在下で、3-アミノ-1-プロパンジオールをアミノ基を介して固定したセンサーチップに対する精製インテレクチンの結合を測定した。インテレクチンとしては、リコンビナントヒトインテレクチン-1を使用した。また、上記化合物はジオール基を含む又は含まない化合物であり、具体的には、グリセロール、1,2-プロパンジオール、1,3-プロパンジオール、1-プロパノール、エチレングリコール、エタノール、2-メトキシエタノール、ガラクトース、2-acetamido-2-deoxy-4-O-beta-D-galactopyranosyl-D-glucopyranose(Galp-GlcNac)、2-acetamido-2-deoxy-4-O-beta-D-galactofuranosyl-D-glucopyranose(Galf-GlcNac)、D-リボース、L-リボースであった。
(インテレクチンとジオール基との解離定数の測定)
ビアコア(GEヘルスケアバイオサイエンス社)を用いて、3-アミノ-1-プロパンジオールをアミノ基を介して固定したセンサーチップに対する精製インテレクチンの結合を測定し、解離定数を求めた。
(モノアシルグリセロールとインテレクチンの結合)
モノパルミチンやモノオレインなどのモノアシルグリセロールはミセルやリポソームの構成成分として使用される。インテレクチンを含む培地500μLに、終濃度100μMの1-モノパルミチン、2-モノパルミチン又は陰性対象としての1-パルミトイルリゾフォスファチジン酸を加え、激しく撹拌し、37℃、15分保温した。インテレクチンとしては、リコンビナントヒトインテレクチン-1を使用した。上記の操作により、外側にジオール基を向け、内側にアシル基を向けたミセル様の構造を持つ不溶性凝集物が形成され、表面上にインテレクチンが結合すると予想される。
(抗体Fab断片とインテレクチンとの結合体の作製)
165SerをAlaに置換し、163AsnのN型糖鎖付加を阻害した変異型リコンビナントヒトインテレクチン-1(S165A)の成熟型のN末端に、スペーサーとしてグリシン残基1残基を介して抗インターロイキン(IL)-6モノクローナル抗体MH166のH鎖のFab領域(VH-CH1領域)を接続したタンパク質をコードする塩基配列断片を作製し、発現プラスミドに組み込んだ。
(TNFR2とインテレクチンとの結合体の作製)
上述した変異型リコンビナントヒトインテレクチン-1(S165A)の成熟型のN末端に、スペーサーとしてのグリシン残基1残基を介してヒトTNF受容体2(TNFR2)の細胞外領域を接続したタンパク質をコードする塩基配列断片を作製し、発現プラスミドに組み込んだ。使用したヒトTNFR2の細胞外領域をコードする塩基配列を配列番号6に示す。上記の発現プラスミドをウサギ腎細胞株RK-13に遺伝子導入し、発現させた。
(抗体Fab断片とインテレクチンとの結合体の反応性の検討)
実験例6で作製した結合体を用いてIL-6タンパク質のELISA解析を行った。
(TNFR2とインテレクチンとの結合体の反応性の検討)
実験例7で作製した結合体を用いて、溶液中のTNF-αの吸着除去試験を行った。ヒトTNF-αを含む培地50μLに、実験例7で作製した結合体を段階希釈して加え、室温で30分反応させた後、ジオールセファロースビーズ3μLを用いてTNF-α及び結合体の複合体を吸収した。続いて、上清中のTNF-αをELISA法にて測定し、残存するTNF-αの存在割合を求めた。実験は独立して2回行った。
(マウスインテレクチン-1及びヒトインテレクチン-1単量体とジオール基との結合)
表面にジオール基を有するビーズ及び表面にジオール基を有しないビーズを準備した。ビーズとしては、実験例1で使用したジオールビーズ、ジオールセファロースビーズ及びヒドロキシビーズを使用した。
図14Aは、マウスインテレクチン-1のSDS-PAGEの結果を示す写真である。図14Bはヒトインテレクチン-1のC31,48S変異体のSDS-PAGEの結果を示す写真である。
(TNFR2とインテレクチンとの結合体とTNF-αとの親和性の測定)
ビアコア(GEヘルスケアバイオサイエンス社)を用いて、実験例7で作製した結合体とTNF-αとの親和性を測定した。対照として、市販の抗TNF製剤(エタネルセプト、可溶性TNF受容体、商品名「エンブレル(登録商標)」、武田薬品工業とTNF-αとの親和性も測定した。
(TNFR2とインテレクチンとの結合体の、TNF-αによる細胞傷害に対する抑制作用の検討)
マウス由来の細胞株であるL929細胞株の培地に終濃度2.12μMのアクチノマイシンD、終濃度1ng/mLのリコンビナントヒトTNF-α(ライフテクノロジー社)、及び実験例7で作製した結合体を段階希釈したものを添加し、37℃で24時間培養した。続いて、市販のキット(型式「CellTiter96(商標)」、プロメガ社)を用いて生細胞数を計測し、細胞生存率を求めた。対照として、実験例7で作製した結合体の代わりに市販の抗TNF製剤(エタネルセプト、可溶性TNF受容体、商品名「エンブレル(登録商標)」、武田薬品工業)を使用した実験も行った。
(TNFR2とインテレクチンとの結合体の、マウスにおけるリコンビナントヒトTNF-α誘発致死に対する抑制作用の検討)
7週齢の雌性C3H/HeNマウス(一群10匹)に実験例7で作製した結合体20μgを含むリン酸緩衝液(PBS)150μLを尾静脈注射により投与し、試験群とした。対照群のマウスには、PBSのみを150μL尾静脈注射により投与した。
(ヒト血清中インテレクチンのジオール修飾シリカゲルへの吸着)
10mM EDTAを添加した又は添加していない、0.1%Tween20-150mM NaCl-50mMリン酸緩衝液(pH7.3)で希釈した、50%新鮮ヒト血清40μLを、ジオール修飾シリカゲルを坦体とするゲル濾過クロマトグラフィカラム(TSKgel G3000 SWXL 東ソー株式会社)に添加し、0.1%Tween20-150mM NaCl-50mMリン酸緩衝液(pH7.3)を溶媒としてゲル濾過を行った。続いて、溶出画分のインテレクチン濃度をELISA法により測定した。
Claims (20)
- 配列番号1に記載のアミノ酸配列からなるタンパク質、又は
配列番号1に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり且つ-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物への結合活性を有するタンパク質と、
機能性分子との結合体。 - 前記機能性分子が標的物質捕捉分子である、請求項1に記載の結合体。
- 前記標的物質捕捉分子が、該標的物質に対する抗体、抗体断片、アプタマー又は受容体である、請求項2に記載の結合体。
- 前記標的物質捕捉分子が腫瘍壊死因子受容体(TNFR)である、請求項2又は3に記載の結合体。
- 前記標的物質がサイトカインである、請求項2~4のいずれか一項に記載の結合体。
- 配列番号1に記載のアミノ酸配列からなるタンパク質、又は
配列番号1に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり且つ-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物への結合活性を有するタンパク質と、
標的物質に対する抗体、抗体断片、ペプチドアプタマー又は受容体との融合タンパク質をコードする核酸を含むことを特徴とするベクター。 - 薬物と、
-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物を含む担体と、
前記化合物に結合した請求項1~5のいずれか一項に記載の結合体と、
を備えたことを特徴とする医薬組成物。 - 請求項1~5のいずれか一項に記載の結合体を有効成分として含有することを特徴とする医薬。
- 請求項1~5のいずれか一項に記載の結合体を、固相に固定された-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物に接触させる工程を有することを特徴とする結合体の精製方法。
- 請求項1~5のいずれか一項に記載の結合体を捕捉するための固相であって、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物が固定されたことを特徴とする結合体捕捉用固相。
- 請求項1~5のいずれか一項に記載の結合体を含む血漿を、固相に固定された-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物に接触させる工程を有することを特徴とする前記結合体の除去方法。
- 標的物質と請求項2~5のいずれか一項に記載の結合体との複合体を含む血漿を、固相に固定された-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物に接触させる工程を有することを特徴とする標的物質除去方法。
- 標的物質を含む血漿を、固相に固定された請求項2~5のいずれか一項に記載の結合体に接触させる工程を有することを特徴とする標的物質除去方法。
- 請求項1~5のいずれか一項に記載の結合体を含む血漿から前記結合体を除去する除去手段を備え、
前記除去手段が、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物が固定された固相を備えることを特徴とする前記結合体の除去装置。 - 標的物質と請求項2~5のいずれか一項に記載の結合体との複合体を含む血漿から前記複合体を除去する除去手段を備え、
前記除去手段が、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物が固定された固相を備えることを特徴とする標的物質除去装置。 - 標的物質を含む血漿から前記標的物質を除去する除去手段を備え、
前記除去手段が、請求項2~5のいずれか一項に記載の結合体が固定された固相を備えることを特徴とする標的物質除去装置。 - 請求項1~5のいずれか一項に記載の結合体を検出するための標識化合物であって、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有することを特徴とする結合体検出用標識化合物。
- 請求項1~5のいずれか一項に記載の結合体に-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する標識化合物を結合させ複合体を形成させる工程と、
該複合体中の標識化合物を検出する工程と、
を有することを特徴とする結合体の検出方法。 - 配列番号1に記載のアミノ酸配列からなるタンパク質、又は
配列番号1に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり且つ-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物への結合活性を有するタンパク質と、
標識物質との結合体であることを特徴とする、
-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物検出用結合体。 - -OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物に請求項19に記載の化合物検出用結合体を結合させ複合体を形成させる工程と、
該複合体中の前記標識物質を検出する工程と、
を有することを特徴とする、-OH及び-OR1[R1は水素原子、アルキル基又は-PO3H2を表す。]を有する化合物の検出方法。
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WO2017141604A1 (ja) * | 2016-02-15 | 2017-08-24 | 地方独立行政法人神奈川県立病院機構 | 膜型ムチン様タンパク質の認識とその医療応用 |
WO2019202994A1 (ja) * | 2018-04-16 | 2019-10-24 | Agc株式会社 | サイズ排除クロマトグラフィ担体、その製造方法、および微小物質の回収方法 |
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WO2019202994A1 (ja) * | 2018-04-16 | 2019-10-24 | Agc株式会社 | サイズ排除クロマトグラフィ担体、その製造方法、および微小物質の回収方法 |
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