WO2020189766A1 - Liposome modifié par un polypeptide ayant une capacité de liaison à un anticorps, et immunoliposome - Google Patents

Liposome modifié par un polypeptide ayant une capacité de liaison à un anticorps, et immunoliposome Download PDF

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WO2020189766A1
WO2020189766A1 PCT/JP2020/012364 JP2020012364W WO2020189766A1 WO 2020189766 A1 WO2020189766 A1 WO 2020189766A1 JP 2020012364 W JP2020012364 W JP 2020012364W WO 2020189766 A1 WO2020189766 A1 WO 2020189766A1
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amino acid
acid sequence
residues
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residue
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PCT/JP2020/012364
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英司 真島
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プロテノバ株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier

Definitions

  • the present invention relates to a polypeptide-modified liposome having an excellent antibody-binding ability. More specifically, the present invention relates to a polypeptide-modified liposome in which a peptide containing a variant of each immunoglobulin binding domain constituting protein A is bound to the surface and has excellent antibody binding ability. The present invention also relates to immunoliposomes in which an antibody or a fragment thereof is bound to the polypeptide-modified liposome.
  • Liposomes can be expected to control the pharmacokinetics of drugs, improve pharmacological effects, improve drug stability, reduce side effects, etc., in addition to being able to encapsulate drugs, and are widely used as carriers for drug delivery systems (DDS). ing.
  • DDS drug delivery systems
  • liposomes having directivity toward molecular targets are attracting attention. If the liposome can be provided with a directivity toward a molecular target, for example, the liposome encapsulating an anticancer agent can be selectively delivered to cancer cells, and selective delivery of a drug, improvement of pharmacological effect, reduction of side effects, etc. This is because it is expected to make a great contribution to.
  • Non-Patent Document 1 immunoliposomes in which polyethylene glycol is used as a linker and bound to a constituent lipid of a liposome as a drug delivery carrier having both reticuloendothelial avoidance ability and active targeting ability are described in Non-Patent Document 1. It is disclosed that it is valid.
  • Patent Document 1 states that "a liposome containing phosphatidylserine as a membrane component, a ligand having an IgG-binding ability to modify the outer surface of the liposome, and both the membrane component of the liposome and the ligand.
  • a liposome composition characterized by having a linker composed of a binding fatty acid can selectively recognize lesion cells or immune cells, and can treat inflammatory autoimmune diseases for a long time and efficiently. It is disclosed that it can be done.
  • Patent Document 2 discloses that "a cationic immunoliposome complex containing a cationic liposome, an antibody or a fragment thereof, and a contrast agent" is useful for the treatment and imaging of diseases including cancerous tumors. ing.
  • An object of the present invention is to provide a polypeptide-modified liposome having an excellent antibody-binding ability and an immunoliposome using the polypeptide-modified liposome.
  • a polypeptide-modified liposome having excellent antibody-binding ability can be obtained by subjecting a polypeptide having the modified immunoglobulin-binding domain as a constituent lipid to the surface of a liposome containing an anionic lipid. I found. Furthermore, the present inventor has found that immunoliposomes having a large amount of antibody binding can be easily produced by using the polypeptide-modified liposome. The present invention has been completed by further studies based on these findings.
  • the present invention provides the inventions of the following aspects.
  • Item 1 On the surface of the liposome containing an anionic lipid as a constituent lipid, the following (A1) to (A3), (B1) to (B3), (C1) to (C3), (D1) to (D3), and (E1) )-(E3), a polypeptide-modified liposome to which a polypeptide containing at least one immunoglobulin binding domain selected from the group consisting of (E3) is bound.
  • (A1) In the amino acid sequence shown in SEQ ID NO: 1, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues. An immunoglobulin binding domain having an amino acid sequence that has been identified.
  • (B1) In the amino acid sequence shown in SEQ ID NO: 2, at least 3 amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues. An immunoglobulin binding domain having an amino acid sequence that has been identified.
  • (C1) In the amino acid sequence shown in SEQ ID NO: 3, at least 3 amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues. An immunoglobulin binding domain having an amino acid sequence that has been identified.
  • (D1) In the amino acid sequence shown in SEQ ID NO: 4, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues. An immunoglobulin binding domain having an amino acid sequence that has been identified.
  • (E1) In the amino acid sequence shown in SEQ ID NO: 5, at least 3 amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues. An immunoglobulin binding domain having an amino acid sequence that has been identified.
  • (A2) In the amino acid sequence shown in SEQ ID NO: 1, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain of (A1) having the same substitution with the introduced lysine residue and / or arginine residue.
  • B2 In the amino acid sequence shown in SEQ ID NO: 2, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain (B1) having the same substitution with the introduced lysine residue and / or arginine residue.
  • C2 In the amino acid sequence shown in SEQ ID NO: 3, at least three amino acid residues in positions 40, 43, 46, 47, 53, and 54 are replaced with lysine residues and / or arginine residues.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain of (C1) in which the substitution with the introduced lysine residue and / or arginine residue is the same.
  • D2 In the amino acid sequence shown in SEQ ID NO: 4, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain of (D1) having the same substitution with the introduced lysine residue and / or arginine residue.
  • E2 In the amino acid sequence shown in SEQ ID NO: 5, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain of (E1) in which the substitution with the introduced lysine residue and / or arginine residue is the same.
  • A3 In the amino acid sequence shown in SEQ ID NO: 1, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • the sequence identity of the region excluding the substituted lysine residue and / or arginine residue is 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 1.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain of (A1) having the same substitution with the introduced lysine residue and / or arginine residue.
  • B3 In the amino acid sequence shown in SEQ ID NO: 2, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • the sequence identity of the region excluding the substituted lysine residue and / or arginine residue is 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 2.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain (B1) having the same substitution with the introduced lysine residue and / or arginine residue.
  • C3 In the amino acid sequence shown in SEQ ID NO: 3, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • the sequence identity of the region excluding the substituted lysine residue and / or arginine residue is 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 3.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain of (C1) in which the substitution with the introduced lysine residue and / or arginine residue is the same.
  • D3 In the amino acid sequence shown in SEQ ID NO: 4, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • the sequence identity of the region excluding the substituted lysine residue and / or arginine residue is 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 4.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain of (D1) having the same substitution with the introduced lysine residue and / or arginine residue.
  • E3 In the amino acid sequence shown in SEQ ID NO: 5, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • the sequence identity of the region excluding the substituted lysine residue and / or arginine residue is 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 5.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain of (E1) in which the substitution with the introduced lysine residue and / or arginine residue is the same.
  • the polypeptide has the 43 and 46 positions in the amino acid sequence shown in any of SEQ ID NOs: 1 to 5 substituted with a lysine residue and / or an arginine residue, and the 40, 47, 53, and 54 positions.
  • polypeptide-modified liposome according to Item 1 wherein at least one of the positions is replaced with a lysine residue and / or an arginine residue.
  • Item 3 In the amino acid sequence shown in any of SEQ ID NOs: 1 to 5, the polypeptide is replaced with a lysine residue and / or an arginine residue at positions 43, 46, and 53, and is among positions 40 and 54.
  • Item 2. The polypeptide-modified liposome according to Item 1 or 2, wherein one or two are replaced with a lysine residue and / or an arginine residue.
  • At least one of the 42, 49, 50, and 56 positions in the amino acid sequence shown in any of SEQ ID NOs: 1 to 5 is replaced with a lysine residue and / or an arginine residue.
  • Item 3. The polypeptide-modified liposome according to any one of Items 1 to 3.
  • Item 5. The polypeptide is selected from the group consisting of (A1) to (A3), (B1) to (B3), (C1) to (C3), (D1) to (D3), and (E1) to (E3).
  • the polypeptide is selected from the group consisting of (A1) to (A3), (B1) to (B3), (C1) to (C3), (D1) to (D3), and (E1) to (E3).
  • Item 8. The polypeptide-modified liposome according to any one of Items 1 to 5, which is a multidomain type polypeptide having at least one immunoglobulin binding domain.
  • Item 7. Item 6.
  • Item 8. Item 3. The polypeptide-modified lipid described in C.
  • Item 8. The polypeptide-modified liposome according to any one of Items 1 to 9, wherein the molar ratio of the anionic lipid: the neutral lipid is 1: 99 to 99: 1.
  • Item 11. An immunoliposome in which an antibody is bound to the polypeptide-modified liposome according to any one of Items 1 to 10.
  • a pharmaceutical composition comprising the immunoliposomes according to Item 11.
  • the polypeptide-modified liposome of the present invention can bind the antibody to the surface by a simple method of mixing with the antibody, it becomes possible to prepare an immunoliposomes in which the liposome has a directivity toward a molecular target. ..
  • immunoliposomes of the present invention can be used as active targeting liposomes, and are useful as drugs, DDS preparations for in-vivo diagnosis, and the like.
  • glycine G
  • alanine A
  • valine Val
  • Leu L
  • isoleucine I
  • Phe phenylalanine
  • Tyr tyrosine
  • Trp Tryptophan
  • Serine Serine
  • Seronine Thr
  • Cysteine Cysteine
  • Methionine Met
  • Glutamine Glu
  • Aspartic acid Aspartic acid (Asn) is N
  • glutamine Gln
  • lysine Lys
  • arginine Arg
  • Histidine Histidine
  • Pro proline
  • the left side indicates the N-terminal and the right side indicates the C-terminal.
  • non-polar amino acids include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan.
  • uncharged amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • acidic amino acids include aspartic acid and glutamic acid.
  • basic amino acids include lysine, arginine, and histidine.
  • polypeptide-modified liposome of the present invention is characterized in that a polypeptide containing an immunoglobulin-binding domain having a specific amino acid sequence is bound to the surface of a liposome containing an anionic lipid as a constituent lipid.
  • polypeptide-modified liposome of the present invention will be described in detail.
  • a liposome in the present invention, as a liposome to which a polypeptide containing an immunoglobulin binding domain is bound, a liposome containing an anionic lipid as a constituent lipid is used.
  • the type of anionic lipid used as a constituent lipid is not particularly limited, and is, for example, dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol (DOPG), dimyristylphosphatidylglycerol (DMPG), and disteroylphosphatidylglycerol.
  • Phosphatidylglycerol (PG) such as glycerol (DSPG); phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylate (PA), tocopherol succinic acid (TS), cholesterol succinic acid (CS), disetylphosphate and the like. ..
  • anionic lipids may be used alone or in combination of two or more.
  • anionic lipids phosphatidylglycerol is preferable, and dipalmitoylphosphatidylglycerol is more preferable, from the viewpoint of further improving the binding efficiency of the polypeptide.
  • the ratio of the anionic lipid to the total amount of the constituent lipids of the liposome is not particularly limited, and examples thereof include 1 to 99 mol%, preferably 10 to 90 mol%, and more preferably 30 to 75 mol%.
  • the liposome used in the present invention preferably contains a neutral lipid in addition to the anionic lipid as a constituent lipid.
  • the type of neutral lipid is not particularly limited, but for example, egg yosphatidylcholine (EPC), soyphosphatidylcholine, dimyristylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), hydrogenated DSPC, etc.
  • Phosphatidylcholine PC
  • sterols such as cholesterol, cholesteryl hemiscusinate, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, phytosterol, phytosterol, stigmasterol, thymosterol, ergosterol, citosterol, campesterol, brush casterol;
  • Digalactosyl phosphatidylglycerides galactosyl diglycerides, glycosyl diglycerides and other glycero lipids and lipids
  • galactosyl phosphatidylside and ganglioside and other sphingo glycoliphatids examples thereof include phosphatidylethanolamine such as amine (PEG-PE); phosphatidylethanolamine having a polyethylene glycol (PEG) chain, ceramide, sphingomierin, cephalin, sterol, and celebroside.
  • neutral lipids may be used alone or in combination of two or more.
  • phosphatidylcholine is preferable, and egg yolk phosphatidylcholine is more preferable, from the viewpoint of further improving the binding efficiency of the polypeptide.
  • the ratio of the anionic lipid to the neutral lipid in the constituent lipid of the liposome used in the present invention is not particularly limited, and for example, the molar ratio of anionic lipid: neutral lipid is 1: 99 to 99: 1. , Preferably from 10:90 to 90:10, and more preferably from 30:70 to 75:25. By satisfying such a ratio, the liposome can be sufficiently negatively charged to bind the polypeptide containing the immunoglobulin binding domain.
  • the ratio of the neutral lipid to the total amount of the constituent lipids of the liposome is not particularly limited, and examples thereof include 1 to 99 mol%, preferably 10 to 90 mol%, and more preferably 25 to 70 mol%.
  • the polydispersity index (PDI) of liposomes used in the present invention is not particularly limited, but is, for example, 0.5 or less, preferably 0.01 to 0.3, and more preferably 0.01. ⁇ 0.1 can be mentioned.
  • the liposomal polydispersity index is a value measured by a dynamic light scattering method.
  • examples of the zeta potential of the liposome used in the present invention include -50 to -1 mV, preferably -50 to -10 mV, and more preferably -50 to -25 mV.
  • the zeta potential of the liposome is a value measured by laser Doppler electrophoresis in a phosphate buffer solution (PBS (-)) using Zeta-sizer nanoZs (Malvern Panasonic). is there.
  • the particle size of the liposome used in the present invention is not particularly limited, but usually, the average particle size is 30 to 1000 nm, preferably 50 to 500 nm.
  • the average particle size of liposomes is a median diameter measured by a dynamic light scattering method using Zeta-sizer nanoZs (Malvern Panalytical).
  • the structure of the liposome used in the present invention is also not particularly limited, and may be any of MLV (multilamellar vesicles), DRV (dehydration-rehydration vesicles), LUV (large unilamellar vesicles), or SUV (small unilamellar vesicles). There may be.
  • MLV multilamellar vesicles
  • DRV dehydration-rehydration vesicles
  • LUV large unilamellar vesicles
  • SUV small unilamellar vesicles
  • the solution contained in the liposome used in the present invention may be a pharmaceutically acceptable aqueous carrier such as water, a buffer solution, or a physiological saline solution.
  • the liposome used in the present invention can contain a drug.
  • the drug contained in the liposome may be any of a nucleic acid drug (antisense oligo DNA, decoy oligo DNA, siRNA, miRNA, etc.), peptide, protein, glycoprotein, polysaccharide, low molecular weight organic compound, inorganic compound and the like. ..
  • drugs contained in the liposomes include anticancer agents, immunosuppressants, immunomodulators, hormone agents, anti-inflammatory agents, steroid agents, antihypertensive agents, antihypertensive agents, antiarrhythmic agents, antipsychotic agents, Painkillers, antipyretics, antiallergic agents, antihistamines, anti-inflammatory agents, antidepressants, sedatives, hypnotics, asthma treatments, anesthetics, antibiotics, antiarrhythmic agents, bronchial dilators, diuretics, antidiuretics , Muscle relaxants, antihyperlipidemic agents, antiemetic agents, anti-infectious agents, parasympathomimetic agents, antibacterial agents, antifungal agents, antiviral agents and other therapeutic agents; X-ray contrast agents, ultrasonic diagnostic agents, Examples thereof include in-vivo diagnostic agents such as diagnostic agents for nuclear magnetic resonance diagnosis.
  • the amount of the drug to be encapsulated in the liposome may be appropriately set according to the type and use of the drug.
  • the liposome used in the present invention uses known liposome production methods such as a hydration method, an ultrasonic treatment method, an ethanol injection method, an ether injection method, a reverse phase evaporation method, a surfactant method, and a freezing / thawing method. Can be made.
  • the particle size distribution of liposomes can be adjusted by passing through a filter having a predetermined pore size.
  • conversion from MLV to monomembrane liposomes and conversion from monomembrane liposomes to MLV can also be performed according to known methods.
  • polypeptide containing immunoglobulin binding domain One aspect of the polypeptide used in the present invention is a polypeptide containing an immunoglobulin binding domain shown in any of the following (A1) to (E1).
  • A1 In the amino acid sequence shown in SEQ ID NO: 1, at least 3 amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • An immunoglobulin binding domain having an amino acid sequence that has been identified.
  • (B1) In the amino acid sequence shown in SEQ ID NO: 2, at least 3 amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues. An immunoglobulin binding domain having an amino acid sequence that has been identified.
  • (C1) In the amino acid sequence shown in SEQ ID NO: 3, at least 3 amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues. An immunoglobulin binding domain having an amino acid sequence that has been identified.
  • (D1) In the amino acid sequence shown in SEQ ID NO: 4, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues. An immunoglobulin binding domain having an amino acid sequence that has been identified.
  • (E1) In the amino acid sequence shown in SEQ ID NO: 5, at least 3 amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues. An immunoglobulin binding domain having an amino acid sequence that has been identified.
  • amino acid sequences shown in SEQ ID NOs: 1 to 5 are the amino acid sequences of the munoglobulin binding domains contained in protein A (amino acid sequence shown in SEQ ID NO: 6, UniProtKB registration number P02976) derived from Staphylococcus aureus, respectively. , Have high sequence identity with each other.
  • amino acid sequence shown in SEQ ID NO: 1 corresponds to the amino acid sequence of the immunoglobulin binding domain C existing in the region at positions 270 to 327 in SEQ ID NO: 6, and the amino acid sequence shown in SEQ ID NO: 2 corresponds to SEQ ID NO:
  • amino acid sequence shown in SEQ ID NO: 3 corresponds to the amino acid sequence of immunoglobulin binding domain E existing in the region at positions 35 to 92 in SEQ ID NO: 6, and the amino acid sequence shown in SEQ ID NO: 6 corresponds to the amino acid binding domain D existing in the region at positions 96 to 153 in SEQ ID NO: 6.
  • amino acid sequence shown in SEQ ID NO: 4 corresponds to the amino acid sequence of the immunoglobulin binding domain A existing in the region at positions 154 to 211 in SEQ ID NO: 6, and the amino acid sequence shown in SEQ ID NO: 5 is It corresponds to the amino acid sequence of the immunoglobulin binding domain B existing in the region at positions 212 to 269 in SEQ ID NO: 6.
  • amino acid residues at positions 40 to 55 in the amino acid sequences shown in SEQ ID NOs: 1 to 5 correspond to ⁇ -helix sites that do not directly contribute to the binding to the Fc region of immunoglobulin.
  • a positive charge is added to the immunoglobulin binding domain by introducing a lysine residue and / or an arginine residue into a site that does not directly contribute to the binding to the antibody in the immunoglobulin binding domain, and the liposome Can be combined with.
  • amino acid residues at positions 40, 43, 46, 47, 53, and 54 are replaced with lysine residues and / or arginine residues.
  • the number of amino acid residues may be 3 or more, and specific examples thereof include 3 to 6, more preferably 3 to 5, and even more preferably 3 or 4.
  • the sites of the amino acid residue substituted with the lysine residue and / or the arginine residue are at positions 40, 43, 46, 47, 53, and At least three of the 54 positions may be used, but from the viewpoint of further improving the binding ability to the liposome, the 43 and 46 positions are preferably lysine residues and / or arginine residues.
  • position is replaced with a lysine residue and / or an arginine residue
  • at least one of the 40th, 53rd, and 54th positions is replaced with a lysine residue and / or an arginine residue; more preferably.
  • the 43rd, 46th, and 53rd positions are replaced with lysine residues and / or arginine residues
  • 0 to 3 of the 40th, 47th, and 54th positions are replaced with lysine residues and / or arginine residues.
  • positions 43, 46, and 53 are replaced with lysine residues and / or arginine residues, and one or two of positions 40 and 54 are lysine residues. And / or an embodiment in which the arginine residue is substituted.
  • immunoglobulin-binding domain corresponding to (A1) above include immunoglobulin-binding domains having the amino acid sequences shown in SEQ ID NOs: 7 to 12 and 60.
  • the amino acid sequence shown in SEQ ID NO: 7 is an amino acid sequence in which positions 40, 43, and 46 are substituted with lysine residues in the amino acid sequence shown in SEQ ID NO: 1.
  • the amino acid sequence shown in SEQ ID NO: 8 is an amino acid sequence in which positions 40, 43, 46 and 53 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 1.
  • the amino acid sequence shown in SEQ ID NO: 9 is an amino acid sequence in which positions 43, 46 and 54 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 1.
  • the amino acid sequence shown in SEQ ID NO: 10 is an amino acid sequence in which positions 43, 46, 53 and 54 are substituted with lysine residues in the amino acid sequence shown in SEQ ID NO: 1.
  • the amino acid sequence shown in SEQ ID NO: 11 is an amino acid sequence in which positions 43, 46, 47 and 54 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 1.
  • the amino acid sequence shown in SEQ ID NO: 12 is an amino acid sequence in which positions 43, 47 and 54 are replaced with arginine residues in the amino acid sequence shown in SEQ ID NO: 1.
  • the amino acid sequence shown in SEQ ID NO: 60 is an amino acid sequence in which positions 43, 47 and 54 are substituted with lysine residues in the amino acid sequence shown in SEQ ID NO: 1.
  • immunoglobulin-binding domain corresponding to the above (B1) include immunoglobulin-binding domains having the amino acid sequences shown in SEQ ID NOs: 13 to 18 and 61.
  • the amino acid sequence shown in SEQ ID NO: 13 is an amino acid sequence in which positions 40, 43, and 46 are substituted with lysine residues in the amino acid sequence shown in SEQ ID NO: 2.
  • the amino acid sequence shown in SEQ ID NO: 14 is an amino acid sequence in which positions 40, 43, 46 and 53 are substituted with lysine residues in the amino acid sequence shown in SEQ ID NO: 2.
  • the amino acid sequence shown in SEQ ID NO: 15 is an amino acid sequence in which positions 43, 46 and 54 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 2.
  • the amino acid sequence shown in SEQ ID NO: 16 is an amino acid sequence in which positions 43, 46, 53 and 54 are substituted with lysine residues in the amino acid sequence shown in SEQ ID NO: 2.
  • the amino acid sequence shown in SEQ ID NO: 17 is an amino acid sequence in which positions 43, 46, 47 and 54 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 2.
  • the amino acid sequence shown in SEQ ID NO: 18 is an amino acid sequence in which positions 43, 47 and 54 are replaced with arginine residues in the amino acid sequence shown in SEQ ID NO: 2.
  • the amino acid sequence shown in SEQ ID NO: 61 is an amino acid sequence in which positions 43, 47 and 54 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 2.
  • immunoglobulin-binding domain corresponding to the above (C1) include immunoglobulin-binding domains having the amino acid sequences shown in SEQ ID NOs: 19 to 24 and 62.
  • the amino acid sequence shown in SEQ ID NO: 19 is an amino acid sequence in which positions 40, 43, and 46 are substituted with lysine residues in the amino acid sequence shown in SEQ ID NO: 3.
  • the amino acid sequence shown in SEQ ID NO: 20 is an amino acid sequence in which positions 40, 43, 46 and 53 are substituted with lysine residues in the amino acid sequence shown in SEQ ID NO: 3.
  • the amino acid sequence shown in SEQ ID NO: 21 is an amino acid sequence in which positions 43, 46 and 54 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 3.
  • the amino acid sequence shown in SEQ ID NO: 22 is an amino acid sequence in which positions 43, 46, 53 and 54 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 3.
  • the amino acid sequence shown in SEQ ID NO: 23 is an amino acid sequence in which positions 43, 46, 47 and 54 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 3.
  • the amino acid sequence shown in SEQ ID NO: 24 is an amino acid sequence in which positions 43, 47 and 54 are replaced with arginine residues in the amino acid sequence shown in SEQ ID NO: 3.
  • the amino acid sequence shown in SEQ ID NO: 62 is an amino acid sequence in which positions 43, 47 and 54 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 3.
  • immunoglobulin-binding domain corresponding to (D1) above include immunoglobulin-binding domains having the amino acid sequences shown in SEQ ID NOs: 25 to 30 and 63.
  • the amino acid sequence shown in SEQ ID NO: 25 is an amino acid sequence in which positions 40, 43, and 46 are substituted with lysine residues in the amino acid sequence shown in SEQ ID NO: 4.
  • the amino acid sequence shown in SEQ ID NO: 26 is an amino acid sequence in which positions 40, 43, 46 and 53 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 4.
  • the amino acid sequence shown in SEQ ID NO: 27 is an amino acid sequence in which positions 43, 46 and 54 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 4.
  • the amino acid sequence shown in SEQ ID NO: 28 is an amino acid sequence in which positions 43, 46, 53 and 54 are substituted with lysine residues in the amino acid sequence shown in SEQ ID NO: 4.
  • the amino acid sequence shown in SEQ ID NO: 29 is an amino acid sequence in which positions 43, 46, 47 and 54 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 4.
  • the amino acid sequence shown in SEQ ID NO: 30 is an amino acid sequence in which positions 43, 47 and 54 are replaced with arginine residues in the amino acid sequence shown in SEQ ID NO: 4.
  • the amino acid sequence shown in SEQ ID NO: 63 is an amino acid sequence in which positions 43, 47 and 54 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 4.
  • immunoglobulin-binding domain corresponding to the above (E1) include immunoglobulin-binding domains having the amino acid sequences shown in SEQ ID NOs: 31 to 36 and 64.
  • the amino acid sequence shown in SEQ ID NO: 31 is an amino acid sequence in which positions 40, 43, and 46 are substituted with lysine residues in the amino acid sequence shown in SEQ ID NO: 5.
  • the amino acid sequence shown in SEQ ID NO: 32 is an amino acid sequence in which positions 40, 43, 46 and 53 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 5.
  • the amino acid sequence shown in SEQ ID NO: 33 is an amino acid sequence in which positions 43, 46 and 54 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 5.
  • the amino acid sequence shown in SEQ ID NO: 34 is an amino acid sequence in which positions 43, 46, 53 and 54 are substituted with lysine residues in the amino acid sequence shown in SEQ ID NO: 5.
  • the amino acid sequence shown in SEQ ID NO: 35 is an amino acid sequence in which positions 43, 46, 47 and 54 are replaced with lysine residues in the amino acid sequence shown in SEQ ID NO: 5.
  • the amino acid sequence shown in SEQ ID NO: 36 is an amino acid sequence in which positions 43, 47 and 54 are replaced with arginine residues in the amino acid sequence shown in SEQ ID NO: 5.
  • the amino acid sequence shown in SEQ ID NO: 64 is an amino acid sequence in which positions 43, 47 and 54 are substituted with lysine residues in the amino acid sequence shown in SEQ ID NO: 5.
  • polypeptide used in the present invention a polypeptide containing an immunoglobulin binding domain shown in any of the following (A2) to (E2) and (A3) to (E3) can be mentioned.
  • A2 In the amino acid sequence shown in SEQ ID NO: 1, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues. And further has an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added other than the substituted lysine residue and / or arginine residue.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain of (A1) having the same substitution with the introduced lysine residue and / or arginine residue.
  • B2 In the amino acid sequence shown in SEQ ID NO: 2, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues. And further has an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added other than the substituted lysine residue and / or arginine residue.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain (B1) having the same substitution with the introduced lysine residue and / or arginine residue.
  • C2 In the amino acid sequence shown in SEQ ID NO: 3, at least three amino acid residues in positions 40, 43, 46, 47, 53, and 54 are replaced with lysine residues and / or arginine residues. And further has an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added other than the substituted lysine residue and / or arginine residue.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain of (C1) in which the substitution with the introduced lysine residue and / or arginine residue is the same.
  • D2 In the amino acid sequence shown in SEQ ID NO: 4, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues. And further has an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added other than the substituted lysine residue and / or arginine residue.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain of (D1) having the same substitution with the introduced lysine residue and / or arginine residue.
  • E2 In the amino acid sequence shown in SEQ ID NO: 5, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues. And further has an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added other than the substituted lysine residue and / or arginine residue.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain of (E1) in which the substitution with the introduced lysine residue and / or arginine residue is the same.
  • (A3) In the amino acid sequence shown in SEQ ID NO: 1, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • the sequence identity of the region excluding the substituted lysine residue and / or arginine residue is 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 1.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain of (A1) having the same substitution with the introduced lysine residue and / or arginine residue.
  • B3 In the amino acid sequence shown in SEQ ID NO: 2, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • the sequence identity of the region excluding the substituted lysine residue and / or arginine residue is 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 2.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain (B1) having the same substitution with the introduced lysine residue and / or arginine residue.
  • C3 In the amino acid sequence shown in SEQ ID NO: 3, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • the sequence identity of the region excluding the substituted lysine residue and / or arginine residue is 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 3.
  • the immunoglobulin binding domain having the same binding ability to the antibody as compared with the immunoglobulin binding domain of (C1) in which the substitution with the introduced lysine residue and / or arginine residue is the same.
  • D3 In the amino acid sequence shown in SEQ ID NO: 4, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • the sequence identity of the region excluding the substituted lysine residue and / or arginine residue is 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 4.
  • E3 In the amino acid sequence shown in SEQ ID NO: 5, at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are replaced with lysine residues and / or arginine residues.
  • the sequence identity of the region excluding the substituted lysine residue and / or arginine residue is 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 5.
  • the immunoglobulin binding domains of (A2) and (A3) are variants of the immunoglobulin binding domain of (A1), and are replaced with lysine residues and / or arginine residues in the amino acid sequence shown in SEQ ID NO: 1. It is an immunoglobulin binding domain in which a region other than the site (hereinafter, also referred to as “arbitrarily modifiable region”) has been modified (substituted, deleted, inserted or added).
  • the immunoglobulin binding domains of (B2) and (B3) are variants of the immunoglobulin binding domain of (B1), and are replaced with lysine residues and / or arginine residues in the amino acid sequence shown in SEQ ID NO: 2. It is an immunoglobulin-binding domain in which a region other than the site (hereinafter, also referred to as “arbitrarily modifiable region”) has been modified (substituted, deleted, inserted or added).
  • the immunoglobulin binding domains of (C2) and (C3) are variants of the immunoglobulin binding domain of (C1), and are replaced with lysine residues and / or arginine residues in the amino acid sequence shown in SEQ ID NO: 3. It is an immunoglobulin-binding domain in which a region other than the site (hereinafter, also referred to as “arbitrarily modifiable region”) has been modified (substituted, deleted, inserted or added).
  • the immunoglobulin binding domains of (D2) and (D3) are variants of the immunoglobulin binding domain of (D1), and are replaced with lysine residues and / or arginine residues in the amino acid sequence shown in SEQ ID NO: 4. It is an immunoglobulin-binding domain in which a region other than the site (hereinafter, also referred to as “arbitrarily modifiable region”) has been modified (substituted, deleted, inserted or added).
  • the immunoglobulin binding domains of (E2) and (E3) are variants of the immunoglobulin binding domain of (E1), and are replaced with lysine residues and / or arginine residues in the amino acid sequence shown in SEQ ID NO: 5. It is an immunoglobulin-binding domain in which a region other than the site (hereinafter, also referred to as “arbitrarily modifiable region”) has been modified (substituted, deleted, inserted or added).
  • Modifications of amino acid residues introduced into any modifiable region in the immunoglobulin binding domains of (A2), (B2), (C2), (D2), and (E2) are substitutions, additions, insertions, and modifications.
  • the deletion may contain only one type of modification (eg, substitution) or may contain two or more types of modification (eg, substitution and addition).
  • the number of amino acid residues to be substituted is It may be one or several, and specific examples thereof include 1 to 10, preferably 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5. ..
  • the number of amino acid residues to be added is It may be one or several, and specific examples thereof include 1 to 10, preferably 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5. ..
  • the number of amino acid residues to be inserted is It may be one or several, specifically 1 to 10, preferably 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5, more preferably. There are 1 to 3, more preferably 1 or 2.
  • the number of amino acid residues deleted when an amino acid is deleted in an arbitrarily modifiable region may be one or several, specifically 1 to 10, preferably 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5. More preferably, 1 to 3, and even more preferably 1 or 2.
  • substitution and / or added lysine residue for each amino acid sequence shown in SEQ ID NOs: 1 to 5 The sequence identity of the site excluding the group and / or the arginine residue may be 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 99. % Or more.
  • the substituted lysine residue for each amino acid sequence shown in SEQ ID NOs: 1 to 5 And / or the sequence identity of the region excluding the arginine residue is the 40th, 43rd, 46th, 47th, 53rd, and 53rd positions from each amino acid sequence shown in SEQ ID NOs: 1 to 5.
  • the sequence identity is calculated by extracting the region excluding the site substituted with the lysine residue and / or the arginine residue in the 54-position and comparing only the region.
  • Sequence identity is the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol.) Of BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)]. The value of the identity of the amino acid sequence obtained by Lett., Vol.174, p247-250, 1999) is shown. The parameters may be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.
  • the modification introduced in the arbitrarily modifiable region of each of the immunoglobulin binding domains (A2) to (E2) and (A3) to (E3) may be appropriately set as long as the binding ability to the antibody can be maintained.
  • the binding efficiency of the polypeptide is further increased.
  • at least one of the amino acid residues at positions 41 to 58 excluding positions 43, 46, 47, 53, and 54 in SEQ ID NOs: 1 to 5 remains lysine.
  • substituting with a group and / or an arginine residue; more preferably, at least one (particularly one to three) of the 42, 49, 50, and 56 positions in SEQ ID NOs: 1 to 5 is used. Examples thereof include substitution with a lysine residue and / or an arginine residue.
  • amino acid substitution site is described above.
  • the 1st, 4th, 7th, 29th, 35th, and 58th positions in SEQ ID NOs: 1 to 5 can be mentioned.
  • the type of amino acid residue to be added is determined.
  • 1 to 10 lysine residues and / or arginine residues are added to the C-terminal, preferably 2 to 5 or more. It is preferable to add 2 to 4 pieces.
  • anionic lipids as compared to the immunoglobulin binding domains of (A1) having the same substitution with the introduced lysine residue and / or arginine residue.
  • the binding ability to liposomes containing anionic lipids is equal to or higher than that means that the binding ability to liposomes containing anionic lipids is the same as the substitution with lysine residue and / or arginine residue introduced in the above-mentioned predetermined region.
  • A1 immunoglobulin binding domain that is, an immunoglobulin binding domain before modification is introduced into an arbitrarily modifiable site
  • the lipid binding rate is ⁇ 50% based on the polypeptide binding rate (100%) measured under the same conditions using the immunoglobulin binding domain of (A1) before the modification was introduced into the arbitrarily modifiable site. It is mentioned that it is within the range, preferably within the range of ⁇ 30%. The same applies to the immunoglobulin binding domains of (B2) to (E2) and (B3) to (E3).
  • the polypeptide used for modifying the liposome has any of the immunoglobulin binding domains (A1) to (E1), (A2) to (E2), and (A3) to (E3). It may be either a single-domain type polypeptide or a multi-domain type polypeptide.
  • one of the immunoglobulin-binding domains (A1) to (E1), (A2) to (E2), and (A3) to (E3) is contained. ..
  • a monodomain-type polypeptide it may consist of any of the immunoglobulin-binding domains (A1) to (E1), (A2) to (E2), and (A3) to (E3).
  • an amino acid residue may be added to the N-terminal and / or the C-terminal.
  • the number of amino acid residues added is not particularly limited, but is, for example, 1 to 100. The number is preferably 1 to 50, more preferably 1 to 20, and even more preferably 1 to 10.
  • a multidomain type polypeptide it is sufficient that two or more immunoglobulin-binding domains are linked, and at least one of the two or more immunoglobulin-binding domains is the above-mentioned (A1) to (E1), (A2). )-(E2) and (A3)-(E3) immunoglobulin binding domains.
  • the number of domains is included, and from two or more domains among the immunoglobulin binding domains (A1) to (E1), (A2) to (E2), and (A3) to (E3).
  • one or more of the immunoglobulin binding domains of (A1) to (E1), (A2) to (E2), and (A3) to (E3) described above may be used. It may contain one or more wild-type immunoglobulin-binding domains and / or variants thereof contained in proteins A, L and G.
  • the total number of linked immunoglobulin-binding domains may be 2 or more, preferably 2 to 10, and more preferably 2 to 6.
  • each of the constituent immunoglobulin-binding domains may have its C-terminal and N-terminal directly linked, and 1 to 40, preferably 1 to 40, are between each immunoglobulin-binding domain. It may be linked via 1 to 10 amino acid residues.
  • the N-terminus of the multidomain polypeptide is the immunoglobulin binding domain of any of the above (A1) to (E1), (A2) to (E2), and (A3) to (E3), or another immunoglobulin binding. It may be the N-terminus of the domain, but it may be further added with an amino acid residue.
  • the number of amino acid residues added is not particularly limited, but is, for example, 1 to 100, preferably 1 to 50, more preferably. Is 1 to 20, more preferably 1 to 10.
  • the C-terminus of the multidomain polypeptide is the immunoglobulin binding domain of any of the above (A1) to (E1), (A2) to (E2), and (A3) to (E3), or another immunoglobulin binding. It may be the C-terminus of the domain, but it may be further added with an amino acid residue.
  • the number of amino acid residues added is not particularly limited, but is, for example, 1 to 100, preferably 1 to 50, more preferably. Is 1 to 20, more preferably 1 to 10.
  • the type of amino acid to be added is not particularly limited.
  • the polypeptide containing any of the immunoglobulin binding domains (A1) to (E1), (A2) to (E2), and (A3) to (E3) is known as a genetic engineering method, a chemical synthesis method, or the like. It can be manufactured by the method of.
  • one of the above-mentioned polypeptides may be used alone, or two or more kinds of the polypeptides may be used in combination.
  • the polypeptide-modified liposome of the present invention is a complex of a polypeptide and a liposome in which the polypeptide is bound to the surface of the liposome.
  • the liposome is subjected to an electrostatic interaction between a negative charge of an anionic lipid contained in the liposome and a positive charge of a lysine residue and / or an arginine residue of the polypeptide.
  • the polypeptide is bound to the surface of the above.
  • the amount of binding of the polypeptide is not particularly limited, but for example, the amount of the polypeptide is about 0.001 to 5.0 mol per 100 mol of the total amount of the constituent lipids of the liposome.
  • the amount is preferably about 0.01 to 1.0 mol, more preferably about 0.05 to 0.5 mol.
  • the polypeptide-modified liposome of the present invention can be obtained by mixing the suspension of the liposome with the polypeptide.
  • the mixing conditions are not particularly limited, but for example, 0.01 to 1 mM of the polypeptide is added to a suspension of liposomes having a total concentration of constituent lipids of about 0.1 to 100 mM, preferably about 1 to 50 mM. Addition so as to be about 0.05 to 0.5 mM, about 4 to 40 ° C., preferably about 10 to 37 ° C., about 0.01 to 36 hours, preferably 0.1 to 24. It may be held for about an hour with stirring as needed.
  • the polypeptide-modified liposome of the present invention can be used as an immunoliposome by binding to an antibody as described later. Further, since the polypeptide binds to the Fc region of an immunoglobulin antibody, the polypeptide-modified liposome of the present invention can be used for various purposes by binding to an Fc fusion protein such as an Fc region fusion cytokine or an Fc region fusion TNFR. It can also be used.
  • an Fc fusion protein such as an Fc region fusion cytokine or an Fc region fusion TNFR. It can also be used.
  • the immunoliposomes of the present invention are a complex of a polypeptide-modified liposome and an antibody formed by binding an antibody to the polypeptide-modified liposome.
  • the isotype of the antibody used in the immunoliposomes of the present invention is not particularly limited, and examples thereof include IgG, IgM, IgA, IgD, and IgE. Among these, IgG is preferably mentioned.
  • the antibody used in the present invention may be a non-human antibody such as a mouse antibody or a rat antibody, but if the immunoglobulin of the present invention is administered in vivo as a drug or an in vivo diagnostic agent, it may be used.
  • Antibodies with reduced antigenicity in the human body specifically, fully human antibodies, humanized antibodies, chimeric antibodies and the like are preferable.
  • the antigen to which the antibody used in the present invention binds may be appropriately set according to the molecular target of the immunoliposomes of the present invention.
  • the immunoliposomes of the present invention contain an anticancer agent, cancer may occur.
  • An antibody capable of binding to an antigen specifically expressed in a cell may be used.
  • the amount of antibody bound is not particularly limited, but for example, the antibody is 0.1 to 100 mmol, preferably 0.5 to 50 mmol, based on 100 mol of the total amount of the constituent lipids of the liposome. Preferably 1 to 10 mmol.
  • the polydispersity index (PDI) of the immunoliposomes of the present invention is not particularly limited, but is, for example, 0.5 or less, preferably 0.01 to 0.3, and more preferably 0.01 to 0. .2 can be mentioned.
  • the polydispersity index of immunoliposomes is a value measured by a dynamic light scattering method using Zeta-sizer nano Zs (Malvern Panasonic).
  • examples of the zeta potential of the immunoliposomes of the present invention include -50 to -1 mV, preferably -50 to -10 mV, and more preferably -50 to -25 mV.
  • the zeta potential of immunoliposomes is measured in phosphate buffer (PBS (-)) by laser Doppler electrophoresis using Zeta-sizer nanoZs (Malvern Panalytical). The value.
  • the particle size of the immunoliposomes of the present invention is not particularly limited, but usually, the average particle size is 30 to 1000 nm, preferably 50 to 500 nm.
  • the average particle size of immunoliposomes is a value measured by a dynamic light scattering method using Zeta-sizer nanoZs (Malvern Panasonic).
  • the immunoliposomes of the present invention can be used as DDS preparations useful as active targeting liposomes and the like. Therefore, the pharmaceutical composition or the diagnostic composition containing the immunoliposomes of the present invention containing a drug (therapeutic agent, in-vivo diagnostic agent, etc.) specifically delivers the drug to the lesion site or the diagnosis target site. Becomes possible.
  • the immunolipolips of the present invention are also useful as a research tool for intracellular uptake of cells via a specific receptor, a research tool for delivery of drugs and the like into cells, and the like. Furthermore, by including the material for genome editing in the immunoliposomes of the present invention, simple and efficient genome editing becomes possible.
  • the immunoliposomes of the present invention can be obtained by mixing a suspension of the polypeptide-modified liposome with an antibody.
  • the mixing conditions are not particularly limited, but for example, the antibody is preferably about 0.1 to 10 ⁇ M with respect to a suspension of liposomes having a total concentration of constituent lipids of about 1 to 100 mM, preferably about 10 to 50 mM. Is added so as to be about 0.5 to 5 ⁇ M, and is required at about 4 to 40 ° C., preferably about 10 to 37 ° C. for about 0.05 to 36 hours, preferably about 0.1 to 24 hours. It may be held with stirring accordingly.
  • EPC egg yolk phosphatidylcholine
  • DPPG dipalmitoylphosphatidylglycerol
  • the mixture was heated at 60 ° C. for 3 minutes and further stirred by vortex.
  • the operation from freezing and thawing to stirring with vortex was performed a total of 3 times, and then the size was adjusted with an extruder to obtain a suspension of liposomes.
  • the average particle size of the liposomes, the polydispersity index (PDI), and the zeta potential were measured.
  • the average particle size and PDI of liposomes were measured by a dynamic light scattering method using Zeta-sizer nanoZs (Malvern Panalytical).
  • the zeta potential of the liposome was measured by laser Doppler electrophoresis using Zeta-sizer nanoZs (Malvern Panalytical). The measurement results are as shown in Table 1.
  • a monodomain-type polypeptide having one immunoglobulin-binding domain C (SEQ ID NO: 1) of protein A or a variant thereof was prepared.
  • the amino acid sequence of the prepared monodomain-type polypeptide is as shown in Table 2.
  • these monodomain type polypeptides were produced by using a genetic engineering technique.
  • monodomain-type polypeptide (2) A monodomain-type polypeptide having one immunoglobulin-binding domain E (SEQ ID NO: 2), immunoglobulin-binding domain B (SEQ ID NO: 5), or a variant thereof of protein A was prepared.
  • the amino acid sequence of the prepared monodomain-type polypeptide is as shown in Table 3.
  • these monodomain type polypeptides were produced by using a genetic engineering technique.
  • a multidomain type polypeptide having two immunoglobulin binding domains was prepared.
  • the amino acid sequence of the prepared multi-domain type polypeptide is as shown in Table 4.
  • the multidomain type polypeptide (Cvar2-1) and two immunoglobulin binding domains corresponding to the above (A2) and (A3) are contained.
  • the multi-domain type polypeptide was produced by using a genetic engineering technique.
  • a multi-domain type polypeptide having four immunoglobulin-binding domains C was prepared.
  • the amino acid sequence of the prepared multi-domain type polypeptide is as shown in Table 5.
  • the multidomain type polypeptides of Cvar4-1 and Cvar4-2 contain two immunoglobulin-binding domains corresponding to the above (A2) and (A3).
  • the multidomain type polypeptides of Cvar4-3 and Cvar4-4 contain one immunoglobulin-binding domain corresponding to the above (A2) and (A3).
  • these multi-domain type polypeptides were produced by using a genetic engineering technique.
  • a multidomain type polypeptide having 6 immunoglobulin binding domains C was prepared.
  • the amino acid sequence of the prepared multi-domain type polypeptide is as shown in Table 6.
  • the multidomain type polypeptides of Cvar6-1 and Cvar6-2 contain one immunoglobulin-binding domain corresponding to the above (A2) and (A3).
  • these multi-domain type polypeptides were produced by using a genetic engineering technique.
  • Protein A Wild type protein A (w protein A, region at positions 35 to 485 of SEQ ID NO: 6, molecular weight 45,000) and protein A consisting of 5 binding domains (rprotein A, 35 of SEQ ID NO: 6) A region at position 327 and a molecular weight of 33,000) were prepared.
  • human IgG manufactured by Japan Blood Products Organization
  • EZ-Link Biotinylation Kit manufactured by Thermo Fisher Scientific Co., Ltd.
  • IgG-fixed by incubating biotinylated IgG prepared to 0.1 mg / mL with 0.1 wt% BSA (bovine serum albumin) -containing PBS and streptavidin sensor chip for 60 minutes at room temperature, and then washing with 0.1 wt% BSA-containing PBS.
  • a chemical chip was prepared.
  • the liposome suspension prepared above was diluted to 800 ⁇ M in terms of lipid concentration to prepare a liposome solution.
  • Each polypeptide was added to the obtained liposome solution so as to have the concentration shown in Table 7, and the mixture was shaken at room temperature for 1 hour.
  • the IgG-immobilized chip prepared above was brought into contact with the liposome solution to which each polypeptide was added, the amount of signal change (nm) for 20 seconds was measured, and the difference in signal between the addition and non-addition of the liposome was measured. Calculated as the amount of liposome binding.
  • the polypeptide containing a modified immunoglobulin binding domain in which two were substituted with a lysine residue and / or an arginine residue had a high binding ability to a liposome containing an anionic lipid.
  • the immunoglobulin binding domain C (SEQ ID NO: 1) of protein A one or two amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions are substituted.
  • Polypeptides containing a modified immunoglobulin binding domain were unable to bind to the surface of liposomes containing anionic lipids.
  • immunoglobulin binding domains E (SEQ ID NO: 2) and B (SEQ ID NO: 5) of protein A at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions.
  • a lysine residue and / or an arginine residue it was confirmed that it could bind to the surface of liposomes containing anionic lipids.
  • the five immunoglobulin-binding domains of protein A show 62.1% sequence identity of domain E and 91.4% of domain B with respect to domain C. Based on this, even in domain E, which has the lowest sequence identity with respect to domain C among the five domains, the binding ability to liposomes containing anionic lipids was recognized by the same modification as domain C. Therefore, the binding ability to liposomes containing anionic lipids can be imparted by making similar modifications to domain D having 72.4% sequence identity and domain A having 79.3% sequence identity to domain C. It can be said that.
  • the suspension to which FITC-labeled IgG was added was subjected to gel filtration (buffer: PBS, flow velocity 0.2 mL / min) using a column (7 ⁇ 160 mm) packed with Sepharose 4 Fast Flow (manufactured by GE Healthcare). It was fractionated into 10 fractions (1 mL per fraction). The fluorescence intensity (Ex: 488 nm, Em: 535 nm) of each of the obtained fractions was measured, and the amount of FITC-labeled IgG contained in each fraction was determined.
  • buffer PBS, flow velocity 0.2 mL / min
  • Sepharose 4 Fast Flow manufactured by GE Healthcare
  • the total value of the amount of the polypeptide in the 3rd and 4th fractions is the total value of all 10 fractions.
  • the IgG binding rate (%) was calculated by dividing by the total amount of the polypeptide.
  • the average particle size, PDI, zeta potential, and IgG binding rate (%) of liposomes were similarly measured for the suspension of liposomes without polypeptide modification and addition of FITC-labeled IgG. was done.
  • the results of the average particle size, PDI, zeta potential, and IgG binding rate (%) are shown in Table 9, and the results of measuring the fluorescence intensity of each fraction for Examples 8, 13, and 14 are shown in FIG.
  • at least three amino acid residues in the 40th, 43rd, 46th, 47th, 53rd, and 54th positions in the immunoglobulin binding domain C (SEQ ID NO: 1) of protein A are lysine residues and / or It was confirmed that the liposome modified with the polypeptide containing the modified immunoglobulin binding domain substituted with the arginine residue can bind IgG and form an immunoliploid.

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Abstract

L'objet de la présente invention est de fournir un liposome modifié par un polypeptide qui présente une excellente capacité de liaison à un anticorps.　Dans chaque domaine de liaison à l'immunoglobuline (numéro de séquence 1 à 4) configurant une protéine A, au moins 3 résidus d'acides aminés choisis parmi la position 40, la position 43, la position 46, la position 47, la position 53 et la position 54 sont modifiés en résidu lysine et/ou en résidu arginine, et par liaison du polypeptide obtenu ayant le domaine de liaison à l'immunoglobuline modifié à la surface du liposome qui contient un lipide anionique en tant que lipide constitutif, il est possible d'obtenir un liposome modifié par un polypeptide qui présente une excellente capacité de liaison à un anticorps.
PCT/JP2020/012364 2019-03-20 2020-03-19 Liposome modifié par un polypeptide ayant une capacité de liaison à un anticorps, et immunoliposome WO2020189766A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60138464A (ja) * 1983-12-27 1985-07-23 Denka Seiken Co Ltd 新規な抗原定量法
JP2007252368A (ja) * 2006-02-21 2007-10-04 Protenova Co Ltd イムノグロブリン親和性リガンド
JP2010215509A (ja) * 2007-07-10 2010-09-30 Katayama Kagaku Kogyo Kk 抗体リポソーム

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60138464A (ja) * 1983-12-27 1985-07-23 Denka Seiken Co Ltd 新規な抗原定量法
JP2007252368A (ja) * 2006-02-21 2007-10-04 Protenova Co Ltd イムノグロブリン親和性リガンド
JP2010215509A (ja) * 2007-07-10 2010-09-30 Katayama Kagaku Kogyo Kk 抗体リポソーム

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