WO2020189766A1

WO2020189766A1 - Polypeptide-modified liposome having antibody binding capacity, and immunoliposome

Info

Publication number: WO2020189766A1
Application number: PCT/JP2020/012364
Authority: WO
Inventors: 英司真島
Original assignee: プロテノバ株式会社
Priority date: 2019-03-20
Filing date: 2020-03-19
Publication date: 2020-09-24
Also published as: JPWO2020189766A1

Abstract

The purpose of the present invention is to provide a polypeptide-modified liposome which has excellent antibody binding capacity.　In each immunoglobulin binding domain (sequence number 1-4) configuring a protein A, at least 3 amino acid residues selected from position 40, position 43, position 46, position 47, position 53 and position 54 are modified to lysine residue and/or arginine residue, and by binding the obtained polypeptide having the modified immunoglobulin binding domain to the surface of the liposome which contains an anionic lipid as a constituent lipid, it is possible to obtain a polypeptide modified liposome which has excellent antibody binding capacity.

Description

Polypeptide-modified liposomes having antibody binding ability and immunoliposomes

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.

In addition, 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.

Conventionally, in order to make liposomes have directivity toward molecular targets, development of immunoliposomes in which an antibody is bound to the surface of liposomes has been attempted. For example, in 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. Further, 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. Further, 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.

However, conventional immunoliposomes have drawbacks such as a small amount of antibody binding and a complicated manufacturing process. In addition, a method of modifying the surface of protein A or protein G in order to impart antibody-binding ability to immunoliplips is also known (for example, Patent Document 1 etc.), but the amino acid sequence of protein A or protein G is modified. Therefore, no technique for improving the antibody-binding ability of immunolipolips has been reported.

JP-A-2015-137273 Special Table 2009-512721

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.

As a result of diligent studies to solve the above problems, the present inventor added a positive charge to the α-helix site that does not directly contribute to the binding to the antibody in each immunoglobulin binding domain constituting the protein A. 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.

That is, 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. 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Item 2. 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. Item 2. The 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.
Item 4. 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). Item 8. The polypeptide-modified liposome according to any one of Items 1 to 4, which is a monodomain-type polypeptide having one kind of immunoglobulin binding domain.
Item 6. 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. The polypeptide-modified liposome according to any one of Items 1 to 6, wherein the polypeptide has at least one immunoglobulin binding domain selected from the group consisting of (A1) to (A3).
Item 8. Item 3. The polypeptide-modified lipid described in C.
Item 9. Item 2. The polypeptide-modified liposome according to any one of Items 1 to 8, further comprising a neutral lipid as a constituent lipid of the liposome.
Item 10. 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.
Item 12. Item 3. A pharmaceutical composition comprising the immunoliposomes according to Item 11.

Since 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. ..

Further, the 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.

It is a figure which shows the result of having fractionated the polypeptide modified liposome suspension to which FITC-labeled IgG was added in Examples 8, 13 and 14, and measured the fluorescence intensity of each fraction.

Hereinafter, the present invention will be described in detail. In addition to the sequence listing, 20 types of amino acid residues in the amino acid sequence may be represented by one-letter abbreviations. That is, glycine (Gly) is G, alanine (Ala) is A, valine (Val) is V, leucine (Leu) is L, isoleucine (Ile) is I, phenylalanine (Phe) is F, and tyrosine (Tyr) is Y. , Tryptophan (Trp) is W, Serine (Ser) is S, Treonine (Thr) is T, Cysteine (Cys) is C, Methionine (Met) is M, Aspartic acid (Asp) is D, Glutamine (Glu) is E. , Aspartic acid (Asn) is N, glutamine (Gln) is Q, lysine (Lys) is K, arginine (Arg) is R, histidine (His) is H, and proline (Pro) is P. Further, in the present invention, when the amino acid sequence is expressed, the left side indicates the N-terminal and the right side indicates the C-terminal.

In the present specification, "non-polar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. In addition, "uncharged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In addition, "acidic amino acids" include aspartic acid and glutamic acid. In addition, "basic amino acids" include lysine, arginine, and histidine.

1. 1. Polypeptide-modified liposome The 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. Hereinafter, the polypeptide-modified liposome of the present invention will be described in detail.

[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. .. These anionic lipids may be used alone or in combination of two or more. Among these 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%.

Further, 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. These neutral lipids may be used alone or in combination of two or more. Among these neutral lipids, 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. In the present specification, the liposomal polydispersity index is a value measured by a dynamic light scattering method.

Further, 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. In the present specification, 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. In the present specification, 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.

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. .. Specific examples of the 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. In addition, the particle size distribution of liposomes can be adjusted by passing through a filter having a predetermined pore size. In addition, 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.

The 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. Specifically, the 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: The 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. The 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.

The amino acid residues at positions 40 to 55 in the amino acid sequences shown in SEQ ID NOs: 1 to 5 correspond to α-helix sites that do not directly contribute to the binding to the Fc region of immunoglobulin. In the present invention, 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.

In the amino acid sequence shown in any of SEQ ID NOs: 1 to 5, the 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.

In the amino acid sequence shown in any of SEQ ID NOs: 1 to 5, 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. A mode in which at least one of the 40th, 47th, 53rd, and 54th positions is substituted with a lysine residue and / or an arginine residue; more preferably, the 43rd position and the 46th position are substituted. An embodiment in which the position is replaced with a lysine residue and / or an arginine residue, and 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, and 0 to 3 of the 40th, 47th, and 54th positions are replaced with lysine residues and / or arginine residues. Substituted Aspects; Particularly preferably, 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.

Specific examples of the 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.

Specific examples of the 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.

Specific examples of the 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.

Specific examples of the 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.

Specific examples of the 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.

In addition, as another embodiment of the 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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 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).

In the immunoglobulin binding domains of (A2), (B2), (C2), (D2), and (E2), when amino acid substitution is performed in an arbitrarily modifiable region, 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. ..

In the immunoglobulin binding domains of (A2), (B2), (C2), (D2), and (E2), when amino acids are added in an arbitrarily modifiable region, 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. ..

In the immunoglobulin binding domains of (A2), (B2), (C2), (D2), and (E2), when amino acids are inserted in an arbitrarily modifiable region, 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.

In the immunoglobulin binding domains of (A2), (B2), (C2), (D2), and (E2), the number of amino acid residues deleted when an amino acid is deleted in an arbitrarily modifiable region. The number 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.

In the immunoglobulin binding domains of (A3), (B3), (C3), (D3), and (E3), the 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.

Here, in the immunoglobulin binding domains of (A3), (B3), (C3), (D3), and (E3), 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 (arbitrarily modifiable region) 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.

For example, in the immunoglobulin binding domains of (A2), (B2), (C2), (D2), and (E2), when amino acids are substituted in an arbitrarily modifiable region, the binding efficiency of the polypeptide is further increased. From the viewpoint of improvement, preferably, 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. Aspects of 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.

Further, when amino acid substitution is performed in an arbitrarily modifiable region in the immunoglobulin binding domains of (A2), (B2), (C2), (D2), and (E2), the amino acid substitution site is described above. In addition to the exemplified sites, for example, the 1st, 4th, 7th, 29th, 35th, and 58th positions in SEQ ID NOs: 1 to 5 can be mentioned.

Further, for example, when an amino acid is added in the immunoglobulin binding domains of (A2), (B2), (C2), (D2), and (E2), the type of amino acid residue to be added is determined. Although not particularly limited, from the viewpoint of further improving the binding efficiency of the polypeptide, as a preferable example, 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.

In the immunoglobulin binding domains of (A2) and (A3) above, "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. It means that it is equal to or higher than a certain (A1) immunoglobulin binding domain (that is, an immunoglobulin binding domain before modification is introduced into an arbitrarily modifiable site), and specifically, it was measured under the following measurement conditions. 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).
(Measurement condition)
(1) Add 30 nmol of immunoglobulin binding domain to 0.2 ml of a suspension (total concentration of EPC and DPPG is 30 mM) of liposome (molar ratio of EPC and DPPG 1: 1) consisting of EPC and DPPG as constituent lipids. And incubate at 37 ° C. for 24 hours to obtain a suspension containing polypeptide-modified liposomes.
(2) The suspension containing the polypeptide-modified liposome obtained in the above procedure (1) is subjected to gel filtration and fractionated into a fraction containing liposomes and a fraction not containing liposomes. (3) After each fraction obtained by the operation of (2) above is subjected to SDS-PAGE, the gel is stained with Coomassie Brilliant Blue (CBB), the density of the staining band is measured by image analysis software, and each fraction The amount of polypeptide contained in (staining band density) is determined.
(4) The polypeptide binding rate (%) is calculated by dividing the total value of the amount of polypeptide of the fraction containing liposomes by the total value of the amount of polypeptide of all fractions.

In the present invention, 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.

In the case of a monodomain-type polypeptide, it is sufficient that one of the immunoglobulin-binding domains (A1) to (E1), (A2) to (E2), and (A3) to (E3) is contained. ..

In the case of a monodomain-type polypeptide, it may consist of any of the immunoglobulin-binding domains (A1) to (E1), (A2) to (E2), and (A3) to (E3). Further, an amino acid residue may be added to the N-terminal and / or the C-terminal. When an amino acid residue is added to the N-terminal and / or C-terminal of the immunoglobulin binding domain in a monodomain-type polypeptide, 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.

Further, in the case of 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. Specifically, in the case of a multidomain type polypeptide, at least one of the immunoglobulin binding domains of (A1) to (E1), (A2) to (E2), and (A3) to (E3) described above. It is sufficient that 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). In addition, 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.

In the case of a multi-domain type polypeptide, the total number of linked immunoglobulin-binding domains may be 2 or more, preferably 2 to 10, and more preferably 2 to 6.

Further, in the case of a multi-domain type polypeptide, 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. When an amino acid residue is added to the N-terminal in a multidomain type polypeptide, 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. When an amino acid residue is added to the C-terminal in a multidomain type polypeptide, 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. When an amino acid residue is added to the C-terminal in a multi-domain type polypeptide, 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.

In the present invention, one of the above-mentioned polypeptides may be used alone, or two or more kinds of the polypeptides may be used in combination.

[Modification of liposomes with polypeptides]
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.

In the polypeptide-modified liposome of the present invention, 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.

In the polypeptide-modified liposome of the present invention, 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.

2. 2. Immunoliposomes 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. For example, when 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.

In the immunoglobulin of the present invention, 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. In the present specification, the polydispersity index of immunoliposomes is a value measured by a dynamic light scattering method using Zeta-sizer nano Zs (Malvern Panasonic).

Further, 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. In the present specification, 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. In the present specification, 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 μM 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 μM, 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.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not construed as being limited to the following examples.

1. 1. Preparation of Lipids 1500 μL of 10 mM egg yolk phosphatidylcholine (EPC) solution dissolved in chloroform and 1500 μL of 10 mM dipalmitoylphosphatidylglycerol (DPPG) solution dissolved in chloroform / methanol (chloroform: methanol volume ratio = 6: 4) were in vitro. The solvent was volatilized to form a lipid thin film (EPC: DPPG molar ratio of 1: 1). Then, 1 mL of phosphate buffer (PBS (-)) was added, and the mixture was hydrated by standing stirring at 60 ° C. for 20 minutes (final lipid concentration was 30 mM). Then, after freeze-thawing, 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.

Using the obtained liposome suspension, 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.

2. 2. Preparation of a polypeptide having an immunoglobulin binding domain
2-1. Preparation of monodomain-type polypeptide (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. In addition, these monodomain type polypeptides were produced by using a genetic engineering technique.

2-2. Preparation of 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. In addition, these monodomain type polypeptides were produced by using a genetic engineering technique.

2-3. Preparation of a multidomain type polypeptide having two immunoglobulin binding domains 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.

2-4. Preparation of a multi-domain type polypeptide having four immunoglobulin-binding domains 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). In addition, these multi-domain type polypeptides were produced by using a genetic engineering technique.

2-5. Preparation of a multidomain type polypeptide having 6 immunoglobulin binding domains 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). In addition, these multi-domain type polypeptides were produced by using a genetic engineering technique.

2-6. Preparation of 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.

3. 3. Preparation and evaluation of polypeptide-modified liposome (1)
The liposome binding property of the polypeptide was evaluated from the interaction analysis with human IgG using the intermolecular interaction analyzer BLItz (manufactured by Molecular Device Co., Ltd.).

First, human IgG (manufactured by Japan Blood Products Organization) was biotinylated using a biotinification kit (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. Separately, using PBS containing 0.1 wt% BSA, the liposome suspension prepared above was diluted to 800 μM 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. After that, 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 results obtained are shown in Table 7. From this result, at least three amino acid residues in the immunoglobulin binding domain C (SEQ ID NO: 1) of protein A at positions 40, 43, 46, 47, 53, and 54 are lysine residues and /. Alternatively, it was confirmed that a polypeptide containing a modified immunoglobulin binding domain substituted with an arginine residue can bind to the surface of a liposome containing an anionic lipid. In particular, in the immunoglobulin binding domain C (SEQ ID NO: 1) of protein A, positions 43, 46, and 53 are replaced with lysine residues and / or arginine residues, and one or one of positions 40 and 54 is used. 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. On the other hand, in 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.

Furthermore, in the 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. When was replaced with 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.

4. Preparation and evaluation of polypeptide-modified liposome (2)
The amount of the polypeptide shown in Table 7 was added to 0.2 mL (6 μmol of lipid) of the liposome suspension of 30 mM in terms of lipid concentration, and the mixture was incubated at 37 ° C. for 24 hours with stirring. Then, the suspension to which the polypeptide 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).

After each fraction is subjected to SDS-PAGE, the gel is stained with Coomassie Brilliant Blue (CBB) to determine the density of the stained band (Image analysis software ImageJ (National Institutes of Health NIH https://imagej.nih.gov). The amount of polypeptide contained in each fraction was determined by measurement by / ij /)). Since the liposomes are eluted in the fractions recovered at the 3rd and 4th fractions (confirmed by measuring the turbidity of the liposomes at a wavelength of 750 nm), the total value of the amounts of the polypeptides of the 3rd and 4th fractions is 10 in total. The polypeptide binding rate (%) was calculated by dividing by the total amount of the polypeptides of the fraction.

The results are shown in Table 8. From this result, at least three amino acid residues in the immunoglobulin binding domain C (SEQ ID NO: 1) of protein A at positions 40, 43, 46, 47, 53, and 54 are lysine residues and / Or the polypeptide containing the modified immunoglobulin binding domain substituted with the arginine residue can bind to the liposome containing an anionic lipid (DPPG) as a constituent lipid, and the polypeptide-modified liposome is formed. confirmed. On the other hand, protein A could not bind to liposomes containing DPPG.

5. Preparation and Evaluation of Immunoliposomes Using the polypeptide-modified liposomes (Examples 7, 8, 12, and 13) evaluated in the above "4. Preparation and evaluation of polypeptide-modified liposomes (2)", the suspension ( The amounts of FITC-labeled IgG (product name: IgG-FITC from human serum, manufactured by SIGMA-ALDRICH) shown in Table 9 were added to 30 mM in terms of lipid concentration), and the mixture was incubated at 37 ° C. for 1 hour with stirring.

Using the suspension to which FITC-labeled IgG was added, the average particle size, PDI, and zeta potential of liposomes were measured by the method shown in the column of "1. Preparation of liposomes".

In addition, 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. Since the liposomes are eluted in the fractions recovered in the 3rd and 4th fractions (confirmed by measuring the absorbance at 750 nm), 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.

Also, for comparison, 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. As a result, 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.

Claims

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. 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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.
Moreover, 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. The polypeptide-modified liposome according to claim 1, wherein at least one of the positions is replaced with a lysine residue and / or an arginine residue.
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. The polypeptide-modified liposome according to claim 1 or 2, wherein one or two are substituted 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. The polypeptide-modified liposome according to any one of claims 1 to 3.
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-modified liposome according to any one of claims 1 to 4, which is a monodomain-type polypeptide having one kind of immunoglobulin binding domain.
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-modified liposome according to any one of claims 1 to 5, which is a multidomain type polypeptide having at least one immunoglobulin binding domain.
The polypeptide-modified liposome according to any one of claims 1 to 6, wherein the polypeptide has at least one immunoglobulin binding domain selected from the group consisting of (A1) to (A3).
Claims 1 to 7, wherein the anionic lipid is at least one phosphatidylglycerol selected from the group consisting of dipalmitoylphosphatidylglycerol, dioleoil phosphatidylglycerol, dimyristylphosphatidylglycerol, and disteroylphosphatidylglycerol. The polypeptide-modified liposome according to any one.
The polypeptide-modified liposome according to any one of claims 1 to 8, further containing a neutral lipid as a constituent lipid of the liposome.
The polypeptide-modified liposome according to any one of claims 1 to 9, wherein the molar ratio of the anionic lipid: the neutral lipid is 1: 99 to 99: 1.
An immunoliposome in which an antibody is bound to the polypeptide-modified liposome according to any one of claims 1 to 10.
A pharmaceutical composition comprising the immunoliposomes according to claim 11.