JP4983148B2 - Glycan-containing albumin, method for producing the same, and use thereof - Google Patents

Description

  The present invention relates to a novel sugar chain-containing albumin protein in which a sugar chain is selectively added to a specific amino acid residue, a method for producing the same, and use thereof. More specifically, the present invention relates to a sugar chain-containing albumin protein in which a sugar chain is selectively added to the partial amino acid sequence of a variant albumin containing a partial amino acid sequence that can undergo sugar chain modification by a host cell, The present invention relates to a DNA encoding mutant albumin, a method for producing the sugar chain-containing albumin protein by culturing eukaryotic cells containing the DNA, and use of the protein as a drug carrier.

  Human serum albumin (hereinafter sometimes referred to as “HSA”) is widely distributed in the body including blood and intercellular fluid. Its primary structure consists of 585 amino acids and is a simple protein with a molecular weight of about 66.5 kDa that has no sugar chain structure. This protein is made in the liver and is primarily responsible for maintaining normal osmotic pressure in the bloodstream and maintaining fluid content in the blood. For this reason, HSA is used in various clinical situations, for example, in the treatment of conditions where there is a loss of fluid from blood vessels, such as surgery, shock, burns, hypoproteinemia causing edema.

  In addition, HSA functions as a carrier for various serum molecules, and is rich in safety, biocompatibility, biodegradability, retention in blood, etc., and thus drug delivery systems for drugs with problems in kinetic properties It is positioned as a preferred carrier in (DDS).

  For DDS by irreversible binding of HSA and drug, there are a method of improving the blood retention of the drug bound by utilizing the long half-life of HSA, and a method of using a modified form of HSA as a carrier for an active transport system. is there. In the former, an attempt is made to express a protein having a short half-life or a physiologically active peptide as a hybrid by a gene fusion technique. On the other hand, in the latter, a method for adjusting the physicochemical properties of HSA such as anionization or cationization, or a recognition element (device) for a receptor existing on the cell surface such as a sugar structure or peptide is introduced. Therefore, attempts to achieve precise dynamic control and cell-specific targeting have been energetically studied (Non-Patent Documents 1 to 6).

  It is known that there are receptors that recognize sugar residues and negative charges in the liver. Utilizing this property, albumin combined with succinic acid, galactose, mannose and the like is used for targeting to the liver.

However, when chemically modifying HSA,
(1) It is not recognized by the liver unless so many sugar residues are attached;
Galactose-modified albumin is not recognized by the liver unless 10 or more galactoses are bound per albumin molecule (see Non-Patent Document 5).
(2) low cell specificity for liver non-parenchymal cells;
Mannose and fucose-modified albumin are known to be taken up by both hepatic endothelial cells and Kupffer cells (see Non-Patent Document 6).
(3) It is difficult to prepare a uniform conjugate, and it is necessary to find appropriate binding conditions;
Such problems are pointed out. Therefore, development of a method for modifying HSA using a non-chemical method has been strongly desired.

Lee YC et al., Biochemisty, 15: 3956-3963, 1976 Opanasopit P et al., Am. J. Physiol. Gastrointest. Liver Physiol. 280: 879-889, 2001 Takakura Y et al., Int. J. Pharm. 105: 19-29, 1994 Yamasaki Y et al., J. Pharmacol. Exp. Ther. 301: 467-477, 2002 Nishikawa M et al., Am. J. Physiol. Gastrointest. Liver Physiol. 268: G849-G856, 1995 Higuchi Y et al., Int. J. Pharm. 287: 147-154, 2004

  An object of the present invention is to provide uniform sugar chain-containing albumin, particularly serum albumin, that specifically migrates to the liver, particularly Kupffer cells, and thus to provide a drug carrier suitable for DDS into the liver. .

  As a result of intensive studies to achieve the above object, the inventors of the present invention converted the N-linked sugar chain consensus sequence (Asn-X-Thr / Ser) into DNA encoding HSA by site-directed mutagenesis. By culturing the introduced Pichia pastoris transformed with an expression vector containing the DNA encoding the mutant HSA thus obtained, a high mannose-type sugar chain having a high ability to migrate to the liver is converted into the consensus sequence. The present inventors have succeeded in producing a sugar chain-containing HSA added to an Asn residue, and completed the present invention.

That is, the present invention
[1] A sugar chain-containing albumin protein in which a sugar chain is selectively added to the partial amino acid sequence of mutant albumin containing one or more partial amino acid sequences capable of undergoing sugar chain modification by eukaryotic cells;
[2] The protein according to [1] above, wherein the sugar chain is a high-mannose sugar chain;
[3] The above-mentioned [1] or [2], wherein at least one of the partial amino acid sequences is Asn-Xaa-Thr or Asn-Xaa-Ser (Xaa represents any genetically encoded amino acid) protein;
[4] The protein according to [3] above, wherein all of the partial amino acid sequences are Asn-Xaa-Thr or Asn-Xaa-Ser (Xaa represents any genetically encoded amino acid);
[5] The protein according to any one of [1] to [4] above, wherein the albumin is human serum albumin;
[6] An amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 to 585 in the amino acid sequence represented by SEQ ID NO: 2, wherein the amino acid represented by amino acid number 63 is Asn and / or amino acid number The protein according to [5] above, wherein the amino acid represented by 320 is Thr or Ser and / or the amino acid represented by amino acid number 494 is Asn;
[7] The protein according to [6] above, wherein at least the amino acid represented by amino acid number 494 is Asn;
[8] DNA encoding mutant albumin comprising one or more partial amino acid sequences capable of undergoing sugar chain modification by eukaryotic cells;
[9] An expression vector comprising the DNA of [8] above, which is under the control of a promoter functional in a host eukaryotic cell;
[10] A transformant obtained by introducing the expression vector according to [9] above into a host eukaryotic cell;
[11] The transformant according to [10] above, wherein the host eukaryotic cell is yeast;
[12] The transformant according to [11] above, wherein the yeast is Pichia yeast;
[13] The protein according to [1], comprising culturing the transformant according to any one of [10] to [12] in a medium, and recovering the sugar chain-containing albumin from the obtained culture. Manufacturing method of
[14] A medicament comprising the protein according to any one of [1] to [7] above;
[15] A drug carrier for the liver comprising the protein according to any one of [1] to [7] above;
[16] A pharmaceutical composition comprising the carrier according to [15] above, wherein the target cell is a Kupffer cell; and [17] a pharmaceutical compound to be delivered to the liver and the carrier according to [15] or [16] above object;
I will provide a.

  Since the sugar chain-containing albumin of the present invention is specifically taken into the liver, particularly non-parenchymal cells of the liver, more specifically, Kupffer cells, it can be used as a drug carrier for the cells. For example, an excellent therapeutic effect can be expected by administering the glycosylated albumin of the present invention combined with an antioxidant or nitric oxide for hepatic ischemia / reperfusion injury. Furthermore, since even one sugar chain is strongly recognized by the liver, it can be used without affecting the original structure and function of albumin. In addition, since it is a recombinant protein, it can be safely used in the human body and the like without the risk of contamination with unknown viruses, which is a problem peculiar to blood-derived preparations.

  Examples of albumin in the present invention include serum albumin and ovalbumin, and serum albumin is preferred. The origin of albumin is not particularly limited. For example, humans or other warm-blooded animals (eg, cattle, monkeys, pigs, horses, sheep, goats, dogs, cats, rabbits, mice, rats, hamsters, guinea pigs, chickens, quails) From the viewpoint of utilization as a carrier for pharmaceuticals or pharmaceutical compounds, human albumin is preferable, and human serum albumin (HSA) is more preferable. Hereinafter, the present invention may be described in detail using HSA as an example. However, those skilled in the art will similarly produce sugar chain-containing albumin based on the description in this specification and other known albumin sequence information. And can be used.

  The sugar chain-containing albumin of the present invention is obtained by selectively adding a sugar chain to the partial amino acid sequence of mutant albumin containing one or more partial amino acid sequences that can undergo sugar chain modification by eukaryotic cells. Examples of the “partial amino acid sequence capable of undergoing sugar chain modification by a eukaryotic cell” (hereinafter also referred to as “glycosylation sequence”) include, for example, Asn-Xaa-Thr or Asn-Xaa which are consensus sequences of N-linked sugar chains. -Ser (Xaa is a genetically encoded amino acid. A sugar chain is added to the Asn residue) (hereinafter abbreviated as "Asn-Xaa-Thr / Ser") or O-linked type Cys-Xaa-Xaa-Gly-Gly-Thr / Ser which is a consensus sequence of O-linked fucose among sugar chains (Xaa is as defined above. A sugar chain is added to a Thr / Ser residue), O Examples include, but are not limited to, Cys-Xaa-Ser-Xaa-Pro-Cys (Xaa has the same meaning as described above. A sugar chain is added to the Ser residue) and the like. N-linked sugar chain consensus sequence Asn-Xaa-Thr / Ser is preferred. There may be one or more glycosylation sequences. The greater the number of sugar chains, the better the targeting efficiency to the liver, especially Kupffer cells, but considering the retention of the original physiological function and antigenicity of albumin, it is advantageous to add fewer sugar chains It is. As will be described later, the targeting function to the liver does not simply depend on the number of added sugar chains, but the position of addition is also important, so that the glycosylation sequence is located at a site that greatly contributes to targeting efficiency. By introducing, excellent targeting efficiency can be achieved with a small number of sugar chains.

  Since natural (wild-type) albumin is a simple protein, it does not have a partial amino acid sequence that can undergo sugar chain modification by eukaryotic cells. Therefore, the sugar chain-containing albumin of the present invention consists of a mutant amino acid sequence containing the glycosylation sequence. The mutant albumin polypeptide of the present invention may be obtained by any method, but in order to selectively add a sugar chain to the glycosylation sequence in the polypeptide, the DNA encoding the same It is preferably provided by culturing eukaryotic cells containing

  For example, eukaryotic cells containing DNA encoding mutant albumin may be treated naturally or artificially (for example, EMS or other mutagenesis agent treatment or UV treatment) on cells that endogenously produce albumin (eg hepatocytes). Etc.) It is also possible to obtain a mutant albumin containing a glycosylated sequence by inducing mutation, but more preferably, the DNA encoding albumin is cloned and genetically manipulated. A nucleotide sequence encoding a glycosylation sequence is introduced therein, and the obtained mutant DNA is inserted into an expression vector containing a promoter functional in an appropriate host eukaryotic cell so as to be under the control of the promoter, It can be produced by transforming the host eukaryotic cell with the obtained mutant albumin expression vector

  Examples of DNA encoding albumin include genomic DNA derived from humans or other warm-blooded animals, cDNA derived from albumin-producing cells (eg, hepatocytes), synthetic DNA, and the like. Genomic DNA and cDNA encoding albumin are prepared by using Polymerase Chain Reaction (hereinafter referred to as “PCR method”) using genomic DNA fraction and total RNA or mRNA fraction prepared from the production cells or tissues (for example, liver) as templates. And a reverse transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”). Alternatively, albumin-encoding genomic DNA and cDNA can be obtained from genomic DNA libraries and cDNA libraries prepared by inserting genomic DNA and total RNA or mRNA fragments prepared from the cells and tissues described above into appropriate vectors. They can also be cloned by colony or plaque hybridization method or PCR method, respectively. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.

  The DNA encoding albumin is, for example, a base sequence encoding the same or substantially the same amino acid sequence as the amino acid sequence (wild type mature HSA) represented by amino acid numbers 1 to 585 in the amino acid sequence represented by SEQ ID NO: 4 And the like containing DNA. “The amino acid sequence substantially the same as the amino acid sequence represented by amino acid numbers 1 to 585 in the amino acid sequence represented by SEQ ID NO: 4” refers to the amino acids represented by amino acid numbers 1 to 585 in the amino acid sequence represented by SEQ ID NO: 4 Examples thereof include amino acid sequences having a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more, particularly preferably about 98% or more. As used herein, “homology” refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably the algorithm uses a sequence of sequences for optimal alignment). The percentage of identical and similar amino acid residues relative to all overlapping amino acid residues in which one or both of the gaps can be considered). "Similar amino acids" means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn) ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).

  The homology of amino acid sequences in the present specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gap; matrix = BLOSUM62; filtering) = OFF). Other algorithms for determining amino acid sequence homology include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithms are NBLAST and XBLAST] Embedded in the program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [The algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [the algorithm is part of the CGC sequence alignment software package Embedded in the ALIGN program (version 2.0), Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [the algorithm is FASTA in the GCG software package. Program It includes built-in 'or the like, may they likewise preferably used.

  More preferably, the amino acid sequence substantially the same as the amino acid sequence represented by amino acid numbers 1 to 585 in the amino acid sequence represented by SEQ ID NO: 4 is represented by amino acid numbers 1 to 585 in the amino acid sequence represented by SEQ ID NO: 4. It is an amino acid sequence having about 80% or more, preferably about 90% or more, more preferably about 95% or more, particularly preferably about 98% or more identity with the amino acid sequence.

The protein containing the amino acid sequence substantially the same as the amino acid sequence represented by amino acid numbers 1 to 585 in the amino acid sequence represented by SEQ ID NO: 4 refers to the amino acid numbers 1 to 585 in the amino acid sequence represented by SEQ ID NO: 4 described above. A protein having substantially the same amino acid sequence as the amino acid sequence represented by (1) and having substantially the same activity as the protein comprising the amino acid sequence represented by amino acid numbers 1 to 585 in the amino acid sequence represented by SEQ ID NO: 4 means.
The substantially homogeneous activity includes the physiological function of albumin (particularly serum albumin), for example, the function as a carrier of serum molecules, the function of maintaining plasma colloid osmotic pressure, and the like. “Substantially the same quality” means that their functions are qualitatively the same. Accordingly, the functions of serum molecules as carriers are preferably equivalent, but quantitative factors such as the degree and molecular weight of proteins may be different.

The DNA encoding albumin includes, for example, (1) one or more of the amino acid sequences represented by amino acid numbers 1 to 585 in the amino acid sequence represented by SEQ ID NO: 4 (preferably about 1 to 30) More preferably, about 1 to 10, more preferably 1 to several (2, 3, 4 or 5) amino acids are deleted, (2) amino acid number 1 in the amino acid sequence shown in SEQ ID NO: 4 1 to 2 or more (preferably about 1 to 30, more preferably about 1 to 10, more preferably 1 to several (2, 3, 4 or 5) amino acids) in the amino acid sequence represented by -585 (3) 1 or more (preferably about 1 to 30, more preferably 1 to 10) in the amino acid sequence represented by amino acid numbers 1 to 585 in the amino acid sequence represented by SEQ ID NO: 4 Degree, more preferably An amino acid sequence in which 1 to several (2, 3, 4 or 5) amino acids are inserted; (4) 1 or 2 of amino acid sequences represented by amino acid numbers 1 to 585 in the amino acid sequence represented by SEQ ID NO: 4 An amino acid sequence in which one or more (preferably about 1 to 30, more preferably about 1 to 10, more preferably 1 to several (2, 3, 4 or 5)) amino acids are substituted with other amino acids, Or (5) DNA encoding a protein containing an amino acid sequence obtained by combining them.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited as long as the activity of the protein is maintained.

More preferably, the DNA encoding albumin (particularly HSA) is a DNA containing the base sequence represented by base numbers 73 to 1827 in the base sequence represented by SEQ ID NO: 3, or the base sequence represented by SEQ ID NO: 3. An activity substantially the same as a protein containing a base sequence that hybridizes under stringent conditions and comprising the amino acid sequence represented by amino acid numbers 1 to 585 in the amino acid sequence represented by SEQ ID NO: 4 (eg, serum) And a DNA encoding a protein having a molecular carrier function). Examples of DNA that can hybridize with the base sequence shown in SEQ ID NO: 3 under stringent conditions include, for example, a region overlapping with the base sequence shown in base numbers 73 to 1827 in the base sequence shown in SEQ ID NO: 3. A DNA containing a base sequence having a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more is used.
The homology of the nucleotide sequences in the present specification uses the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (expected value = 10; allow gap; filtering = ON; match) It can be calculated by score = 1; mismatch score = -3). As another algorithm for determining the homology of a base sequence, the above-mentioned algorithm for calculating homology of amino acid sequences is also preferably exemplified.

Hybridization should be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can do. When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can be preferably performed according to highly stringent conditions.
High stringent conditions include, for example, a hybridization reaction at 45 ° C. in 6 × SSC (sodium chloride / sodium citrate), followed by one or more washings at 65 ° C. in 0.2 × SSC / 0.1% SDS. Can be mentioned. A person skilled in the art may appropriately change the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the washing temperature, etc. Thus, the desired stringency can be easily adjusted.

  DNA encoding albumin (particularly HSA) is amplified by PCR using a synthetic DNA primer having a part of the base sequence encoding albumin, or DNA incorporated into an appropriate expression vector is used as a part of albumin or Cloning can be performed by hybridization with a DNA fragment encoding the entire region or a labeled synthetic DNA.

  As a method for introducing a nucleotide sequence encoding a glycosylation sequence into the DNA encoding albumin (especially HSA) obtained as described above, a site-directed mutagenesis method known per se (for example, see Examples below) ) Etc. The glycosylated sequence coding sequence may be introduced into any part of the DNA encoding albumin, but, for example, by site-directed mutagenesis using the PCR method, the consensus sequence Asn-Xaa- When a base sequence encoding Thr / Ser is introduced, it is preferably introduced into a portion encoding Asn residue of DNA encoding albumin or a portion encoding Thr or Ser residue. That is, using DNA encoding albumin as a template, (1) an oligonucleotide complementary to a region containing a base sequence encoding any Asn-Xaa1-Xaa2 portion in albumin (provided that the codon corresponding to Xaa2 is Thr or Substituted with a codon encoding Ser), or (2) an oligonucleotide complementary to a region containing a base sequence encoding any Xaa1-Xaa2-Thr / Ser part in albumin (corresponding to Xaa1) By performing PCR using one primer as a codon substituted with a codon encoding Asn, a base sequence encoding a consensus sequence of an N-linked sugar chain can be introduced. The glycosylation sequence is not only based on amino acid substitution as described above, but also by inserting a nucleotide sequence encoding amino acid (or amino acid sequence) into DNA encoding albumin, or using amino acid from the DNA using the same method. It can also be present in the DNA by deleting the base sequence encoding (or amino acid sequence).

For example, in the case of HSA, more preferably, the N-linked sugar chain consensus sequence is obtained by substituting the Asp residue represented by amino acid number 494 in the amino acid sequence represented by SEQ ID NO: 4 with an Asn residue (Asn ⁴⁹⁴ ). Introduced (see SEQ ID NO: 2). The sugar chain-containing HSA in which a sugar chain is added to Asn ⁴⁹⁴ has a sugar chain-containing albumin (having many sugar chains) obtained by a conventionally known chemical modification, even though the number of sugar chains in the molecule is one. ) Can be targeted to the liver with an efficiency equal to or greater than In another preferred embodiment, the N-linked sugar chain consensus sequence is obtained by substituting the Asp residue represented by amino acid number 63 in the amino acid sequence represented by SEQ ID NO: 4 with an Asn residue (Asn ⁶³ ), or It is introduced by replacing the Ala residue represented by amino acid number 320 with a Thr or Ser residue (Thr / Ser ³²⁰ ) (see SEQ ID NO: 2). In a particularly preferred embodiment, the sugar chain-containing HSA of the present invention can further contain one or more glycosylation sequences in addition to Asn ⁴⁹⁴ , preferably an N-linked sugar chain consensus sequence Asn-Xaa-Thr / Ser. . Additional glycosylation sites include Asn ³¹⁸ resulting from the above Asn ⁶³ and / or Thr / Ser ³²⁰ substitution.

Using the restriction enzyme and DNA ligase, a suitable expression vector for the DNA encoding mutant albumin containing one or more partial amino acid sequences that can be subjected to sugar chain modification by eukaryotic cells, cloned as described above. An expression vector containing the DNA can be produced by ligating downstream of the promoter.
Expression vectors include plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), λ phage, etc. Bacteriophages, retroviruses, vaccinia viruses, baculoviruses and other animal (insect) viruses, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like.
The promoter may be any promoter as long as it is appropriate for the host used for gene expression. The host cell in the present invention is not particularly limited as long as it has a sugar chain modification mechanism capable of adding a sugar chain to the glycosylation sequence contained in the mutant albumin of the present invention. And other eukaryotic cells such as animal cells, insect cells, plant cells, yeast cells, and fungal cells, transgenic animals and plants, and insects.
For example, when the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable.
When the host is an animal cell, a cytomegalovirus (CMV) -derived promoter (eg, CMV immediate early promoter), a human immunodeficiency virus (HIV) -derived promoter (eg, HIV LTR), a rous sarcoma virus (RSV) -derived promoter ( Example: RSV LTR), mouse mammary tumor virus (MMTV) derived promoter (example: MMTV LTR), Moloney murine leukemia virus (MoMLV) derived promoter (example: MMTV LTR), herpes simplex virus (HSV) derived promoter (example: HSV thymidine Kinase (TK) promoter), SV40 promoter (eg, SV40 early promoter), Epstein Barr virus (EBV) promoter, adeno-associated virus (AAV) promoter (eg, AAV p5 promoter), adenovirus (AdV) promoter (Ad2 or Ad5 major late promoter) is used .
When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 origin of replication, and the like can be used as desired. Examples of the selection marker include dihydrofolate reductase (dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance (Amp ^r ) gene, neomycin resistance (Neo ^r ) gene (G418 resistance), and the like. In particular, when dhfr gene-deficient Chinese hamster (CHO-dhfr ⁻ ) cells are used and the dhfr gene is used as a selection marker, the target gene can also be selected using a medium not containing thymidine. Further, when the inserted DNA does not contain a start codon and a stop codon, a start codon (ATG or GTG) and a stop codon (TAG, TGA, TAA) are included downstream of the promoter region and upstream of the terminator region, respectively. Vectors are preferably used.

  If necessary, a base sequence (signal codon) encoding a signal sequence suitable for the host may be added to the 5 'end of the DNA encoding mutant albumin. For example, when the host is yeast, MFα signal sequence, SUC2 signal sequence and the like are used, and when the host is animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence and the like are used. However, it is known that the native prepro sequence of HSA (amino acid sequence represented by amino acid numbers -24 to -1 in the amino acid sequence represented by SEQ ID NO: 4) functions as a secretion signal in most heterologous eukaryotic cells. Therefore, DNA encoding preproHSA can be inserted into an expression vector as it is.

As described above, for example, yeast, insect cells, insects, animal cells, animals, etc. are used as the host.
Examples of yeast include Saccharomyces cerevisiae ^{(Saccharomyces cerevisiae) AH22, AH22R -} , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe (Schizosaccharomyces pombe) NCYC1913, NCYC2036 and the like are used.

Insect cells include, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five ^TM cells derived from eggs of Trichoplusia ni , Cells derived from Mamestra brassicae, cells derived from Estigmena acrea, and the like are used. When the virus is BmNPV, insect-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used as insect cells. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217 (1977)).
Examples of insects include silkworm larvae.

Examples of animal cells include monkey-derived cells (eg, COS-1, COS-7, CV-1, Vero), hamster-derived cells (eg, BHK, CHO, CHO-K1, CHO-dhfr ⁻ ), mouse-derived cells. Cells (eg, NIH3T3, L, L929, CTLL-2, AtT-20), rat-derived cells (eg, H4IIE, PC-12, 3Y1, NBT-II), human-derived cells (eg, HEK293, A549, HeLa, HepG2, HL-60, Jurkat, U937) are used.

Transformation can be performed according to a known method depending on the type of host.
Yeast can be transformed according to the method described in, for example, Methods in Enzymology, 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).
Insect cells and insects can be transformed according to the method described in, for example, Bio / Technology, 6, 47-55 (1988).
Animal cells can be transformed, for example, according to the method described in Cell Engineering Supplement 8 New Cell Engineering Experimental Protocol, 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973).

The culture of the transformant can be performed according to a known method depending on the type of the host.
A liquid medium is preferable as the medium. Moreover, it is preferable that a culture medium contains a carbon source, a nitrogen source, an inorganic substance, etc. which are required for the growth of a transformant. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose; examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, Inorganic or organic substances such as potato extract; examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added to the medium. The pH of the medium is preferably about 5-8.
Examples of the medium for culturing a transformant whose host is yeast include a Burkholder minimum medium and an SD medium containing 0.5% casamino acid. The pH of the medium is preferably about 5-8. The culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours. Aeration and agitation may be performed as necessary.
As a medium for culturing a transformant whose host is an insect cell or an insect, for example, a medium obtained by appropriately adding an additive such as 10% bovine serum inactivated to Grace's Insect Medium is used. The pH of the medium is preferably about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days. You may perform ventilation | gas_flowing and stirring as needed.
As a medium for culturing a transformant whose host is an animal cell, for example, a minimum essential medium (MEM) containing about 5 to 20% fetal bovine serum, Dulbecco's modified Eagle medium (DMEM), RPMI1640 medium, 199 A medium or the like is used. The pH of the medium is preferably about 6-8. The culture is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours. You may perform ventilation | gas_flowing and stirring as needed.
As described above, sugar chain-containing albumin can be produced inside or outside the transformant.

  Since the sugar chain-containing albumin of the present invention can be preferably used as a carrier molecule that can specifically migrate to the liver, particularly Kupffer cells, a high-mannose sugar chain having a high affinity for the receptor on the cell surface is added. More preferred is. Here, “high mannose type” means that one or more, preferably 2, or more, more preferably 3 or more, particularly preferably 5 or more mannose molecules are further added to the core sugar chain (including 3 mannose molecules). It means the sugar chain made. From such a viewpoint, in addition to the high mannose type, in addition to the animal cell and insect cell that can be modified with different glycoforms of complex type and hybrid type, only the high mannose type sugar chain is added, and more than the animal cell etc. Yeast cells that can add hypermannose-type sugar chains containing a large amount of mannose molecules are more preferred as host cells. Among them, Pichia yeast can grow using methanol as the sole carbon source. When grown in methanol, enzymes required for the treatment of methanol and its metabolic intermediates are derepressed and expressed. To do. It is known that when this methanol assimilation pathway is used, the amount of secreted expression of heterologous proteins greatly exceeds that of Saccharomyces yeasts. In fact, HSA production using this system has already been put into practical use (see, for example, JP-A-6-22784), and 10 g HSA can be produced from 1 L of medium. Hereinafter, as one of particularly preferred embodiments of the present invention, a method for producing a sugar chain-containing albumin of the present invention using Pichia yeast as a host cell will be described.

  The vector to be used is not particularly limited as long as it is autonomously replicated in the cells of Pichia yeast or integrated into the yeast genome to be genetically stable. Examples of autonomously replicable vectors include YEp vectors, YRp vectors, YCp vectors, and the like. Examples of vectors that can be integrated into the yeast genome include YIp vectors and YRp vectors.

  Examples of promoters that can function in Pichia yeast include promoters derived from yeast, such as S. cerevisiae. Cerevisiae-derived PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. Examples include Pastoris-derived alcohol oxidase (AOX) 1 promoter, AOX 2 promoter, dihydroxyacetone synthase promoter, P40 promoter, ADH promoter, folate dehydrogenase promoter, and the like. In addition, the above yeast-derived promoter is modified so that the gene expression efficiency is further improved, for example, a mutant AOX2 (mAOX2) promoter [Ohi et al., Mol. Gen. Genet., 243, 489- 499 (1994); JP-A-4-99984]. Preferably, the promoter is a promoter of an enzyme gene required for treatment of methanol or a metabolic intermediate thereof, such as the AOX1 promoter and the mAOX2 promoter, in order to utilize the methanol metabolic system of Pichia yeast.

  The expression vector containing the DNA encoding the mutant albumin of the present invention further comprises a transcription termination sequence (terminator) (for example, AOX1 terminator, etc.) that can function in Pichia yeast, an enhancer sequence, and a selection marker gene that can be used for yeast selection (An auxotrophic gene such as HIS4, LEU2, ARG4, URA3 gene etc. derived from P. pastoris or S. cerevisiae, or antibiotic resistance gene such as cycloheximide, G-418, chloramphenicol, bleomycin, hygromycin And the like, and a replicable unit capable of functioning in yeast may be included if desired. Furthermore, for mass preparation of the vector, it preferably contains a replicable unit that can function in E. coli and a selectable marker gene (for example, a gene resistant to ampicillin or tetracycline) that can be used for selection of E. coli.

  When the expression vector is a type of vector that is integrated into the yeast genome, the vector preferably further contains a sequence homologous to the yeast genome required for homologous recombination. Examples of such homologous sequences include the sequences of the above-mentioned auxotrophic genes. Accordingly, in a preferred embodiment, the expression vector of the present invention comprises the above-mentioned mutant albumin expression cassette (herein, the “expression cassette” means a unit that enables gene expression in the auxotrophic gene, The one in which the protein coding sequence is placed under the control of the promoter is defined as the minimum unit, but is preferably a unit comprising a promoter-protein coding region-terminator).

  The expression vectors obtained as described above are, for example, competent cell method, protoplast method, calcium phosphate coprecipitation method, polyethylene glycol method, lithium method, electroporation method, microinjection method, liposome fusion method, particle gun It can be introduced into the target Pichia yeast using known transformation techniques such as the method.

The Pichia yeast used in the present invention is not particularly limited. Pastoris, Pichia acaciae, Pichia angusta, Pichia anomala, Pichia capsulata, Pichia ciferrii, Pichia hell, Pichia etcs Pichia fabianii, Pichia farinosa, Pichia guilliermondii, Pichia inositovora, Pichia jadinii, Pichia methanolica (Pichia methanolica) , Pichia norvegensis, Pichia ofunaensis, Pichia pinus and the like. Preferably, P.I. Pastoris, especially auxotrophic mutation Pastoris strain (e.g., P pastoris GTS115 strain ^{(HIS4 -.) [NNRL Y} -15851], P pastoris GS190 strain ^{(ARG4 -.) [NNRLY-} 1801], P pastoris ^{PPF1 (HIS4 -., URA4 -} ) [NNRL Y -18017]).

  The transformed Pichia yeast can produce sugar chain-containing albumin by culturing it by a method usually used in the art. The medium used should contain at least the carbon source and inorganic or organic nitrogen source necessary for the growth of the host cells. Examples of the carbon source include methanol, glycerol, glucose, sucrose, dextran, soluble starch and the like. Examples of inorganic or organic nitrogen sources include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, yeast extract, soybean meal, potato extract and the like. Furthermore, other nutrients, for example, inorganic salts such as calcium chloride, sodium dihydrogen phosphate and magnesium chloride, vitamins such as biotin, antibiotics and the like may be contained as desired.

  Examples of the medium to be used include a normal natural medium (for example, YPD medium, YPM medium, YPG medium, etc.) or a synthetic medium. The pH and the culture temperature of the medium are appropriately selected from pH and temperature suitable for yeast growth and albumin production. For example, a pH of about 5 to about 8 and a culture temperature of about 20 to about 30 ° C. are preferably exemplified. . Further, aeration and agitation can be performed as necessary. The culture is usually performed for about 48 to about 120 hours.

  For example, when a promoter that is induced by methanol, such as an AOX1 promoter or mAOX2 promoter, is used as a promoter that can function in Pichia yeast cells, glycerol is included as a carbon source for cell growth, and albumin expression induction The most preferred example is a method in which liquid aeration and agitation culture is performed using a natural medium containing methanol as a factor and controlled to a pH of about 6.0. When the expression of albumin is unfavorable for the growth of bacterial cells, a culture method in which the amount of bacterial cells is first increased with a carbon source other than methanol and then methanol is added to induce the expression of albumin is more preferable. Moreover, in the culture | cultivation by a jar fermenter, a high-density culture method is illustrated as a suitable method for production of albumin. The culture may be performed by any of batch culture, fed-batch culture, and continuous culture, and a fed-batch culture method is preferable. That is, for a certain period of time, the host cell is cultured in a medium (initial medium) containing a carbon energy source (for example, glucose) and / or a nutrient source suitable for its growth. A method is used in which albumin is not extracted out of the system until the end of the culture while a substrate (that is, methanol) that governs the growth of the cells is additionally supplied to the medium (see, for example, JP-A-3-83595). ).

  For albumin produced in the culture, the culture after completion of the culture is centrifuged and / or filtered to recover the culture supernatant (when secreted) or yeast cells (when expressed in the body). Then, it can be isolated and purified from the supernatant or the bacterial cells according to a method known per se. As such methods, methods utilizing the solubility such as salting out and solvent precipitation method; mainly using differences in molecular weight such as dialysis method, ultrafiltration method, gel filtration method, and SDS-polyacrylamide gel electrophoresis method. A method utilizing a difference in charge such as ion exchange chromatography; a method utilizing a specific affinity such as affinity chromatography; a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography; A method using a difference in isoelectric point, such as point electrophoresis, is used. These methods can be combined as appropriate.

  As a method for confirming the isolated and purified albumin containing sugar chain, a known Western blotting method and the like can be mentioned. The purified sugar chain-containing albumin can be clarified by amino acid analysis, N-terminal amino acid sequence, primary structure analysis, sugar chain analysis, and the like.

The sugar chain-containing albumin thus obtained is a uniform glycoprotein in which a sugar chain, preferably a high mannose sugar chain, is selectively added to the glycosylation sequence of mutant albumin. High migration to parenchymal cells, especially Kupffer cells. Therefore, the present invention also provides a drug carrier for the liver comprising the aforementioned sugar chain-containing albumin of the present invention.
It is prophylactically and / or therapeutically effective that the drug carrier of the present invention based on the sugar chain-containing albumin (especially HSA) of the present invention is delivered to the liver, preferably non-parenchymal cells of the liver, particularly Kupffer cells. Any pharmaceutical compound can be used to target the organ or cell. Examples of such pharmaceutical compounds include antioxidant substances (eg, N-acetylcysteine and ascorbic acid) and nitric oxide. The preparation in which the pharmaceutical compound is bound to the sugar chain-containing albumin of the present invention can be used for the treatment of hepatic ischemia-reperfusion injury. Examples of other pharmaceutical compounds include liver drugs such as the therapeutic drug for liver fibrosis OK432. Moreover, since albumin itself has an antioxidant effect, it can be used as it is as an antioxidant drug.

  The binding mode between the sugar chain-containing albumin and the pharmaceutical compound is not particularly limited, and examples thereof include a covalent bond, a hydrogen bond, and a hydrophobic bond, and preferably a covalent bond. A method for binding albumin and a pharmaceutical compound is well known, and for example, “Drug Delivery System” (1986, issued by CMC) can be referred to.

  The pharmaceutical compound-sugar chain-containing albumin conjugate can be formulated by a known method (ultrafiltration, sterilization filtration, dispensing, lyophilization, etc.). Specifically, a liquid preparation containing 5 to 25% of the conjugate, having a pH of about 6.4 to 7.4, and an osmotic pressure ratio of about 1 is exemplified. If necessary, the preparation may contain acetyltryptophan or a salt thereof (for example, sodium salt) and sodium caprylate as a stabilizer. Examples of the added amount of the stabilizer include 0.01 to 0.2M, preferably about 0.02 to 0.05M. The sodium content is exemplified by 3.7 mg / ml or less. The stabilizer is added before processing such as ultrafiltration, sterilization filtration, dispensing, and freeze-drying.

  Although it is considered that the medical preparation of the present invention obtained through the above steps is very unlikely to be contaminated with various microorganisms, aseptic means for ensuring the sterility of the preparation more positively You may inactivate the contaminating microorganisms by heat treatment (pastration) later.

  The heat treatment may be performed in a water bath of about 50 to about 70 ° C. (preferably about 60 ° C.), for example, regardless of the formulation filled in the container per dosage unit. By holding for 30 minutes or more, contaminating microorganisms are sufficiently inactivated. The heating time is preferably about 30 minutes to about 2 hours.

  The pharmaceutical preparation can be administered, for example, as an injection to humans or other mammals. The dosage of the preparation varies depending on the type of pharmaceutical compound, administration route, severity of disease, species of animal to be administered, drug acceptability of the subject to be administered, body weight, age, etc. In the case of a therapeutic agent for hepatic ischemia / reperfusion injury containing nitrogen, the daily amount of nitric oxide is usually 0.1 to 30 μg / kg, preferably 0.5 to 3 μg / kg, and the amount of albumin containing sugar chain is about 0.1 to about 0.1 to It is in the range of 30 mg / kg, preferably 0.5-3 mg / kg, and this amount is contained in about 5 to about 10 ml of solution and slowly administered by intravenous injection or intravenous infusion.

  Albumin (especially HSA) is itself used as a medicine, for example, mainly for the purpose of rapid plasma increase during shock, supplementation of circulating blood volume, improvement of hypoproteinemia, maintenance of colloid osmotic pressure, and the like. Specifically, it is effective for albumin loss (burn, nephrotic syndrome, etc.) and hypoalbuminemia due to decreased albumin synthesis (cirrhosis, etc.), hemorrhagic shock, and the like. Therefore, the sugar chain-containing albumin of the present invention can also be used as a medicament for improving such diseases and conditions. Also in this case, it can be formulated as an injection similar to the above.

  The dosage of the albumin preparation varies depending on the administration route, the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, the body weight, the age, etc., but usually 20 to 25 ml of an HSA 25% solution once per adult (5 to 12.5 g as HSA) is slowly administered by intravenous injection or intravenous infusion.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Examples Production of glycan-containing albumin (1) Mutation of albumin gene For the preparation of Asn ^63- linked glycan-containing albumin using plasmid pPIC9 with a plasmid containing human serum albumin gene (hereinafter pPIC9-HSA) as a template SEQ ID NO: 5 (5'-GAGTCAGCTGAAAATTGTAACAAATCACTTCATACCC-3 ' ) D63N antisense primer of D63N sense primer SEQ ID NO: 6 (5'-GGGTATGAAGTGATTTGTTACAATTTTCAGCTGACTC-3' ), Asn 318 linked sugar chain-containing albumin prepared in SEQ ID NO: 7 ( 5'-GGATGTTTGCAAAAACTATACTGAGGCAAAGG-3 A320T antisense primer ') of A320T sense primer SEQ ID NO: 8 (5'-CCTTTGCCTCAGTATAGTTTTTGCAAACATCC-3' ), Asn 494 linked sugar chain-containing albumin prepared in SEQ ID NO: 9 (5'-GCTCTGGAAGTCAATGAAACATACGTTCCC -3 ') and D494N antisense primer of SEQ ID NO: 10 (5'-GGGAACGTATGTTTCATTGACTTCCAGAGC-3') As a primer, N-linked sugar chain mutation (QuikChange XL Site-Directed Mutagenesis Kit, Stratagene) was performed. Mutation reaction conditions include treatment of DNA at 95 ° C for 30 seconds, followed by 12 cycles of denaturation (95 ° C, 30 seconds), annealing (55 ° C, 1 minute), and extension (68 ° C, 10 minutes). It was. After the reaction, the template plasmid was digested with Dpn I, and the resulting pPIC9-HSA (D63N), pPIC9-HSA (A320T) and pPIC9-HSA (D494N) were introduced into XL-10-Gold ultracompetent cells, respectively. Made a conversion. Transformants introduced with the target plasmids pPIC9-HSA (D63N), pPIC9-HSA (A320T) and pPIC9-HSA (D494N) were screened in an ampicillin-supplemented medium, and from the resulting transformants, plasmids Was purified (QIAprep Spin Miniprep Kit, manufactured by QIAGEN). For confirmation of mutation, pPIC9-HSA (D63N) is SEQ ID NO: 11 (5'-GAAAATTTCGACGCCTTGGTGTTGATTGCC-3 ') D63N sequence primer, and pPIC9-HSA (A320T) is SEQ ID NO: 12 (5'-GGCGGACCTTGCCGACTATATCTGTGA-3') The A320T sequencing primer of pPIC9-HSA (D494N) was subjected to ABI Prism 310 Genetic Analyzer (Applied Biosystems) using the D494N sequencing primer of SEQ ID NO: 13 (5′-GGTCTCAAGAAACCTAGGAAAAGTGGG-3 ′). In addition, human serum albumin in which sugar chains are bound to all three sites of Asn ⁶³ , Asn ³¹⁸ and Asn ⁴⁹⁴ was prepared using the A320T sense primer of SEQ ID NO: 7 using pPIC9-HSA (D63N) prepared above as a template. Using the A320T antisense primer of SEQ ID NO: 8 as a synthetic primer, mutation (QuikChange XL Site-Directed Mutagenesis Kit, Stratagene) of N-linked sugar chain was similarly performed. Using the prepared pPIC9-HSA (D63N / A320T) as a template, using the D494N sense primer of SEQ ID NO: 9 and the D494N antisense primer of SEQ ID NO: 10 as a synthetic primer, mutation (QuikChange XL Site-Directed Mutagenesis Kit) , Stratagene) to prepare pPIC9-HSA (D63N / A320T / D494N).

(2) Expression of sugar chain-containing human serum albumin
pPIC9-HSA (D63N), pPIC9-HSA (A320T), pPIC9-HSA (D494N) and pPIC9-HSA (D63N / A320T / D494N) are digested with restriction enzyme Sal I, phenol extracted, and purified by ethanol precipitation. Subsequently, transformation was performed by homologous recombination into the HIS4 locus of Pichia yeast (GS115 strain) using an electroporation apparatus (Gene Pulser II Electroporation System, manufactured by BIO-RAD). The obtained transformant was cultured in a BMMY liquid medium, and after confirming the expression of albumin, glycerol was stocked.

(3) Purification of sugar chain-containing albumin The transformed Pichia yeast was cultured in a BMGY liquid medium for 48 hours, and then cultured in BMMY medium for 96 hours while adding 1% methanol every 12 hours. After separating the yeast by centrifugation (6,000 g × 10 minutes), the culture supernatant was dialyzed against 200 mM acetate buffer. Thereafter, albumin was bound to a Blue Sepharose CL-6B column (manufactured by Amersham Biosciences), and albumin was eluted with a concentration gradient of 0 → 3 M NaCl. Thereafter, this eluate was dialyzed against 0.65 M ammonium sulfate / 100 mM sodium phosphate buffer (pH 7.0), and then passed through a HiTrap Phenyl HP column (manufactured by Amersham Biosciences) to collect the non-adsorbed fraction. Then, degreasing with activated carbon was performed.

Comparative Example 1 Production of sugar chain-free (wild-type) human serum albumin The same procedure as in Example was carried out except that the N-linked sugar chain was not changed to the consensus sequence. To obtain human serum albumin (HSA).

(4) Test example 1
Sugar chain-containing albumin prepared in the same manner as in Example was labeled with radioactive indium isotope ( ¹¹¹ In) to prepare ¹¹¹ In sugar chain-containing albumin (D63N, A320T, D494N and D63N / A320T / D494N). ¹¹¹ In sugar chain-containing albumin was administered via tail vein to mice (dose; 1 mg / kg), blood and liver were collected at regular intervals after administration, and albumin concentration and liver migration were measured with a radiation dose meter. It was measured. As a control, ¹¹¹ In human serum albumin obtained by labeling human serum albumin obtained in Comparative Example 1 with ¹¹¹ In was administered to mice and measured in the same manner. FIG. 1 shows the ratio of the glycan-containing albumin concentration in plasma and liver to the elapsed time and dose after administration, that is, the hepatic accumulation (% of dose).
From the results in FIG. 1, it was revealed that sugar chain-containing albumin, particularly D494N and D63N / A320T / D494N, disappeared rapidly from the blood and actively taken into the liver. In addition, since the pharmacokinetics of D63N, A320T, and D494N are significantly different, the transferability of sugar chain-containing albumin to the liver is not limited to the sugar density of the molecular surface that has been proposed so far. It was suggested that it also depends heavily.

(5) Test example 2
The charge state of ¹¹¹ In sugar chain-containing albumin (D63N, A320T, D494N and D63N / A320T / D494N) of Example and human serum albumin of Comparative Example 1 was evaluated using a laser electrophoresis-zeta potential analyzer (LEZA-500T). did. As shown in Table 1, no significant difference in charge was observed in all the mutants produced this time, compared to albumin containing no sugar chain (HSA). Therefore, albumin that has undergone sugar chain modification by eukaryotic cells has less difference in protein charge compared to those that have not undergone sugar chain modification, and sufficiently retains the properties of the original protein. It can be said.
On the other hand, when the transferability to the liver (Hepatic accumulation (% of dose)) at 60 minutes is read from FIG. 1 (Table 1), glycan-containing albumin is 6 to 65 in comparison with glycan-free albumin (HSA). Doubled. This indicates that the ability to migrate to the liver was enhanced while maintaining the properties of albumin protein.

Comparative Example 2
Chemically modified albumin was compared with reference to the figure presented in Non-Patent Document 3 (FIG. 2, Table 1). Table 1 presents the values read from FIG. The chemical modification is due to succinic acid (Suc) modification (imido bond with ε-amino group of Lys residue of bovine serum albumin (BSA)), and “Suc _n -BSA” is BSA to which n succinic acids are bound. It represents that.
So far, experiments using albumin (BSA) by chemical modification have shown that the negative charge density on the surface of the modified molecule is important for the transferability to the liver. It has been said that the higher the level of recognition by the liver (see Non-Patent Document 3). However, albumin modified with as many as 20 succinic acid molecules can be gradually imparted to the liver.
On the other hand, unmodified BSA has a charge of about −0.35, whereas succinic acid-modified BSA has a value of −0.5 or more. It is thought that it has a great influence on the structure and function of.

  The sugar chain-containing albumin of the present invention can be used as a drug carrier for DDS targeting liver non-parenchymal cells, particularly Kupffer cells. In addition, since the recombinant protein and the host sugar chain modification mechanism are used, it can be produced as a uniform protein compared to the chemical modification method, and the modification operation can be omitted. Furthermore, since there is no risk of contamination with viruses or the like, it can be safely administered to a living body for medical purposes.

4 is a graph showing the change over time in the transferability of the sugar chain-containing human serum albumin of the present invention in Test Example 1 to plasma (upper) and liver (lower). The vertical axis represents the ratio (%) to the dose, and the horizontal axis represents the time (minutes) after administration. The graph which shows the transfer property to the plasma (upper), a liver (middle), and a kidney (lower) when the ¹¹¹ In labeled body of a succinic acid modification (Suc-) bovine serum albumin (BSA) is intravenously administered to a mouse | mouth. Yes (see Non-Patent Document 3). _{N in} Suc _n -BSA represents the number of succinic acid bound to BSA, ● represents 0.1 mg / kg, ○ represents 1 mg / kg, ▼ represents 10 mg / kg, and ▽ represents 20 mg / kg BSA dose. The presented data is obtained by extracting a necessary part from Non-Patent Document 3 and adding a vertical axis.

Claims (10)

Glycan-containing serum albumin in which a high mannose sugar chain is selectively added by yeast cells to the partial amino acid sequence of mutant serum albumin containing one or more partial amino acid sequences capable of undergoing sugar chain modification by eukaryotic cells A drug carrier for a liver containing a protein, wherein at least one of the partial amino acid sequences is Asn-Xaa-Thr or Asn-Xaa-Ser (Xaa represents any genetically encoded amino acid) There is a drug carrier. Mutant serum albumin comprises two or more partial amino acid sequences capable of undergoing sugar chain modification by eukaryotic cells, according to claim 1 Symbol placement of the carrier. The carrier according to claim 2 , wherein all of the partial amino acid sequences are Asn-Xaa-Thr or Asn-Xaa-Ser (Xaa represents any genetically encoded amino acid). The carrier according to any one of claims 1 to 3 , wherein the serum albumin is human serum albumin. Human serum albumin, an amino acid sequence represented by amino acid sequence SEQ ID NO: 1 to 585 shown in SEQ ID NO: 2, amino acids represented by amino acid number 63 is represented by Asn and / or amino acid number 320 is Thr Or the carrier of Claim 4 which has an amino acid sequence whose amino acid shown by Ser and / or amino acid number 494 is Asn. The carrier according to claim 5 , wherein at least the amino acid represented by amino acid number 494 is Asn. The carrier according to claim 5 , wherein the amino acid represented by amino acid number 63 is Asn and the amino acid represented by amino acid number 320 is Thr or Ser and the amino acid represented by amino acid number 494 is Asn. The carrier according to any one of claims 1 to 7 , wherein the yeast is Pichia yeast. The carrier according to any one of claims 1 to 8 , wherein the target cell is a Kupffer cell. Pharmaceutical compound to be delivered to the liver and in the claims 1 to pharmaceutical compositions comprising the carrier according to any one of 9.

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