Lyophilized preparation

Abstract

The present invention relates to a lyophilized preparation containing, as an active ingredient, a fusion protein of an antibody and heparan N-sulfatase (SGSH), the lyophilized preparation further comprising an isotonic agent, a nonionic surfactant, and a buffering agent, wherein the nonionic surfactant contains polysorbate and poloxamer, and the buffering agent contains histidine.

Description

Lyophilized formulation

[0001] The present invention relates to a freeze-dried preparation containing a fusion protein of an antibody and heparan N-sulfatase (SGSH).

[0002] As a fusion protein of an antibody and heparan N-sulfatase (SGSH), for example, Patent Document 1 discloses a fusion protein comprising the amino acid sequence of an immunoglobulin heavy chain and SGSH. Furthermore, for example, Patent Document 2 discloses a protein as an enzyme for enzyme replacement therapy (ERT), comprising (a) a first Fc polypeptide linked to SGSH, and (b) a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide, wherein the first Fc polypeptide and / or the second Fc polypeptide does not contain an immunoglobulin heavy chain and / or light chain variable region sequence or an antigen-binding portion thereof.

[0003] Special table 2016-525545 publication Special table 2021-500857 publication

[0004] Regarding a fusion protein of an antibody and heparan N-sulfatase (SGSH), no pharmaceutical formulation with excellent storage stability is known. An object of the present invention is to provide a pharmaceutical formulation with excellent storage stability that contains a fusion protein of an antibody and heparan N-sulfatase (SGSH) as an active ingredient.

[0005] The present inventors have found that a lyophilized formulation containing a fusion protein of an antibody and heparan N-sulfatase (SGSH), an isotonicity agent, a nonionic surfactant, and a buffer, and further containing polysorbate and poloxamer as nonionic surfactants and histidine as a buffer, has excellent storage stability. The present invention is based on this finding and includes, for example, the following inventions.

[0006] [1] A freeze-dried preparation containing a fusion protein of an antibody and heparan N-sulfatase (SGSH) as an active ingredient, further containing an isotonicity agent, a nonionic surfactant, and a buffer, wherein the nonionic surfactant comprises polysorbate and poloxamer, and the buffer comprises histidine. [2] The freeze-dried preparation according to [1], wherein the nonionic surfactant is polysorbate 80 and polyoxyethylene (160) polyoxypropylene (30) glycol. [3] The freeze-dried preparation according to [1] or [2], wherein the buffer is L-histidine. [4] The freeze-dried preparation according to any of [1] to [3], wherein the isotonicity agent comprises at least one selected from a neutral salt and a disaccharide. [5] The freeze-dried preparation according to [4], wherein the neutral salt is sodium chloride, and the disaccharide is sucrose. [6] The freeze-dried formulation according to any one of [1] to [5], wherein the contents of the isotonic agent, the polysorbate, the poloxamer, and the buffering agent are 2.515 to 202.5 (w / w), 0.001 to 1.5 (w / w), 0.005 to 6 (w / w), and 0.05 to 60 (w / w), respectively, relative to the content of the fusion protein. [6-1] The freeze-dried formulation according to [4] or [5], wherein the contents of the neutral salt, the disaccharide, the polysorbate, the poloxamer, and the buffering agent are 0.015 to 2.5 (w / w), 2.5 to 200 (w / w), 0.001 to 1.5 (w / w), 0.005 to 6 (w / w), and 0.05 to 60 (w / w), respectively, relative to the content of the fusion protein. [7] The freeze-dried formulation according to any one of [1] to [6], wherein the contents of the isotonic agent, the polysorbate, the poloxamer, and the buffer are 5.05 to 50.5 (w / w), 0.005 to 0.05 (w / w), 0.02 to 0.2 (w / w), and 0.2 to 2 (w / w), respectively, relative to the content of the fusion protein.[7-1] The freeze-dried formulation according to [4] or [5], wherein the contents of the neutral salt, the disaccharide, the polysorbate, the poloxamer, and the buffering agent are 0.05 to 0.5 (w / w), 5 to 50 (w / w), 0.005 to 0.05 (w / w), 0.02 to 0.2 (w / w), and 0.2 to 2 (w / w), respectively, relative to the content of the fusion protein. [8] The freeze-dried formulation according to any of [1] to [7], wherein the contents of the isotonicity agent, the polysorbate, the poloxamer, and the buffering agent are 10.1 to 25.25 (w / w), 0.01 to 0.025 (w / w), 0.04 to 0.1 (w / w), and 0.4 to 1 (w / w), respectively, relative to the content of the fusion protein. [8-1] The freeze-dried formulation according to [4] or [5], wherein the contents of the neutral salt, the disaccharide, the polysorbate, the poloxamer, and the buffering agent are 0.1 to 0.25 (w / w), 10 to 25 (w / w), 0.01 to 0.025 (w / w), 0.04 to 0.1 (w / w), and 0.4 to 1 (w / w), respectively, relative to the content of the fusion protein. [9] The freeze-dried formulation according to any of [1] to [8], wherein the contents of the isotonicity agent, the polysorbate, the poloxamer, and the buffering agent are 15.16 (w / w), 0.015 (w / w), 0.065 (w / w), and 0.62 (w / w), respectively, relative to the content of the fusion protein. [9-1] The freeze-dried formulation according to [4] or [5], wherein the contents of the neutral salt, the disaccharide, the polysorbate, the poloxamer, and the buffer are 0.16 (w / w), 15 (w / w), 0.015 (w / w), 0.065 (w / w), and 0.62 (w / w), respectively, relative to the content of the fusion protein.

[10] The freeze-dried formulation according to any of [1] to [9], which has a pH of 5.0 to 5.7 when dissolved in pure water.

[11] The freeze-dried formulation according to any of [1] to

[10] , which has a pH of 5.2 to 5.7 when dissolved in pure water.

[12] The freeze-dried formulation according to any of [1] to

[11] , which has a pH of 5.4 when dissolved in pure water.

[13] The freeze-dried preparation according to any one of [1] to

[12] , wherein the fusion protein comprises the heparan N-sulfatase (SGSH) bound via a peptide bond to either the C-terminus or the N-terminus of either the light chain or the heavy chain of the antibody, directly or via a linker.

[14] The freeze-dried preparation according to any one of [1] to

[13] , wherein the fusion protein comprises the heparan N-sulfatase (SGSH) bound via a peptide bond to the C-terminus of the heavy chain of the antibody, directly or via a linker.

[15] The freeze-dried preparation according to any one of [1] to

[14] , wherein the fusion protein comprises the heparan N-sulfatase (SGSH) bound via a peptide bond to the C-terminus of the heavy chain of the antibody, via a linker.

[16] The freeze-dried preparation according to any one of

[13] to

[15] , wherein the linker comprises three consecutive amino acid sequences represented by SEQ ID NO: 3.

[17] The freeze-dried preparation according to any one of [1] to

[16] , wherein the heparan N-sulfatase (SGSH) is human heparan N-sulfatase (hSGSH).

[18] The freeze-dried preparation according to any one of [1] to

[17] , wherein the antibody is a human antibody or a humanized antibody.

[19] The freeze-dried preparation according to any one of [1] to

[18] , wherein the antibody is a Fab.

[20] The freeze-dried preparation according to any one of [1] to

[19] , wherein the antibody recognizes a molecule present on the surface of a vascular endothelial cell as an antigen.

[21] The freeze-dried preparation according to

[20] , wherein the vascular endothelial cell is a human vascular endothelial cell.

[22] The freeze-dried preparation according to

[20] or

[21] , wherein the vascular endothelial cell is a cerebrovascular endothelial cell.

[23] The freeze-dried preparation according to any one of

[20] to

[22] , wherein the molecule present on the surface of the cerebrovascular endothelial cells is selected from the group consisting of transferrin receptor (TfR), insulin receptor, leptin receptor, lipoprotein receptor, IGF receptor, OATP-F, organic anion transporter, MCT-8, and monocarboxylate transporter.

[24] The freeze-dried preparation according to any one of [1] to

[23] , wherein the antibody is a humanized anti-human transferrin receptor (hTfR) antibody.

[25] The freeze-dried preparation according to any of [1] to

[24] , wherein the antibody is a humanized anti-hTfR antibody and is a Fab, the heparan N-sulfatase (SGSH) is human heparan N-sulfatase (hSGSH), the light chain of the humanized anti-hTfR antibody has the amino acid sequence shown in SEQ ID NO: 2, and the heavy chain of the humanized anti-hTfR antibody is bound to the human heparan N-sulfatase (hSGSH) at its C-terminus via a linker comprising three consecutive amino acid sequences shown in SEQ ID NO: 3, thereby forming the amino acid sequence shown in SEQ ID NO: 1.

[26] The freeze-dried preparation according to any of [1] to

[25] , which is sealed in a container made of borosilicate glass or a hydrophobic resin.

[27] The freeze-dried preparation according to any one of [1] to

[26] , wherein the container is formed using a cycloolefin copolymer, a cycloolefin ring-opening polymer, or a hydrogenated cycloolefin ring-opening polymer.

[0007] The present invention provides a preparation that has excellent storage stability and contains, as an active ingredient, a fusion protein of an antibody and heparan N-sulfatase (SGSH). The freeze-dried preparation of the present invention suppresses the formation of aggregates, polymers, and degradation products over a period of 12 months or more, and is able to sufficiently maintain the affinity of the antibody for the antigen and the enzymatic activity of the heparan N-sulfatase.

[0008] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following describes in detail the embodiments of the present invention, but the present invention is not limited to the following embodiments.

[0009] The freeze-dried preparation of the present invention contains a fusion protein of an antibody and heparan N-sulfatase (SGSH) as an active ingredient. The freeze-dried preparation further contains an isotonicity agent, a nonionic surfactant, and a buffering agent, wherein the nonionic surfactant comprises polysorbate and poloxamer, and the buffering agent comprises histidine.

[0010] The antibody to be bound to heparan N-sulfatase is preferably a human antibody or a humanized antibody, but there are no particular limitations on the animal species of the antibody as long as it has the property of specifically binding to an antigen. For example, the antibody may be an antibody from a mammal other than human, or may be a chimeric antibody composed of a human antibody and an antibody from another mammal other than human.

[0011] A human antibody is an antibody that is entirely encoded by a gene of human origin. However, an antibody encoded by a gene in which a mutation has been added to an original human gene for the purpose of increasing gene expression efficiency, etc., is also a human antibody. An antibody in which two or more genes encoding a human antibody are combined and a part of one human antibody is replaced with a part of another human antibody is also a human antibody. The same applies to humanized antibodies, which will be described later.

[0012] In principle, a human antibody has three complementarity-determining regions (CDRs) in the variable region of the immunoglobulin light chain and three complementarity-determining regions (CDRs) in the variable region of the immunoglobulin heavy chain. The three CDRs in the immunoglobulin light chain are called CDR1, CDR2, and CDR3, starting from the N-terminus. The three CDRs in the immunoglobulin heavy chain are called CDR1, CDR2, and CDR3, starting from the N-terminus. An antibody in which the antigen specificity, affinity, etc. of a human antibody have been modified by replacing the CDR of one human antibody with the CDR of another human antibody is also a human antibody. The same applies to humanized antibodies, which will be described later.

[0013] The heavy and light chain variable regions of a human antibody each generally contain four framework regions 1 to 4 (FR1 to FR4). FR1 is the region adjacent to CDR1 on the N-terminus, and consists of the amino acid sequence from its N-terminus to the amino acid adjacent to the N-terminus of CDR1 in each peptide constituting the heavy chain and light chain. FR2 consists of the amino acid sequence between CDR1 and CDR2 in each peptide constituting the heavy chain and light chain. FR3 consists of the amino acid sequence between CDR2 and CDR3 in each peptide constituting the heavy chain and light chain. FR4 consists of the amino acid sequence from the amino acid adjacent to the C-terminus of CDR3 to the C-terminus of the variable region. However, this is not limited thereto, and in the present invention, the framework region can also be a region obtained by excluding 1 to 5 amino acids on the N-terminus and / or 1 to 5 amino acids on the C-terminus of each of the above FR regions. The same applies to the humanized antibodies described below.

[0014] In the present invention, antibodies obtained by modifying the genes of original human antibodies to add mutations such as substitutions, deletions, and additions to the amino acid sequence of the original antibody are also referred to as human antibodies. When amino acids in the amino acid sequence of the original antibody are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. When amino acids in the amino acid sequence of the original antibody are deleted, the number of amino acids to be deleted is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. Furthermore, antibodies that have been subjected to mutations that combine these amino acid substitutions and deletions are also human antibodies. When amino acids are added, preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or the N-terminus or C-terminus of the original antibody. Antibodies that have been subjected to mutations that combine these amino acid additions, substitutions, and deletions are also human antibodies. The amino acid sequence of the mutated antibody preferably exhibits 80% or more sequence identity, more preferably 90% or more sequence identity, even more preferably 95% or more sequence identity, and even more preferably 98% or more sequence identity, to the amino acid sequence of the original antibody. In other words, in the present invention, the term "human-derived gene" includes not only the original human-derived gene, but also a gene obtained by modifying the original human-derived gene. The same applies to the humanized antibody described below.

[0015] For example, the above rules apply when mutations are added to the light chain of the humanized anti-hTfR antibody shown in SEQ ID NO: 2 and the heavy chain (Fab heavy chain) of the humanized anti-hTfR antibody shown in SEQ ID NO: 4.

[0016] When mutations are introduced into a gene encoding all or part of the light chain variable region of an original human antibody, the gene after mutation preferably has 80% or more sequence identity, more preferably 90% or more sequence identity, with the original gene. However, there are no particular limitations on the sequence identity as long as the antibody after mutation has specific affinity for the antigen. When amino acids in the amino acid sequence of the light chain variable region are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. When amino acids in the amino acid sequence of the light chain variable region are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be introduced. When amino acids are added to the light chain variable region, preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence of the light chain variable region or to the N-terminus or C-terminus thereof. Mutations combining these amino acid additions, substitutions, and deletions can also be added. The amino acid sequence of the mutated light chain variable region preferably exhibits 80% or more sequence identity, more preferably 90% or more sequence identity, and even more preferably 95% or more sequence identity, with the amino acid sequence of the original light chain variable region. In particular, when amino acids in the CDR amino acid sequence are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. When amino acids in the CDR amino acid sequence are deleted, the number of amino acids to be deleted is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. Mutations combining these amino acid substitutions and deletions can also be added. When amino acids are added, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added.The amino acid sequence of each mutated CDR preferably has 80% or more sequence identity, more preferably 90% or more sequence identity, and even more preferably 95% or more sequence identity with the amino acid sequence of the original CDR. The same applies to the humanized antibody described below.

[0017] For example, when mutations are added to the variable region of the light chain of the humanized anti-hTfR antibody shown in SEQ ID NO: 6, the above rules are applied.

[0018] The light chain variable region shown in SEQ ID NO: 6 comprises the amino acid sequence of SEQ ID NO: 8 or 9 in CDR1, the amino acid sequence of SEQ ID NO: 10 or 11 in CDR2, and the amino acid sequence of SEQ ID NO: 12 in CDR3. When mutations are made to these CDRs, the above rules apply.

[0019] When mutations are introduced into a gene encoding all or part of the heavy chain variable region of an original human antibody, the gene after mutation preferably has 80% or more sequence identity, more preferably 90% or more sequence identity, with the original gene. However, there are no particular limitations on the sequence identity, as long as the antibody after mutation has specific affinity for the antigen. When amino acids in the amino acid sequence of the heavy chain variable region are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. When amino acids in the amino acid sequence of the heavy chain variable region are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be introduced. When amino acids are added to the heavy chain variable region, preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence of the heavy chain variable region or to the N-terminus or C-terminus thereof. Mutations combining these amino acid additions, substitutions, and deletions can also be added. The amino acid sequence of the mutated heavy chain variable region preferably exhibits 80% or more sequence identity, more preferably 90% or more sequence identity, and even more preferably 95% or more sequence identity, with the amino acid sequence of the original heavy chain variable region. In particular, when amino acids in the CDR amino acid sequence are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. When amino acids in the CDR amino acid sequence are deleted, the number of amino acids to be deleted is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. Mutations combining these amino acid substitutions and deletions can also be added. When amino acids are added, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus. Mutations that combine addition, substitution, and deletion of these amino acids can also be added.The amino acid sequence of each mutated CDR preferably has 80% or more sequence identity, more preferably 90% or more sequence identity, and even more preferably 95% or more sequence identity with the amino acid sequence of the original CDR. The same applies to the humanized antibody described below.

[0020] For example, when mutations are introduced into the heavy chain variable region of the humanized anti-hTfR antibody shown in SEQ ID NO:5, the above rules are applied.

[0021] The heavy chain variable region shown in SEQ ID NO: 5 comprises the amino acid sequence of SEQ ID NO: 17 or 18 in CDR1, the amino acid sequence of SEQ ID NO: 13 or 14 in CDR2, and the amino acid sequence of SEQ ID NO: 15 or 16 in CDR3. When mutations are made to these CDRs, the above rules are applied.

[0022] Examples of substitutions of amino acids in an amino acid sequence with other amino acids include substitutions between amino acids in the same group, 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), and amino acids with small side chains (GIy, AIa, Ser, Thr, Met). Such substitutions with similar amino acids are predicted to not result in changes in the phenotype of the protein (i.e., conservative amino acid substitutions). Specific examples of conservative amino acid substitutions are well known in the art and have been described in various publications (see, for example, Bowie et al., Science, 247:1306-1310 (1990)).

[0023] As used herein, "sequence identity" refers to the percentage (%) of the total number of matching residues between two sequences, based on the total number (including gaps) of residues (bases or amino acid residues) in the region that is the subject of the alignment, in optimal alignment when two sequences (base sequences or amino acid sequences) are compared using a homology calculation algorithm. Comparing two sequences based on sequence identity expressed as such a percentage is well known in the technical field of the present invention and is easily understood by those skilled in the art.

[0024] Well-known homology calculation algorithms include BLAST (Altschul S. F. J. Mol. Biol. 215.403-10, (1990)), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA. 85.2444 (1988)), and the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2.482-9 (1981)). In addition, blastp, one of the BLAST programs provided on the Internet by the National Institutes of Health, is well-known as a means for calculating the sequence identity of two amino acid sequences.

[0025] In the present invention, the term "humanized antibody" refers to an antibody in which the amino acid sequence of a portion of the variable region (e.g., particularly all or part of the CDRs) is derived from a mammal other than human, and the remaining regions are derived from human. Examples of humanized antibodies include antibodies produced by replacing three complementarity-determining regions (CDRs) of an immunoglobulin light chain and three complementarity-determining regions (CDRs) of an immunoglobulin heavy chain that constitute a human antibody with CDRs from another mammal. The species of other mammal from which the CDRs to be grafted into appropriate positions in a human antibody are derived is not particularly limited as long as it is a mammal other than human, but is preferably a mouse, rat, rabbit, horse, or non-human primate, and more preferably a mouse or rat, such as a mouse.

[0026] In the present invention, the term "chimeric antibody" refers to an antibody formed by linking fragments of two or more different antibodies derived from two or more different species.

[0027] A chimeric antibody between a human antibody and an antibody from another mammal is an antibody in which parts of a human antibody are replaced with parts of an antibody from a mammal other than human. The antibody consists of an Fc region, a Fab region, and a hinge region, as described below. Specific examples of such chimeric antibodies include chimeric antibodies in which the Fc region is derived from a human antibody and the Fab region is derived from an antibody from another mammal. The hinge region is derived from either a human antibody or an antibody from another mammal. Conversely, examples of chimeric antibodies include chimeric antibodies in which the Fc region is derived from another mammal and the Fab region is derived from a human antibody. The hinge region may be derived from either a human antibody or an antibody from another mammal.

[0028] Alternatively, an antibody can be said to be composed of a variable region and a constant region. Another specific example of a chimeric antibody is a chimeric antibody in which the constant region of the heavy chain (C Ｈ ) and the light chain constant region (C Ｌ ) is derived from a human antibody, while the variable region of the heavy chain (V Ｈ ) and the light chain variable region (V Ｌ ) derived from antibodies of other mammals, conversely, the heavy chain constant region (C Ｈ ) and the light chain constant region (C Ｌ ) are derived from antibodies of other mammals, while the variable region of the heavy chain (V Ｈ ) and the light chain variable region (V Ｌ Here, the other mammalian species is not particularly limited as long as it is a mammal other than a human, but is preferably a mouse, rat, rabbit, horse, or non-human primate, more preferably a mouse.

[0029] Chimeric antibodies of a human antibody and a mouse antibody are particularly called "human / mouse chimeric antibodies." Examples of human / mouse chimeric antibodies include chimeric antibodies in which the Fc region is derived from a human antibody and the Fab region is derived from a mouse antibody, and conversely, chimeric antibodies in which the Fc region is derived from a mouse antibody and the Fab region is derived from a human antibody. The hinge region is derived from either a human antibody or a mouse antibody. Another specific example of a human / mouse chimeric antibody is a chimeric antibody in which the heavy chain constant region (C Ｈ ) and the light chain constant region (C Ｌ ) is derived from a human antibody, while the variable region of the heavy chain (VＨ ) and the light chain variable region (V Ｌ ) derived from a mouse antibody, and conversely, the heavy chain constant region (C Ｈ ) and the light chain constant region (C Ｌ ) is derived from a mouse antibody, while the variable region of the heavy chain (V Ｈ ) and the light chain variable region (V Ｌ ) derived from a human antibody.

[0030] An antibody originally has a basic structure consisting of four polypeptide chains: two immunoglobulin light chains and two immunoglobulin heavy chains. However, in the present invention, the term "antibody" also includes, in addition to those having this basic structure, (1) those consisting of two polypeptide chains: one immunoglobulin light chain and one immunoglobulin heavy chain; (2) single-chain antibodies consisting of an immunoglobulin light chain with a linker sequence attached to the C-terminus thereof, and (3) single-chain antibodies consisting of an immunoglobulin heavy chain with a linker sequence attached to the C-terminus thereof, and (4) single-chain antibodies consisting of an Fab region in which the Fc region has been deleted from the basic structure of an antibody in the true sense, and those consisting of an Fab region and all or part of a hinge region (Fab, F(ab') and F(ab')). ２ Furthermore, scFv, which is a single-chain antibody formed by linking the light chain variable region and the heavy chain variable region via a linker sequence, is also included in the "antibody" of the present invention.

[0031] Here, Fab refers to a region consisting of a variable region and a C Ｌ one light chain containing a variable region and a C region (light chain constant region) Ｈ Fab is a molecule in which one heavy chain containing one region (part 1 of the constant region of the heavy chain) is bound by disulfide bonds between cysteine ​​residues present in each heavy chain. ＨIn addition to the region 1 (part 1 of the heavy chain constant region), a part of the hinge region may also be included, but in this case the hinge region lacks the cysteine ​​residues present in the hinge region that bind the heavy chains of the antibody. Ｌ The cysteine ​​residues present in the heavy chain constant region (C Ｈ The heavy chains that make up Fab are called Fab heavy chains. Fab lacks the cysteine ​​residues that bind the heavy chains of antibodies in the hinge region, so it consists of one light chain and one heavy chain. The light chains that make up Fab are composed of a variable region and a C Ｌ The heavy chain that constitutes Fab contains a variable region and a C Ｈ It may consist of one region, a variable region, C Ｈ In this case, the hinge region is selected so as not to contain a cysteine ​​residue that connects the heavy chains, so that disulfide bonds are not formed between the two heavy chains at the hinge region. In F(ab'), the heavy chain consists of a variable region and a C Ｈ In addition to the F(ab') region, it also contains all or part of the hinge region containing the cysteine ​​residues that link the heavy chains. ２ refers to a molecule in which two F(ab)s are linked by disulfide bonds between cysteine ​​residues in the hinge region of each other. F(ab') or F(ab') ２A heavy chain that forms the Fab' heavy chain is called a Fab' heavy chain. Furthermore, polymers such as dimers and trimers formed by linking multiple antibodies directly or via a linker are also antibodies. Furthermore, without being limited to these, any entity that contains a portion of an immunoglobulin molecule and has the property of specifically binding to an antigen is included in the "antibody" referred to in the present invention. That is, in the present invention, the term "immunoglobulin light chain" includes those derived from an immunoglobulin light chain and having all or part of the amino acid sequence of its variable region. Furthermore, the term "immunoglobulin heavy chain" includes those derived from an immunoglobulin heavy chain and having all or part of the amino acid sequence of its variable region. Therefore, as long as it has all or part of the amino acid sequence of the variable region, even those lacking, for example, the Fc region, are immunoglobulin heavy chains.

[0032] Here, Fc or Fc region refers to the C region in an antibody molecule. Ｈ 2 region (part 2 of the constant region of the heavy chain), and C Ｈ It refers to a region containing a fragment consisting of three regions (part 3 of the heavy chain constant region).

[0033] Furthermore, in the present invention, the term "antibody" refers to (5) the Fab, F(ab') or F(ab') shown in (4) above. ２ The light chain and heavy chain constituting the above are linked via a linker sequence to form single-chain antibodies, namely scFab, scF(ab'), and scF(ab'). ２ Also included are scFab, scF(ab'), and scF(ab'). ２ In the case of scFv, a linker sequence may be attached to the C-terminus of the light chain, and a heavy chain may be attached to the C-terminus of that, or a linker sequence may be attached to the C-terminus of the heavy chain, and a light chain may be attached to the C-terminus of that. Furthermore, the antibodies of the present invention also include scFvs, which are single-chain antibodies formed by linking the light chain variable region and the heavy chain variable region via a linker sequence. In the case of scFv, a linker sequence may be attached to the C-terminus of the light chain variable region, and a heavy chain variable region may be attached to the C-terminus of that, or a linker sequence may be attached to the C-terminus of the heavy chain variable region, and a light chain variable region may be attached to the C-terminus of that.

[0034] In the present invention, the term "single-chain antibody" refers to a protein that comprises an amino acid sequence comprising all or part of the variable region of an immunoglobulin light chain, to which a linker sequence is attached at the C-terminus, and to which an amino acid sequence comprising all or part of the variable region of an immunoglobulin heavy chain is further attached at the C-terminus thereof, and that is capable of specifically binding to a specific antigen. For example, the antibodies described in (2), (3), and (5) above are included in single-chain antibodies. Furthermore, a protein that comprises an amino acid sequence comprising all or part of the variable region of an immunoglobulin heavy chain, to which a linker sequence is attached at the C-terminus thereof, and to which an amino acid sequence comprising all or part of the variable region of an immunoglobulin light chain is further attached at the C-terminus thereof, and that is capable of specifically binding to a specific antigen is also a "single-chain antibody" in the present invention. In single-chain antibodies in which an immunoglobulin light chain is attached at the C-terminus of the immunoglobulin heavy chain via a linker sequence, the immunoglobulin heavy chain usually lacks an Fc region. The variable region of the immunoglobulin light chain has three complementarity-determining regions (CDRs) that are involved in the antigen specificity of the antibody. Similarly, the variable region of an immunoglobulin heavy chain also has three CDRs. These CDRs are the main regions that determine the antigen specificity of an antibody. Therefore, a single-chain antibody preferably contains all three CDRs of an immunoglobulin heavy chain and all three CDRs of an immunoglobulin light chain. However, as long as the antigen-specific affinity of the antibody is maintained, a single-chain antibody can also be obtained by deleting one or more CDRs.

[0035] In a single-chain antibody, the linker sequence disposed between the light and heavy chains of an immunoglobulin is a peptide chain composed of preferably 2 to 50, more preferably 8 to 50, even more preferably 10 to 30, and even more preferably 12 to 18 or 15 to 25, for example, 15 or 25 amino acid residues. The amino acid sequence of such a linker sequence is not limited as long as the antibody formed by linking both chains thereby retains affinity for the antigen. Preferably, the linker sequence is composed of only glycine or glycine and serine, and examples thereof include the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly-Gly, the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 19), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 20), or sequences in which these amino acid sequences are repeated 2 to 10 times, or 2 to 5 times. For example, when an scFv is prepared by linking an immunoglobulin light chain variable region via a linker sequence to the C-terminus of an amino acid sequence consisting of the entire region of an immunoglobulin heavy chain variable region, a preferred linker sequence is one consisting of a total of 15 amino acids corresponding to three consecutive amino acids of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3).

[0036] In the present invention, antigens specifically recognized by antibodies are, for example, molecules (surface antigens) present on the surface of vascular endothelial cells. Examples of such surface antigens include, but are not limited to, transferrin receptors (TfR), insulin receptors, leptin receptors, lipoprotein receptors, IGF receptors, organic anion transporters such as OATP-F, monocarboxylic acid transporters such as MCT-8, and Fc receptors. The antigens are preferably these molecules (surface antigens) present on the surface of human vascular endothelial cells.

[0037] Among the above-mentioned surface antigens, transferrin receptor (TfR), insulin receptor, leptin receptor, lipoprotein receptor, IGF receptor, organic anion transporters such as OATP-F, and monocarboxylic acid transporters such as MCT-8 are present on the surface of brain capillary endothelial cells that form the blood-brain barrier. Antibodies that can recognize these antigens can bind to brain capillary endothelial cells (cerebral vascular endothelial cells) via the antigens. Antibodies that bind to brain capillary endothelial cells can then pass through the blood-brain barrier and reach the central nervous system. Therefore, by binding heparan N-sulfatase to such antibodies, it can pass through the blood-brain barrier and reach the central nervous system, thereby improving central nervous system disorders.

[0038] In the present invention, the term "human transferrin receptor" or "hTfR" refers to a membrane protein having the amino acid sequence shown in SEQ ID NO: 21. In one embodiment, the anti-hTfR antibody of the present invention specifically binds to the portion of the amino acid sequence shown in SEQ ID NO: 21 from the 89th cysteine ​​residue from the N-terminus to the C-terminal phenylalanine (extracellular domain of hTfR), but is not limited thereto.

[0039] A method for producing an antibody will be explained below, taking an antibody against hTfR as an example. A common method for producing an antibody against hTfR is to produce a recombinant human transferrin receptor (rhTfR) using cells transfected with an expression vector incorporating the hTfR gene, and then immunize an animal such as a mouse with this rhTfR to obtain the antibody. Antibody-producing cells against hTfR are extracted from the immunized animal, and hybridoma cells capable of producing antibodies against hTfR are then fused with myeloma cells.

[0040] Alternatively, cells producing antibodies against hTfR can be obtained by immunizing immune system cells obtained from animals such as mice with rhTfR by ex vivo immunization. When immunization is performed by ex vivo immunization, the animal species from which the immune system cells are derived is not particularly limited; however, preferred are mice, rats, rabbits, guinea pigs, dogs, cats, horses, and primates including humans, more preferably mice, rats, and humans, and even more preferably mice and humans. Examples of mouse immune system cells that can be used include spleen cells prepared from mouse spleens. Examples of human immune system cells that can be used include cells prepared from human peripheral blood, bone marrow, spleens, etc. When human immune system cells are immunized by ex vivo immunization, human antibodies against hTfR can be obtained.

[0041] When the antibody specifically recognizes a molecule (surface antigen) present on the surface of vascular endothelial cells, heparan N-sulfatase (SGSH) bound to the antibody can be used as a therapeutic agent for central nervous system disorders in Sanfilippo syndrome.

[0042] Sanfilippo syndrome, also known as mucopolysaccharidosis type III (MPS type III), is a disease caused by the accumulation of intracellular heparan sulfate associated with a deficiency in intralysosomal heparan N-sulfatase activity. However, deficiencies of other enzymes, such as α-N-acetylglucosaminidase, can also be a cause of the disease. Patients with Sanfilippo syndrome may also suffer from central nervous system disorders. Therefore, the fusion protein of this antibody and hSGSH can cross the BBB and degrade heparan sulfate accumulated in brain tissue, and therefore can be administered to patients with Sanfilippo syndrome who suffer from central nervous system disorders and used as a therapeutic agent for central nervous system disorders. Furthermore, it can also be administered prophylactically to patients with Sanfilippo syndrome who do not exhibit central nervous system disorders.

[0043] As used herein, the terms "human heparan N-sulfatase (SGSH)," "human SGSH," or "hSGSH" particularly refer to hSGSH having the same amino acid sequence as wild-type hSGSH. Wild-type hSGSH has an amino acid sequence consisting of 482 amino acids as set forth in SEQ ID NO: 7. However, without being limited thereto, hSGSH also includes mutations such as substitutions, deletions, and additions in the amino acid sequence of wild-type hSGSH, as long as they have SGSH activity. When amino acids in the amino acid sequence of hSGSH are substituted with other amino acids, the number of amino acids to be substituted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. When amino acids in the amino acid sequence of hl2S are deleted, the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. Mutations that combine these amino acid substitutions and deletions can also be made. When amino acids are added to hSGSH, preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2 amino acids are added within the amino acid sequence of hSGSH or to the N-terminus or C-terminus. Mutations that combine these amino acid additions, substitutions, and deletions can also be made. The amino acid sequence of the mutated hSGSH preferably exhibits 80% or more sequence identity, more preferably 90% or more sequence identity, even more preferably 95% or more sequence identity, and even more preferably 98% or more sequence identity with the amino acid sequence of the original hSGSH.

[0044] In this specification, when hSGSH is said to have SGSH activity, it means that when hSGSH is fused with an antibody to form a fusion protein, the fusion protein has 3% or more of the activity inherent in natural hSGSH. However, the activity is preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more of the activity inherent in natural hSGSH. The same applies when the hSGSH fused with the antibody is a mutated one. The antibody is, for example, an anti-hTfR antibody.

[0045] In the present invention, the term "fusion protein" refers to a substance in which an antibody and heparan N-sulfatase are linked via a non-peptide linker, a peptide linker, or directly. Methods for linking an antibody and heparan N-sulfatase are described in detail below.

[0046] Methods for linking an antibody to heparan N-sulfatase include linking via a non-peptide linker or a peptide linker. Examples of non-peptide linkers that can be used include biotin-streptavidin, polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitins, and hyaluronic acid, as well as derivatives or combinations of these. A peptide linker is a peptide chain or derivative thereof consisting of 1 to 50 peptide-bonded amino acids, and its N-terminus and C-terminus form covalent bonds with either the antibody or heparan N-sulfatase, respectively, thereby linking the antibody to heparan N-sulfatase.

[0047] When biotin-streptavidin is used as the non-peptide linker, the antibody may be bound to biotin, the heparan N-sulfatase may be bound to streptavidin, and the antibody and heparan N-sulfatase may be bound via the bond between the biotin and streptavidin, or conversely, the antibody may be bound to streptavidin, the heparan N-sulfatase may be bound to biotin, and the antibody and heparan N-sulfatase may be bound via the bond between the biotin and streptavidin. Biotin and streptavidin can be bound to proteins by well-known techniques.

[0048] The antibody of the present invention conjugated to heparan N-sulfatase using PEG as a non-peptide linker is particularly referred to as antibody-PEG-SGSH. Antibody-PEG-SGSH can be produced by conjugating an antibody to PEG to prepare antibody-PEG, and then conjugating the antibody-PEG to heparan N-sulfatase. Alternatively, antibody-PEG-SGSH can be produced by conjugating heparan N-sulfatase to PEG to prepare SGSH-PEG, and then conjugating the SGSH-PEG to the antibody. When conjugating PEG to an antibody and heparan N-sulfatase, PEG modified with a functional group such as carbonate, carbonylimidazole, active ester of carboxylic acid, azlactone, cyclic imidothione, isocyanate, isothiocyanate, imidate, or aldehyde is used. The functional groups introduced into the PEG react primarily with amino groups in the antibody and heparan N-sulfatase molecules, thereby covalently bonding the PEG to the antibody and heparan N-sulfatase. The molecular weight and shape of the PEG used are not particularly limited, but its average molecular weight (MW) is preferably 300 to 60,000, and more preferably 500 to 20,000. For example, PEGs with average molecular weights of about 300, about 500, about 1,000, about 2,000, about 4,000, about 10,000, or about 20,000 can be suitably used as non-peptide linkers.

[0049] For example, antibody-PEG is prepared by mixing an antibody with polyethylene glycol (ALD-PEG-ALD) having an aldehyde group as a functional group so that the molar ratio of ALD-PEG-ALD to the antibody is 11, 12.5, 15, 110, 120, etc., and then adding NaCNBH ３ The antibody-PEG is then reacted with a reducing agent such as NaCNBH ３ Antibody-PEG-SGSH can be obtained by reacting the antibody with heparan N-sulfatase in the presence of a reducing agent such as PEG-ALD or PEG-ALD-PEG-ALD. Conversely, antibody-PEG-SGSH can also be obtained by first conjugating heparan N-sulfatase with ALD-PEG-ALD to prepare SGSH-PEG, and then conjugating the SGSH-PEG to the antibody.

[0050] The antibody and heparan N-sulfatase can also be linked by peptide bond to the N- or C-terminus of the antibody heavy or light chain, either directly or via a linker sequence. Fusion proteins in which an antibody and heparan N-sulfatase are linked in this manner can be obtained by incorporating a DNA fragment in which a cDNA encoding heparan N-sulfatase is located in-frame at the 3' or 5' end of the cDNA encoding the antibody heavy or light chain, either directly or via a DNA fragment encoding a linker sequence, into an expression vector for eukaryotes such as mammalian cells or yeast, and then culturing mammalian cells into which this expression vector has been introduced. When a DNA fragment encoding heparan N-sulfatase is linked to a heavy chain, an expression vector for mammalian cells incorporating a cDNA fragment encoding the antibody light chain is also introduced into the same mammalian host cell. When a DNA fragment encoding heparan N-sulfatase is linked to a light chain, an expression vector for mammalian cells incorporating a cDNA fragment encoding the antibody heavy chain is also introduced into the same host cell. When the antibody is a single-chain antibody, a fusion protein combining the antibody and heparan N-sulfatase can be obtained by incorporating a DNA fragment in which a cDNA encoding a single-chain antibody is linked to the 5'- or 3'-end of the cDNA encoding heparan N-sulfatase, either directly or via a DNA fragment encoding a linker sequence, into an expression vector for eukaryotes such as mammalian cells or yeast, and expressing the DNA fragment in the cells transfected with the expression vector.

[0051] A fusion protein in which heparan N-sulfatase is linked to the C-terminus of an antibody light chain comprises an antibody comprising an amino acid sequence containing all or part of the light chain variable region and an amino acid sequence containing all or part of the heavy chain variable region, and heparan N-sulfatase is linked to the C-terminus of the antibody light chain. The antibody light chain and heparan N-sulfatase may be linked directly or via a linker.

[0052] A fusion protein in which heparan N-sulfatase is linked to the C-terminus of an antibody heavy chain comprises an antibody comprising an amino acid sequence containing all or part of a light chain variable region and an amino acid sequence containing all or part of a heavy chain variable region, and heparan N-sulfatase linked to the C-terminus of the antibody heavy chain. The antibody heavy chain and heparan N-sulfatase may be linked directly or via a linker.

[0053] A fusion protein in which heparan N-sulfatase is bound to the N-terminus of an antibody light chain comprises an antibody comprising an amino acid sequence containing all or part of the light chain variable region and an amino acid sequence containing all or part of the heavy chain variable region, and heparan N-sulfatase is bound to the N-terminus of the antibody light chain. The antibody light chain and heparan N-sulfatase may be bound directly or via a linker.

[0054] A fusion protein in which heparan N-sulfatase is bound to the N-terminus of an antibody heavy chain comprises an antibody comprising an amino acid sequence containing all or part of a light chain variable region and an amino acid sequence containing all or part of a heavy chain variable region, and heparan N-sulfatase is bound to the N-terminus of the antibody heavy chain. The antibody heavy chain and heparan N-sulfatase may be bound directly or via a linker.

[0055] In this case, when a linker sequence is placed between the antibody and heparan N-sulfatase, the sequence preferably consists of 1 to 50 amino acids, more preferably 1 to 17, even more preferably 1 to 10, and even more preferably 1 to 5 amino acids. The number of amino acids constituting the linker sequence can be appropriately adjusted to 1, 2, 3, 1 to 17, 1 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, etc. Such a linker sequence is not limited to its amino acid sequence, as long as the antibody linked thereto retains its affinity for the antigen and the heparan N-sulfatase linked via the linker sequence can exhibit physiological activity under physiological conditions. However, a linker sequence consisting of glycine and serine is preferred, for example, a linker consisting of one amino acid of either glycine or serine, such as the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, or the amino acid sequence Gly The linker sequence may be a sequence of 1 to 50 amino acids, such as Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 19), or Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 20), or a sequence of 1 to 10 or 2 to 5 consecutive amino acids, or a sequence of 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, or 25 to 29 amino acids. For example, a sequence having the amino acid sequence Gly-Ser can be suitably used as the linker sequence. The same applies to a single-chain antibody.

[0056] In the present invention, when one peptide chain contains multiple linker sequences, for convenience, the linker sequences are named, in order from the N-terminus, as the first linker sequence, the second linker sequence, and so on.

[0057] When the antibody is a humanized antibody and an anti-human transferrin receptor antibody, a preferred form of the antibody is an antibody comprising a light chain having the amino acid sequence shown in SEQ ID NO:2 and a heavy chain having the amino acid sequence shown in SEQ ID NO:4.

[0058] However, when the antibody is a humanized antibody and an anti-human transferrin receptor antibody, the preferred form of the antibody is not limited to the above. For example, an antibody whose light chain amino acid sequence has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and whose heavy chain amino acid sequence has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4 can also be used as the antibody of the present invention, as long as it has affinity for hTfR. Furthermore, an antibody whose light chain amino acid sequence has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and whose heavy chain amino acid sequence has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4 can also be used as the antibody of the present invention, as long as it has affinity for hTfR. Furthermore, an antibody whose light chain amino acid sequence has 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and whose heavy chain amino acid sequence has 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4 can also be used as the antibody of the present invention, as long as it has affinity for hTfR.

[0059] In addition, antibodies whose light chain amino acid sequence is the amino acid sequence shown in SEQ ID NO: 2 with 1 to 10 substitutions, deletions, or additions and / or whose heavy chain amino acid sequence is the amino acid sequence shown in SEQ ID NO: 4 with 1 to 10 substitutions, deletions, or additions can also be used as antibodies in the present invention, so long as they have affinity for hTfR. In addition, antibodies whose light chain amino acid sequence is the amino acid sequence shown in SEQ ID NO: 2 with 1 to 5 substitutions, deletions, or additions and / or whose heavy chain amino acid sequence is the amino acid sequence shown in SEQ ID NO: 4 with 1 to 5 substitutions, deletions, or additions can also be used as antibodies in the present invention, so long as they have affinity for hTfR. Furthermore, antibodies whose light chain amino acid sequence is the amino acid sequence shown in SEQ ID NO: 2 with 1 to 3 substitutions, deletions, or additions and / or whose heavy chain amino acid sequence is the amino acid sequence shown in SEQ ID NO: 4 with 1 to 3 substitutions, deletions, or additions can also be used as antibodies in the present invention, so long as they have affinity for hTfR.

[0060] In the light chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence shown in SEQ ID NO: 6 is the variable region, and in the heavy chain amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 5 is the variable region. Substitutions, deletions, or additions in the amino acid sequences constituting the heavy and / or light chain amino acid sequences are introduced particularly into these variable regions.

[0061] The freeze-dried preparation according to this embodiment contains a fusion protein of an antibody and heparan N-sulfatase (SGSH) as an active ingredient, and further contains an isotonic agent, a nonionic surfactant, and a buffering agent.

[0062] The isotonic agent contained in the freeze-dried preparation is not particularly limited as long as it is pharmaceutically acceptable, but neutral salts and disaccharides are preferred, and a combination of a neutral salt and a disaccharide is particularly preferred.

[0063] The content of the isotonic agent in the freeze-dried formulation is preferably 2.515 to 202.5 (w / w), more preferably 5.05 to 50.5 (w / w), even more preferably 10.1 to 25.25 (w / w), for example, 15.16 (w / w), relative to the content of the fusion protein.

[0064] The neutral salt contained in the freeze-dried preparation is not particularly limited as long as it is pharmaceutically acceptable, but sodium chloride and magnesium chloride are preferred, with sodium chloride being particularly preferred.

[0065] The content of the neutral salt in the freeze-dried preparation is preferably 0.015 to 2.5 (w / w), more preferably 0.05 to 0.5 (w / w), even more preferably 0.1 to 0.25 (w / w), for example, 0.16 (w / w), relative to the content of the fusion protein.

[0066] The disaccharide contained in the freeze-dried preparation is not particularly limited as long as it is pharmaceutically acceptable, but trehalose, sucrose, maltose, lactose, or a combination thereof is preferred, with sucrose being particularly preferred.

[0067] The content of the disaccharide in the freeze-dried preparation is preferably 2.5 to 200 (w / w), more preferably 5 to 50 (w / w), even more preferably 10 to 25 (w / w), for example, 15 (w / w), relative to the content of the fusion protein.

[0068] Nonionic surfactants contained in the lyophilized formulation include polysorbates and poloxamers. The polysorbates and poloxamers are not particularly limited as long as they are pharmaceutically acceptable, and examples thereof include polysorbate 20, polysorbate 80, and polyoxyethylene (160) polyoxypropylene (30) glycol, with polysorbate 80 and polyoxyethylene (160) polyoxypropylene (30) glycol being particularly preferred. Polyoxyethylene (160) polyoxypropylene (30) glycol is synonymous with poloxamer 188. The lyophilized formulation may contain other nonionic surfactants in addition to polysorbates and poloxamers, as long as they do not impair the effects of the present invention.

[0069] The content of the nonionic surfactant in the lyophilized formulation is preferably 0.006 to 7.5 (w / w), more preferably 0.025 to 0.25 (w / w), and even more preferably 0.05 to 0.125 (w / w), for example, 0.08 (w / w), relative to the content of the fusion protein.

[0070] The content of polysorbate in the freeze-dried preparation is preferably 0.001 to 1.5 (w / w), more preferably 0.005 to 0.05 (w / w), even more preferably 0.01 to 0.025 (w / w), for example, 0.015 (w / w), relative to the content of the fusion protein.

[0071] The content of poloxamer in the freeze-dried preparation is preferably 0.005 to 6 (w / w), more preferably 0.02 to 0.2 (w / w), even more preferably 0.04 to 0.1 (w / w), for example, 0.065 (w / w), relative to the content of the fusion protein.

[0072] The buffer contained in the lyophilized preparation includes histidine. There are no particular limitations on the histidine as long as it can be used as a pharmaceutical excipient. Histidine may be in the D-form, the L-form, or a mixture of the D-form and the L-form (e.g., racemic), with L-histidine being particularly preferred. The lyophilized preparation may contain other buffers in addition to histidine, as long as they do not impair the effects of the present invention.

[0073] The content of the buffer in the lyophilized formulation is preferably 0.05 to 60 (w / w), more preferably 0.2 to 2 (w / w), even more preferably 0.4 to 1 (w / w), for example, 0.62 (w / w), relative to the content of the fusion protein.

[0074] Examples of suitable compositions of the freeze-dried preparation according to this embodiment include: (A) a preparation containing 0.5 to 40 mg of the fusion protein of an antibody and heparan N-sulfatase, and the contents of the isotonic agent, nonionic surfactant, and buffer relative to the fusion protein are 2.515 to 202.5 (w / w), 0.006 to 7.5 (w / w), and 0.05 to 60 (w / w), respectively; (B) a preparation containing 0.5 to 40 mg of the fusion protein of an antibody and heparan N-sulfatase, and the contents of the isotonic agent, nonionic surfactant, and buffer relative to the fusion protein are 5.05 to 50.5 (w / w), 0.025 to 0.25 (w / w), and 0.2 to 2 (w / w), respectively; (C) The content of the fusion protein of an antibody and heparan N-sulfatase is 0.5 to 40 mg, and the contents of the isotonic agent, nonionic surfactant, and buffer relative to the content of the fusion protein are 10.1 to 25.25 (w / w), 0.05 to 0.125 (w / w), and 0.4 to 1 (w / w), respectively.

[0075] Examples of more suitable compositions of the freeze-dried preparation according to this embodiment include: (C) a preparation in which the content of the fusion protein of an antibody and heparan N-sulfatase is 0.5 to 40 mg, and the contents of the neutral salt, disaccharide, nonionic surfactant, and buffer relative to the content of the fusion protein are 0.015 to 2.5 (w / w), 2.5 to 200 (w / w), 0.006 to 7.5 (w / w), and 0.05 to 60 (w / w), respectively; (D) a preparation in which the content of the fusion protein of an antibody and heparan N-sulfatase is 0.5 to 40 mg, and the contents of the neutral salt, disaccharide, nonionic surfactant, and buffer relative to the content of the fusion protein are 0.05 to 0.5 (w / w), 5 to 50 (w / w), 0.025 to 0.25 (w / w), and 0.2 to 2 (w / w), respectively; (E) The content of the fusion protein of an antibody and heparan N-sulfatase is 0.5 to 40 mg, and the contents of the neutral salt, disaccharide, nonionic surfactant, and buffer relative to the content of the fusion protein are 0.1 to 0.25 (w / w), 10 to 25 (w / w), 0.05 to 0.125 (w / w), and 0.4 to 1 (w / w), respectively.

[0076] More preferred examples of the composition of the freeze-dried preparation according to this embodiment include: (F) a freeze-dried preparation in which the content of the fusion protein of an antibody and heparan N-sulfatase is 0.5 to 40 mg, and the contents of sodium chloride, sucrose, polysorbate 80, polyoxyethylene (160) polyoxypropylene (30) glycol, and histidine relative to the content of the fusion protein are 0.015 to 2.5 (w / w), 2.5 to 200 (w / w), 0.001 to 1.5 (w / w), 0.005 to 6 (w / w), and 0.05 to 60 (w / w), respectively; (G) The content of the fusion protein of an antibody and heparan N-sulfatase is 0.5 to 40 mg, and the contents of sodium chloride, sucrose, polysorbate 80, polyoxyethylene (160) polyoxypropylene (30) glycol, and histidine relative to the content of the fusion protein are 0.05 to 0.5 (w / w), 5 to 50 (w / w), 0.005 to 0.05 (w / w), 0.02 to 0.2 (w / w), and 0.2 to 2 (w / w), respectively. (H) The content of the fusion protein of an antibody and heparan N-sulfatase is 0.5 to 40 mg, and the contents of sodium chloride, sucrose, polysorbate 80, polyoxyethylene (160) polyoxypropylene (30) glycol, and histidine relative to the content of the fusion protein are 0.1 to 0.25 (w / w), 10 to 25 (w / w), 0.01 to 0.025 (w / w), 0.04 to 0.1 (w / w), and 0.4 to 1 (w / w), respectively.

[0077] More specific examples of the composition of the freeze-dried preparation according to this embodiment include: (I) a freeze-dried preparation containing 20 mg of the fusion protein of an antibody and heparan N-sulfatase, and the contents of sodium chloride, sucrose, polysorbate 80, polyoxyethylene (160) polyoxypropylene (30) glycol, and histidine relative to the amount of the fusion protein are 0.16 (w / w), 15 (w / w), 0.015 (w / w), 0.065 (w / w), and 0.62 (w / w), respectively.

[0078] In the lyophilized preparations (A) to (I) above, the fusion protein of an antibody and heparan N-sulfatase is, for example, a fusion protein of a humanized anti-hTfR antibody and hSGSH. A preferred form of the fusion protein of a humanized anti-hTfR antibody and hSGSH is a fusion protein in which the light chain of the humanized anti-hTfR antibody has the amino acid sequence shown in SEQ ID NO: 2, the heavy chain of the humanized anti-hTfR antibody has the amino acid sequence shown in SEQ ID NO: 4, and human heparan N-sulfatase is linked to the C-terminus of the heavy chain via a linker sequence. In this fusion protein, the human heparan N-sulfatase preferably has the amino acid sequence shown in SEQ ID NO: 7, and the linker sequence preferably has an amino acid sequence consisting of a total of 15 amino acids corresponding to three consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 3. Furthermore, this fusion protein typically consists of one light chain and one heavy chain linked to human heparan N-sulfatase.

[0079] A further preferred embodiment of the fusion protein of a humanized anti-hTfR antibody and hSGSH in the lyophilized preparations (A) to (I) above is a fusion protein (fusion protein (1)) in which the humanized anti-hTfR antibody is a Fab, the light chain of the humanized anti-hTfR antibody has the amino acid sequence shown in SEQ ID NO: 2, and the heavy chain of the humanized anti-hTfR antibody is linked to human heparan N-sulfatase at the C-terminus via a linker having an amino acid sequence consisting of a total of 15 amino acids corresponding to three consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 3, thereby forming the amino acid sequence shown in SEQ ID NO: 1.

[0080] In the lyophilized preparations shown in (A) to (I) above, when the fusion protein of an antibody and heparan N-sulfatase is the above-mentioned fusion protein (1) of a humanized anti-hTfR antibody and hSGSH, the content of the fusion protein is preferably 0.5 to 40 mg, for example, 3 to 30 mg, 5 to 25 mg, etc., and is appropriately adjusted to 10 mg, 20 mg, etc.

[0081] Examples of suitable compositions of the freeze-dried preparation of the fusion protein (1) include: (J) a freeze-dried preparation containing 0.5 to 40 mg of the fusion protein, and the contents of sodium chloride, sucrose, polysorbate 80, polyoxyethylene (160) polyoxypropylene (30) glycol, and histidine relative to the amount of the fusion protein are 0.015 to 2.5 (w / w), 2.5 to 200 (w / w), 0.001 to 1.5 (w / w), 0.005 to 6 (w / w), and 0.05 to 60 (w / w), respectively; (K) The content of the fusion protein is 0.5 to 40 mg, and the contents of sodium chloride, sucrose, polysorbate 80, polyoxyethylene (160) polyoxypropylene (30) glycol, and histidine relative to the content of the fusion protein are 0.05 to 0.5 (w / w), 5 to 50 (w / w), 0.005 to 0.05 (w / w), 0.02 to 0.2 (w / w), and 0.2 to 2 (w / w), respectively. (L) The content of the fusion protein is 0.5 to 40 mg, and the contents of sodium chloride, sucrose, polysorbate 80, polyoxyethylene (160) polyoxypropylene (30) glycol, and histidine relative to the content of the fusion protein are 0.1 to 0.25 (w / w), 10 to 25 (w / w), 0.01 to 0.025 (w / w), 0.04 to 0.1 (w / w), and 0.4 to 1 (w / w), respectively. More specifically, (M) contains 20 mg of the fusion protein, and the contents of sodium chloride, sucrose, polysorbate 80, polyoxyethylene (160) polyoxypropylene (30) glycol, and histidine relative to the amount of the fusion protein are 0.16 (w / w), 15 (w / w), 0.015 (w / w), 0.065 (w / w), and 0.62 (w / w), respectively.

[0082] The lyophilized preparation according to this embodiment preferably has a pH of 5.0 to 5.7 when dissolved in pure water, more preferably a pH of 5.2 to 5.7 when dissolved in pure water, and even more preferably a pH of 5.4 when dissolved in pure water.

[0083] The lyophilized formulation of the present invention, which contains a fusion protein of an antibody and heparan N-sulfatase as an active ingredient, can be supplied in a sealed or filled form in a container such as a double-chamber syringe or vial. The lyophilized formulation of the present invention can also be supplied as a kit together with a dedicated solution for dissolving it. For example, the lyophilized formulation is dissolved in a dedicated solution, pure water, Ringer's solution, or the like before use, and then diluted with physiological saline to form an infusion solution, which is then administered intravenously. Alternatively, the lyophilized formulation can be administered intramuscularly, intraperitoneally, subcutaneously, or the like to a patient. There are no particular limitations on the material of the containers, such as syringes and vials, used to seal or fill the lyophilized formulation. However, borosilicate glass is preferred. Hydrophobic resins, such as cycloolefin copolymers (copolymers of cyclic olefins and olefins), cycloolefin ring-opening polymers, and hydrogenated cycloolefin ring-opening polymers, are also suitable.

[0084] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0085] Example 1: Preparation of fusion protein of humanized anti-hTfR antibody and hSGSH A fusion protein of humanized anti-hTfR antibody and hSGSH (hereinafter, this fusion protein will also be referred to as "humanized anti-hTfR antibody-hSGSH") was prepared by a standard method and used in the following tests. The humanized anti-hTfR antibody-hSGSH consists of a light chain having the amino acid sequence shown in SEQ ID NO: 2 and a heavy chain (Fab heavy chain) having the amino acid sequence shown in SEQ ID NO: 4, to which hSGSH having the amino acid sequence shown in SEQ ID NO: 7 is linked via a linker consisting of three consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 3 at the C-terminus, resulting in a Fab heavy chain-hSGSH having the amino acid sequence shown in SEQ ID NO: 1 as a whole.

[0086] Example 2: Investigation of formulation of humanized anti-hTfR antibody-hSGSH (1), investigation of buffer and isotonicity agent Citric acid (citric acid hydrate and sodium citrate hydrate) and L-histidine (L-histidine and hydrochloric acid) were selected as candidate buffers. Sucrose alone and a combination of sucrose and sodium chloride were selected as isotonicity agents.

[0087] Aqueous solutions containing humanized anti-hTfR antibody-hSGSH (formulations 1 to 4) with the compositions shown in Table 1 were prepared, and 2.0 mL of each was filled into a 2 mL glass vial to serve as samples. These samples were stored in a dark, upright position at 5°C, 25°C, and 40°C for one week, and then subjected to various tests.

[0088]

[0089] (Test 2-1) Appearance Inspection: For the appearance inspection, each formulation was visually inspected before and after storage against a white and black background to check for color tone, clarity, and the presence or absence of precipitate. As a result, no changes in appearance such as discoloration or precipitate were observed in any of the formulations.

[0090] (Test 2-2) pH Stability Test The pH stability test was carried out by measuring the pH of each formulation before and after storage. As a result, no change in pH was observed in any of the formulations.

[0091] (Test 2-3) Molecular Weight Stability Test The molecular weight stability test was performed by measuring the content of high molecular weight species, including aggregates, and low molecular weight species, including decomposition products, by size exclusion chromatography (SE-HPLC) for each formulation before and after storage. A size exclusion chromatography column, a TSKgel UltraSW Aggregate 3 μm column (7.8 mm diameter x 30 cm length, TOSOH Corporation), was installed in a Shimadzu HPLC system LC-20A (Shimadzu Corporation). In addition, an absorption spectrophotometer was installed downstream of the column to enable continuous measurement of the absorbance (measurement wavelength 215 nm) of the effluent from the column. After equilibrating the column by flowing 0.2 M sodium phosphate buffer solution at a flow rate of 0.5 mL / min, the sample was loaded onto the column, and then 0.2 M sodium phosphate buffer solution was flowed at the same flow rate. During this time, the absorbance (measurement wavelength: 215 nm) of the effluent from the column was measured to obtain an elution profile. From the obtained elution profile, the peak area of ​​the humanized anti-hTfR antibody-hSGSH monomer (monomer peak area), the peak area of ​​the high molecular weight species appearing before this monomer peak (high molecular weight species peak area), and the peak area of ​​the low molecular weight species appearing after this monomer peak (low molecular weight species peak area) were determined. The contents (%) of high molecular weight species and low molecular weight species were then calculated using the following formulas: Content (%) of high molecular weight species = {high molecular weight species peak area / (monomer peak area + high molecular weight species peak area + low molecular weight species peak area)} × 100 Content (%) of low molecular weight species = {low molecular weight species peak area / (monomer peak area + high molecular weight species peak area + low molecular weight species peak area)} × 100 The results are shown in Table 2. In Table 2, the relative amount (%) indicates a relative value of the content when the content before storage was taken as 100%. The contents of high molecular weight species and low molecular weight species increased in all formulations. The increase rates of the low molecular weight species content were similar in each formulation, but the increase rates of the high molecular weight species content were lower in formulations 3 and 4, which used histidine as a buffer. Furthermore, no difference was observed between the presence and absence of sodium chloride.

[0092]

[0093] (Test 2-4) Colloidal Stability Test The colloidal stability test was performed by measuring the zeta potential and second virial coefficient of each formulation before storage. The zeta potential and second virial coefficient were measured using a zeta potential measuring device (Zetasizer Nano ZSP, manufactured by Malvern Instruments) according to a standard protocol. The results are shown in Table 3. Compared with Formulations 1 and 2, which used citric acid as a buffer, Formulations 3 and 4, which used histidine as a buffer, had higher absolute zeta potential values ​​and second virial coefficients. Therefore, it is determined that the formulations using histidine as a buffer have superior colloidal stability. Furthermore, no difference was observed between the presence and absence of sodium chloride.

[0094]

[0095] (Test 2-5) Structural Stability Test The structural stability test was carried out using a protein physical property evaluation device (UNcle, manufactured by Unchained Labs) for each formulation before and after storage, and measuring the denaturation midpoint temperature (T ｍ ) and aggregation initiation temperature (T ａｇｇ The results are shown in Table 4. In Table 4, T ａｇｇ The wavelengths listed next to the mean wavelengths of the lasers used for the measurements. Compared with Formulations 1 and 2, which use citric acid as a buffer, Formulations 3 and 4, which use histidine as a buffer, have a T ｍ and T ａｇｇ Therefore, it is considered that the formulation using histidine as a buffer has superior structural stability. Furthermore, no difference was observed between the presence and absence of sodium chloride.

[0096]

[0097] (Summary) It was revealed that the formulation using histidine as a buffering agent is superior in molecular weight stability, colloidal stability, and structural stability compared to the formulation using citric acid as a buffering agent.

[0098] Example 3: Study of formulations of humanized anti-hTfR antibody-hSGSH (2), study of pH Aqueous solutions containing humanized anti-hTfR antibody-hSGSH (formulations 5 to 9) with the compositions shown in Table 5 were prepared, and 2 mL glass vials were filled with 2.0 mL of each solution to prepare samples. These samples were stored in a dark, upright position at 5°C, 25°C, or 40°C for one week or one month, and then subjected to various tests.

[0099]

[0100] (Test 3-1) Visual Inspection Visual inspection was carried out for each formulation before and after storage in the same manner as in Test 2-1. As a result, no changes in appearance such as discoloration or precipitates were observed in any of the formulations.

[0101] (Test 3-2) pH Stability Test The pH stability test was carried out by measuring the pH of each formulation before and after storage. As a result, no change in pH was observed in any of the formulations.

[0102] (Test 3-3) Molecular Weight Stability Test A molecular weight stability test was performed on each formulation before and after storage in the same manner as in Test 2-3. The results are shown in Table 6. In Table 6, the relative amount (%) indicates the relative value of the content when the content before storage was set to 100%. In all formulations, the content of high molecular weight species and the content of low molecular weight species increased depending on the storage period and storage temperature. Comparing the formulations, the lower the pH of the formulation, the higher the rate of increase in the content of low molecular weight species, and the higher the pH of the formulation, the higher the rate of increase in the content of high molecular weight species.

[0103]

[0104] (Test 3-4) Colloidal Stability Test The colloidal stability test was carried out by measuring the zeta potential and second virial coefficient for each formulation before storage. The zeta potential and second virial coefficient were measured in the same manner as in Test 2-4. The results are shown in Table 7. The absolute value of the zeta potential and the second virial coefficient were highest for Formulation 5, but no significant differences were observed among Formulations 6 to 9.

[0105]

[0106] (Test 3-5) Structural stability test The structural stability test was carried out for each formulation before and after storage in the same manner as in Test 2-5. The results are shown in Table 8. In Table 8, T ａｇｇ The wavelengths listed next to the mean excitation wavelengths of the laser used for the measurements. ｍ and T ａｇｇ was high.

[0107]

[0108] (Summary) It has been revealed that formulations using histidine as a buffering agent maintain colloidal stability and structural stability over a wide pH range. Taking the results of Tests 3-1 to 3-5 together, it is believed that formulations using histidine as a buffering agent have a pH of 5.2 to 5.6, with pH 5.4 being optimal.

[0109] Example 4: Study of formulations of humanized anti-hTfR antibody-hSGSH (3), study of surfactants Aqueous solutions containing humanized anti-hTfR antibody-hSGSH with the compositions shown in Table 9 (formulations 10 to 14) were prepared, and 2 mL of each was filled into a 2 mL glass vial to serve as samples. These samples were shaken at room temperature for 24 hours using a shaker, and then the quality was evaluated before and after shaking. The inverted vials were shaken vertically at a rate of 240 strokes / min. To clearly detect the effects of surfactants, formulations using citric acid as a buffer, which has low colloidal and structural stability, were used in the study.

[0110]

[0111] (Test 4-1) Visual Inspection Visual inspection was carried out on each formulation before and after shaking in the same manner as in Test 2-1. As a result, cloudiness was observed in formulations 10 and 11.

[0112] (Test 4-2) Molecular weight stability test The molecular weight stability test was carried out for each formulation before and after shaking in the same manner as in Test 2-3. As a result, no generation of high molecular weight species or low molecular weight species was observed in any of the formulations.

[0113] (Test 4-3) Evaluation of Particle Count The number of particles contained in the sample was evaluated using a flow cytometer particle image analyzer (FlowCAM: VS-1, manufactured by FLUID IMAGING TECHNOLOGIES) according to a standard protocol, by measuring the number of particles with a particle size of 1 to 10 μm, the number of particles with a particle size of 10 μm or more, and the number of particles with a particle size of 25 μm or more for particles with a particle size of 1000 μm or less. The results are shown in Table 10. In Formulations 10 and 11, the number of particles of all particle sizes increased after shaking. Furthermore, film-like particles were observed as particles with a particle size of 10 μm or more. In Formulations 12 to 14, regardless of the concentration of polysorbate 80, the number of particles with a particle size of 1 to 10 μm increased, the number of particles with a particle size of 10 μm or more remained unchanged, and the number of particles with a particle size of 25 μm or more decreased.

[0114]

[0115] (Test 4-4) Particle size evaluation The particle size was evaluated by measuring the average particle size using a zeta potential measuring device (Zetasizer Nano ZSP, manufactured by Malvern Instruments) according to a standard protocol. As a result, no change in particle size was observed in any of the formulations.

[0116] (Summary) A formulation containing only poloxamer 188 was unable to suppress an increase in the number of fine particles. On the other hand, a formulation containing both poloxamer 188 and polysorbate 80 suppressed an increase in the number of fine particles due to shaking. Furthermore, in the case of a formulation containing both poloxamer 188 and polysorbate 80, it is estimated that fine particle formation can be suppressed by setting the concentrations to 0.25 to 0.325 mg / mL and 0.05 to 0.5 mg / mL, respectively. On the other hand, a preliminary formulation containing polysorbate 80 at a concentration of 1.0 to 3.0 mg / mL suppressed an increase in the number of fine particles, but the problem of easy foaming during operation arose.

[0117] Example 5: Study of formulation of humanized anti-hTfR antibody-hSGSH (4), freeze-drying. The formulations determined from the results of Examples 2 to 4 were examined to determine whether they could be properly freeze-dried. An aqueous solution containing humanized anti-hTfR antibody-hSGSH (formulation 15) with the composition shown in Table 11 was prepared, and 4.4 mL was filled into a borosilicate glass vial (φ24.5 mm × 53 mm) to prepare a sample. This sample was freeze-dried under the conditions shown in Table 12, stored in a dark, upright position at 40°C for one month, and then subjected to various tests.

[0118]

[0119]

[0120] (Test 5-1) Visual Inspection Visual inspection was carried out on the formulation after storage in the same manner as in Test 2-1. The lyophilized product in the vial was a white dry cake with good appearance.

[0121] (Test 5-2) Solubility The solubility test was carried out by adding 4.4 mL of water for injection to the lyophilized product in the vial to dissolve it. As a result, the lyophilized product in the vial was easily redissolved in water for injection.

[0122] (Test 5-3) Quality after dissolution The formulation (lyophilized product) before and after storage was dissolved in water for injection, and the solution was subjected to an appearance inspection (conducted in the same manner as in Test 2-1), pH measurement, molecular weight stability test (conducted in the same manner as in Test 2-3), and particle number evaluation (conducted in the same manner as in Test 4-3). As a result, no abnormalities were observed in appearance, pH, generation of high molecular weight species and low molecular weight species, generation of particles, or other abnormalities.

[0123] Example 6: Long-term stability test of lyophilized formulation Various properties after long-term storage were confirmed for the lyophilized product containing humanized anti-hTfR antibody-hSGSH obtained in Example 5. The lyophilized product prepared and filled as described in Example 5 was used as the sample. This sample was stored in a dark place upright at 2 to 8°C for 36 months and then subjected to various tests.

[0124] (Test 6-1) Appearance Inspection: The appearance inspection was performed by visually observing the color and shape of the sample after storage against a white background. The freeze-dried product in the vial was a white dry cake with a good appearance.

[0125] (Test 6-2) Solubility The solubility test was carried out by adding 4.3 mL of water for injection to the lyophilized product in the vial to dissolve it. As a result, the lyophilized product in the vial was easily redissolved in water for injection.

[0126] (Test 6-3) Quality after Dissolution Samples (lyophilized products) before and after storage were dissolved in water for injection, and the solutions were subjected to visual inspection (conducted by comparing color and clarity with a reference solution using a method similar to Test 2-1), pH measurement, molecular weight stability test (conducted using the same method as Test 2-3), and particle count evaluation (conducted using the same method as Test 4-3). As a result, no changes in appearance were observed. Other results are shown in Table 13. No changes in pH, generation of high-molecular-weight and low-molecular-weight species, generation of particles, or other abnormalities were observed. These results indicate that the lyophilized product containing humanized anti-hTfR antibody-hSGSH obtained by lyophilization of Formulation 15 is stable for at least 36 months at 2-8°C in a dark place.

[0127]

[0128] SEQ ID NO: 1: Amino acid sequence of humanized anti-hTfR antibody Fab heavy chain to which hSGSH is linked via a linker at the C-terminus via a linker SEQ ID NO: 2: Amino acid sequence of humanized anti-hTfR antibody light chain SEQ ID NO: 3: Amino acid sequence of linker example 1 SEQ ID NO: 4: Amino acid sequence of humanized anti-hTfR antibody Fab heavy chain SEQ ID NO: 5: Amino acid sequence of humanized anti-hTfR antibody heavy chain variable region SEQ ID NO: 6: Amino acid sequence of humanized anti-hTfR antibody light chain variable region SEQ ID NO: 7: Amino acid sequence of human SGSH SEQ ID NO: 8: Amino acid sequence 1 of humanized anti-hTfR antibody light chain CDR1 SEQ ID NO: 9: Amino acid sequence 2 of humanized anti-hTfR antibody light chain CDR1 SEQ ID NO: 10: Amino acid sequence 1 of humanized anti-hTfR antibody light chain CDR2 SEQ ID NO: 11: Amino acid sequence 2 of humanized anti-hTfR antibody light chain CDR2 SEQ ID NO: 12: Amino acid sequence of light chain CDR3 of humanized anti-hTfR antibody SEQ ID NO: 13: Amino acid sequence 1 of heavy chain CDR2 of humanized anti-hTfR antibody SEQ ID NO: 14: Amino acid sequence 2 of heavy chain CDR2 of humanized anti-hTfR antibody SEQ ID NO: 15: Amino acid sequence 1 of heavy chain CDR3 of humanized anti-hTfR antibody SEQ ID NO: 16: Amino acid sequence 2 of heavy chain CDR3 of humanized anti-hTfR antibody SEQ ID NO: 17: Amino acid sequence 1 of heavy chain CDR1 of humanized anti-hTfR antibody SEQ ID NO: 18: Amino acid sequence 2 of heavy chain CDR1 of humanized anti-hTfR antibody SEQ ID NO: 19: Amino acid sequence of linker example 2 SEQ ID NO: 20: Amino acid sequence of linker example 3 SEQ ID NO: 21: Amino acid sequence of human TfR

Claims

1. A freeze-dried preparation containing as an active ingredient a fusion protein of an antibody and human heparan N-sulfatase (hSGSH), further containing an isotonicity agent, a non-ionic surfactant, and a buffering agent, wherein the antibody is a human antibody or a humanized antibody, the isotonicity agent comprises at least one selected from a neutral salt and a disaccharide, the neutral salt is sodium chloride, the disaccharide is sucrose, the non-ionic surfactant is polysorbate 80 and polyoxyethylene (160) polyoxypropylene (30) glycol, and the buffering agent is L-histidine.

2. The freeze-dried formulation according to claim 1, wherein the contents of the isotonic agent, the polysorbate, the poloxamer, and the buffering agent relative to the content of the fusion protein are 5.05 to 50.5 (w / w), 0.005 to 0.05 (w / w), 0.02 to 0.2 (w / w), and 0.2 to 2 (w / w), respectively.

3. The freeze-dried formulation according to claim 1, wherein the contents of the isotonic agent, the polysorbate, the poloxamer, and the buffering agent relative to the content of the fusion protein are 10.1 to 25.25 (w / w), 0.01 to 0.025 (w / w), 0.04 to 0.1 (w / w), and 0.4 to 1 (w / w), respectively.

4. The freeze-dried formulation according to claim 1, wherein the contents of the isotonicity agent, the polysorbate, the poloxamer, and the buffering agent are 15.16 (w / w), 0.015 (w / w), 0.065 (w / w), and 0.62 (w / w), respectively, relative to the content of the fusion protein.

5. The freeze-dried preparation according to claim 1, which has a pH of 5.0 to 5.7 when dissolved in pure water.

6. The freeze-dried preparation according to any one of claims 1 to 5, wherein the fusion protein comprises the human heparan N-sulfatase (hSGSH) bound to the C-terminus of the heavy chain of the antibody via a linker via a peptide bond, and the linker consists of three consecutive amino acid sequences set forth in SEQ ID NO:

3.

7. The lyophilized formulation of claim 6, wherein the antibody is a Fab.

8. The lyophilized formulation of claim 6, wherein the antibody is a humanized anti-human transferrin receptor (hTfR) antibody.

9. The lyophilized formulation according to any one of claims 1 to 5, wherein the antibody is a humanized anti-hTfR antibody and is in the form of a Fab; the heparan N-sulfatase (SGSH) is human heparan N-sulfatase (hSGSH); the light chain of the humanized anti-hTfR antibody has the amino acid sequence shown in SEQ ID NO: 2; and the heavy chain of the humanized anti-hTfR antibody is linked to the human heparan N-sulfatase (hSGSH) at its C-terminus via a linker consisting of three consecutive amino acid sequences shown in SEQ ID NO: 3, thereby forming the amino acid sequence shown in SEQ ID NO:

1.

10. The freeze-dried preparation according to any one of claims 1 to 5, which is sealed in a container made of borosilicate glass or a hydrophobic resin.

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