Method for treating retinal disease

Abstract

To provide a pharmaceutical composition for treatment or prevention of diseases associated with retinal abnormalities.SOLUTION: The present invention provides a pharmaceutical composition for treatment or prevention of diseases associated with retinal abnormalities, the pharmaceutical composition containing as an active ingredient a conjugate composed of an antibody or a fragment thereof specifically binding to a protein present on a cell forming the blood-retinal barrier and a substance capable of delivering pharmacological effects in the retina, wherein the antibody or fragment thereof possesses specific affinity, for example, to a transferrin receptor.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to a therapeutic agent for diseases accompanied by retinal abnormalities, which comprises as an active ingredient a conjugate of an antibody or a fragment thereof that specifically binds to a protein present on cells that form the blood-retinal barrier and a substance having activity to exert a pharmacological effect in the retina, and in particular to a therapeutic agent for retinal diseases accompanied by the accumulation of glycosaminoglycans (GAGs) such as heparan sulfate and dermatan sulfate in cells that constitute the retina, which comprises as an active ingredient a protein that combines an anti-human transferrin receptor antibody (anti-hTfR antibody) with human iduronate-2-sulfatase (hI2S). [Background technology]

[0002] It has been reported that a protein formed by binding an anti-human transferrin receptor antibody (anti-hTfR antibody) and human iduronate-2-sulfatase (hI2S) is effective as a therapeutic agent for Hunter syndrome (Patent Document 1). The therapeutic agent can reach the brain through the blood-brain barrier (BBB) ​​by binding to the transferrin receptor (hTfR) present on vascular endothelial cells that constitute the BBB. Therefore, by administering the therapeutic agent to a patient with Hunter syndrome, glycosaminoglycans (GAGs) such as heparan sulfate and dermatan sulfate that have accumulated in the brain of the patient can be degraded and removed. This alleviates the brain abnormalities observed in patients with Hunter syndrome (Non-Patent Document 1).

[0003] The retina is a membranous tissue with a layered structure in which nerve cells are regularly arranged. The retina has a structure in which the transport of substances from the blood is restricted by the blood retinal barrier (BRB). Therefore, even if a polymeric substance, such as a protein, is administered by intravenous injection, it is almost unable to pass through the BRB and therefore cannot reach the retina. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US20180171012 [Non-patent literature]

[0005] [Non-Patent Document 1] Okuyama T. et al., Mol Ther. 29. 671-9 (2021)Her. 67, 46-53 (1988) Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide a therapeutic agent for retinal diseases accompanied by accumulation of glycosaminoglycans (GAGs) such as heparan sulfate and dermatan sulfate in cells constituting the retina, which comprises as an active ingredient a protein combining an anti-human transferrin receptor antibody (anti-hTfR antibody) and human iduronate-2-sulfatase (hI2S), which can pass through the BRB and exert its function in the retina. [Means for solving the problem]

[0007] In the research aimed at the above-mentioned object, the present inventors conducted intensive studies and discovered that the amount of GAG accumulated in cells constituting the retina can be reduced by administering a protein in which an anti-hTfR antibody and hI2S are bound, and thus completed the present invention. That is, the present invention includes the following. 1. A pharmaceutical composition for treating or preventing diseases accompanied by retinal abnormalities, comprising as an active ingredient a conjugate formed by combining an antibody or a fragment thereof that specifically binds to a protein present on cells that form the blood-retinal barrier with a substance having an activity that is to exert a medicinal effect in the retina. 2. The pharmaceutical composition described in 1 above, wherein the protein present on the cells that form the blood-retinal barrier is selected from the group consisting of transferrin receptor (TfR), insulin receptor, leptin receptor, lipoprotein receptor, IGF receptor, organic anion transporters such as OATP-F, monocarboxylate transporters, and Fc receptors. 3. The pharmaceutical composition according to 1 above, wherein the antibody is an anti-human transferrin receptor antibody or an anti-human insulin receptor antibody. 4. The pharmaceutical composition according to any one of 1 to 4 above, wherein the substance having an activity to exert a pharmacological effect in the retina is a lysosomal enzyme. 5. The pharmaceutical composition according to 4 above, wherein the lysosomal enzyme is human iduronate-2-sulfatase or human α-L-iduronidase. 6. A pharmaceutical composition for treating or preventing a disease accompanied by accumulation of glycosaminoglycan (GAG) in the retina, comprising as an active ingredient a conjugate of an antibody or fragment thereof having affinity for the human transferrin receptor and human iduronate-2-sulfatase (hI2S). 7. Use of an antibody or a fragment thereof that specifically binds to a protein present on cells that form the blood-retinal barrier, in order to allow a substance having an activity that is to exert a pharmacological effect in the retina to pass through the blood-retinal barrier. Effect of the Invention

[0008] According to the present invention, for example, glycosaminoglycans (GAGs) such as heparan sulfate and dermatan sulfate that have accumulated within the cells that make up the retina can be broken down and reduced, making it possible to provide a treatment for retinopathies accompanied by accumulation of GAGs within the retina. [Brief description of the drawings]

[0009] [Figure 1] This figure shows the results of measuring the concentration of hI2S-anti-hTfR antibody and hI2S in the retina after administration to mice via the tail vein. The vertical axis shows the concentration of hI2S-anti-hTfR antibody in the retina (μg / g tissue protein). (1) shows the concentration of hI2S-anti-hTfR antibody, and (2) shows the concentration of hI2S. [Diagram 2] Figure showing the results of measuring the concentration of heparan sulfate in the retina of mice administered hI2S-anti-hTfR antibody via the tail vein. The vertical axis shows the concentration of heparan sulfate in the retina (ng / g tissue protein). (1) Wild-type mouse, (2) Pathological model mouse, (3) Pathological model mouse administered 0.5 mg / kg hI2S-anti-hTfR antibody, (4) Pathological model mouse administered 2.0 mg / kg hI2S-anti-hTfR antibody, (5) Pathological model mouse administered 0.5 mg / kg hI2S. [Diagram 3] (a) Measurement results of the ERG a-wave (signal derived from photoreceptor cells) in wild-type and I2S-KO / hTfR-KI mice. (1) is the measurement result of a wild-type mouse, and (2) is the measurement result of a pathological model mouse. The vertical axis shows the intensity of the a-wave (μV). (b) Measurement results of the ERG b-wave (signal derived from amacrine cells / bipolar cells) in wild-type and I2S-KO / hTfR-KI mice. (1) is the measurement result of a wild-type mouse, and (2) is the measurement result of a pathological model mouse. The vertical axis shows the intensity of the b-wave (μV). [Figure 4] (a) Measurement results of ERG a-wave (signal derived from photoreceptor cells) in mice administered hI2S-anti-hTfR antibody via the tail vein. The vertical axis shows the intensity of the a-wave (μV). (b) Measurement results of ERG b-wave (signal derived from amacrine cells / bipolar cells) in mice administered hI2S-anti-hTfR antibody via the tail vein. The vertical axis shows the intensity of the b-wave (μV). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] In the present invention, a human antibody refers to an antibody that is entirely encoded by a gene derived from a human. However, an antibody encoded by a gene in which a mutation has been added to the original human gene for the purpose of increasing gene expression efficiency, etc., is also a human antibody. In addition, an antibody in which two or more genes encoding human antibodies 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.

[0011] 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 of an immunoglobulin light chain are called CDR1, CDR2, and CDR3, starting from the N-terminus. The three CDRs of an 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.

[0012] Both the heavy and light chain variable regions of a human antibody generally contain four framework regions 1 to 4 (FR1 to 4). FR1 is a region adjacent to CDR1 at its N-terminus, and consists of an amino acid sequence from the N-terminus to the amino acid adjacent to the N-terminus of CDR1 in each peptide constituting the heavy and light chains. FR2 consists of an amino acid sequence between CDR1 and CDR2 in each peptide constituting the heavy and light chains. FR3 consists of an amino acid sequence between CDR2 and CDR3 in each peptide constituting the heavy and light chains. FR4 consists of an amino acid sequence from the amino acid adjacent to the C-terminus of CDR3 to the N-terminus of the variable region. However, without being limited thereto, in the present invention, a region excluding 1 to 5 amino acids on the N-terminus side and / or 1 to 5 amino acids on the C-terminus side in each of the above FR regions can also be a framework region. The same applies to a humanized antibody described later.

[0013] In the present invention, an antibody in which mutations such as substitution, deletion, and addition are added to the amino acid sequence of the original antibody by modifying the gene of the original human antibody is also called a human antibody. When an amino acid in the amino acid sequence of the original antibody is replaced with another amino acid, the number of amino acids to be replaced is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3. When an amino acid in the amino acid sequence of the original antibody is 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. An antibody in which a mutation that combines these amino acid substitutions and deletions is added is also a human antibody. When an amino acid is 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. An antibody in which a mutation that combines these amino acid additions, substitutions, and deletions is added is also a human antibody. The amino acid sequence of the mutated antibody preferably exhibits 80% or more homology with the amino acid sequence of the original antibody, more preferably 90% or more homology, even more preferably 95% or more homology, and even more preferably 98% or more homology. 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 humanized antibodies, etc., described below.

[0014] When a mutation is added to a gene encoding all or a part of the light chain variable region of an original human antibody, the gene after the mutation has preferably 80% or more homology with the original gene, more preferably 90% or more homology, but there is no particular limit to the homology as long as the antibody after the mutation has a specific affinity for the antigen. When an amino acid in the amino acid sequence of the light chain variable region is replaced with another amino acid, the number of amino acids to be replaced is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. When an amino acid in the amino acid sequence of the light chain variable region is 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 the replacement and deletion of these amino acids can also be added. When amino acids are added to the light chain variable region, preferably 1 to 10 amino acids, more preferably 1 to 5 amino acids, even more preferably 1 to 3 amino acids, 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-terminal or C-terminal side of the light chain variable region. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of the mutated light chain variable region preferably has a homology of 80% or more, more preferably a homology of 90% or more, and even more preferably a homology of 95% or more, with the amino acid sequence of the original light chain variable region.

[0015] In particular, when a mutation is added to the variable region of the light chain, if an amino acid in the amino acid sequence of the CDR is replaced with another amino acid, the number of amino acids to be replaced is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. When an amino acid in the amino acid sequence of the CDR is 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 the substitution and deletion of these amino acids can also be added. When an amino acid is 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 combining the addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of each mutated CDR preferably has a homology of 80% or more, more preferably 90% or more, and even more preferably 95% or more with the amino acid sequence of the original CDR. When a mutation is added to a CDR, a mutation can also be added to the framework region. The same applies to the humanized antibodies described below.

[0016] In particular, when a mutation is added to the variable region of the light chain, when an amino acid in the amino acid sequence of the framework region (FR) is replaced with another amino acid, the number of amino acids to be replaced is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. When an amino acid in the amino acid sequence of the FR is 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 the substitution and deletion of these amino acids can also be added. When an amino acid is added, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2 amino acids are added in the amino acid sequence or on the N-terminal side or C-terminal side. Mutations combining the addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of each FR to which a mutation has been added has preferably 80% or more homology with the amino acid sequence of the original FR, more preferably 90% or more homology, and even more preferably 95% or more homology. When a mutation is added to the FR, a mutation can also be added to the CDR. The same applies to the humanized antibodies described below.

[0017] When a mutation is introduced into a gene encoding all or a part of the heavy chain variable region of an original human antibody, the gene after the mutation has preferably 80% or more homology with the original gene, more preferably 90% or more homology, but there is no particular limit to the homology as long as the antibody after the mutation has a specific affinity for the antigen. When an amino acid in the amino acid sequence of the heavy chain variable region is replaced with another amino acid, the number of amino acids to be replaced is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. When an amino acid in the amino acid sequence of the heavy chain variable region is 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 the replacement and deletion of these amino acids can also be introduced. When amino acids are added to the heavy chain variable region, preferably 1 to 10 amino acids, more preferably 1 to 5 amino acids, even more preferably 1 to 3 amino acids, 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-terminal or C-terminal side. Mutations that combine addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of the mutated heavy chain variable region preferably has a homology of 80% or more, more preferably a homology of 90% or more, and even more preferably a homology of 95% or more, with the amino acid sequence of the original heavy chain variable region.

[0018] In particular, when a mutation is added to the variable region of the heavy chain, if an amino acid in the amino acid sequence of the CDR is replaced with another amino acid, the number of amino acids to be replaced is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. When an amino acid in the amino acid sequence of the CDR is 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 the substitution and deletion of these amino acids can also be added. When an amino acid is 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 combining the addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of each mutated CDR preferably has a homology of 80% or more, more preferably 90% or more, and even more preferably 95% or more with the amino acid sequence of the original CDR. When a mutation is added to a CDR, a mutation can also be added to the framework region. The same applies to the humanized antibodies described below.

[0019] In particular, when a mutation is added to the variable region of the heavy chain, when an amino acid in the amino acid sequence of the framework region (FR) is replaced with another amino acid, the number of amino acids to be replaced is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. When an amino acid in the amino acid sequence of the FR is 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 the substitution and deletion of these amino acids can also be added. When an amino acid is 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 combining the addition, substitution, and deletion of these amino acids can also be added. The amino acid sequence of each FR to which a mutation has been added has preferably 80% or more homology with the amino acid sequence of the original FR, more preferably 90% or more homology, and even more preferably 95% or more homology. When a mutation is added to the framework region, a mutation can also be added to the CDR. The same applies to the humanized antibodies described below.

[0020] In the present invention, "homology" means the ratio (%) of homologous amino acid residues to all amino acid residues in optimal alignment when two amino acid sequences are compared using a homology calculation algorithm. Comparing two amino acid sequences based on the homology expressed as such a ratio is well known in the technical field of the present invention and is easily understood by those skilled in the art. As homology calculation algorithms for calculating the homology between the amino acid sequence of an original protein (including an antibody) and the amino acid sequence of a mutated protein, BLAST (Altschul SF. J Mol. Biol. 215. 403-10, (1990)), Pearson and Lipman's similarity search method (Proc. Natl. Acad. Sci. USA. 85. 2444 (1988)), Smith and Waterman's local homology algorithm (Adv. Appl. Math. 2. 482-9 (1981)), etc. are well known. 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 homology between two amino acid sequences.

[0021] In addition, the substitution of an amino acid in the amino acid sequence of an original protein (including an antibody) with another amino acid occurs within an amino acid family that is related, for example, by the side chains and chemical properties of the amino acids. Substitutions within such an amino acid family are predicted not to significantly change the function of the original protein (i.e., they are conservative amino acid substitutions). Examples of such amino acid families include the following: (1) The acidic amino acids aspartic acid and glutamic acid, (2) The basic amino acids histidine, lysine, and arginine, (3) the aromatic amino acids phenylalanine, tyrosine, and tryptophan, (4) Serine and threonine, which are amino acids that have hydroxyl groups (hydroxyamino acids), (5) The hydrophobic amino acids methionine, alanine, valine, leucine, and isoleucine, (6) The neutral hydrophilic amino acids cysteine, serine, threonine, asparagine, and glutamine, (7) Glycine and proline, which are amino acids that affect the orientation of peptide chains, (8) Asparagine and glutamine, which are amide amino acids (polar amino acids), (9) The aliphatic amino acids alanine, leucine, isoleucine, and valine, (10) The amino acids with small side chains, alanine, glycine, serine, and threonine, (11) Alanine and glycine, which are amino acids with particularly small side chains, (12) The branched-chain amino acids valine, leucine, and isoleucine.

[0022] In the present invention, the term "humanized antibody" refers to an antibody in which the amino acid sequence of a part of the variable region (e.g., all or part of the CDR in particular) is derived from a mammal other than human, and the other regions are derived from human. For example, a humanized antibody can be an antibody produced by replacing three complementarity determining regions (CDRs) of an immunoglobulin light chain constituting a human antibody and three complementarity determining regions (CDRs) of an immunoglobulin heavy chain with CDRs of another mammal. The species of other mammal from which the CDRs to be grafted to the appropriate positions of 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 a non-human primate, and more preferably a mouse or rat, for example a mouse.

[0023] 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.

[0024] A chimeric antibody between a human antibody and an antibody of another mammal is an antibody in which a part of a human antibody is replaced by a part of an antibody of a mammal other than a human. The antibody consists of an Fc region, a Fab region, and a hinge region, which are described below. A specific example of such a chimeric antibody is a chimeric antibody in which the Fc region is derived from a human antibody while the Fab region is derived from an antibody of another mammal. The hinge region is derived from either a human antibody or an antibody of another mammal. Conversely, a chimeric antibody in which the Fc region is derived from another mammal while the Fab region is derived from a human antibody is included. The hinge region may be derived from either a human antibody or an antibody of another mammal.

[0025] 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 heavy chain constant region (C H ) and the light chain constant region (C L ) are derived from human antibodies, while the heavy chain variable region (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 heavy chain variable region (V H ) and the light chain variable region (V L ) derived from a human antibody. 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, a rat, a rabbit, a horse, or a non-human primate, and more preferably a mouse.

[0026] Chimeric antibodies of human and mouse antibodies are specifically 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. Other specific examples of human / mouse chimeric antibodies include chimeric antibodies in which the constant region of the heavy chain (C H ) and the light chain constant region (C L) are derived from human antibodies, while the heavy chain variable region (V H ) and the light chain variable region (V L ) derived from mouse antibodies, whereas the heavy chain constant region (C H ) and the light chain constant region (C L ) is derived from a mouse antibody, while the heavy chain variable region (V H ) and the light chain variable region (V L ) derived from a human antibody.

[0027] When mutations are added to the heavy and light chains of humanized and chimeric antibodies, the rules for adding mutations to human antibodies described above also apply.

[0028] An antibody originally has a basic structure consisting of four polypeptide chains: two light immunoglobulin chains and two heavy immunoglobulin chains. However, in the present invention, the term "antibody" refers to an antibody having this basic structure, as well as: (1) Consists of two polypeptide chains, one light immunoglobulin chain and one heavy immunoglobulin chain, or, as described in more detail below, (2) A single-chain antibody, which is composed of an immunoglobulin light chain bound to a linker at its C-terminus and an immunoglobulin heavy chain bound to the C-terminus of the linker. (3) A single-chain antibody, which is composed of an immunoglobulin heavy chain bound to a linker at its C-terminus and an immunoglobulin light chain bound to the C-terminus of the linker. (4) A single-chain antibody (scFv) which is composed of a linker attached to the C-terminus of the variable region of an immunoglobulin heavy chain and a variable region of an immunoglobulin light chain attached to the C-terminus of the linker, and (5) Single chain antibodies (scFv) are also included, which are formed by linking a linker to the C-terminus of the variable region of an immunoglobulin light chain and further linking the variable region of an immunoglobulin heavy chain to the C-terminus of the linker. (6) Those consisting of a Fab region in which the Fc region has been deleted from the basic structure of an antibody in the original sense, and those consisting of a Fab region and all or part of the hinge region (including Fab, F(ab') and F(ab')2), and (7) Single domain antibodies are also included in the "antibody" of the present invention. Furthermore, scFv, which is a single-chain antibody formed by linking the variable region of the light chain and the variable region of the heavy chain via a linker, is also included in the antibody of the present invention.

[0029] In the present invention, the term "linker" refers to, for example, a peptide chain in which multiple amino acids are linked by peptide bonds. A linker consisting of such a peptide chain can also be called a "peptide linker." In the context of this specification, "linker" can also be called a "linker sequence." The N-terminus of this linker and the C-terminus of another protein are linked by a peptide bond, and the N-terminus of another protein is further linked to the C-terminus of the linker, whereby the two proteins form a conjugate via the linker.

[0030] In one embodiment of the present invention, Fab is a polypeptide that is a mixture of a variable region and a C L One light chain containing the constant region (the light chain constant region) and one variable region and C H A molecule in which one heavy chain containing one region (the constant region of the heavy chain) is bound by disulfide bonds between the cysteine ​​residues present in each heavy chain. In Fab, the heavy chain is composed of a variable region and a C H In addition to the region 1 (part 1 of the heavy chain constant region), a portion of the hinge region may also be included, but in this case the hinge region lacks the cysteine ​​residues that are present in the hinge region and that bind the heavy chains of the antibody. In the Fab, the light chain and the heavy chain are bound by the light chain constant region (C L The cysteine ​​residues in the heavy chain constant region (C H 1 region) or hinge region. The heavy chains that form Fab are called Fab heavy chains. Fab consists of one light chain and one heavy chain because it lacks the cysteine ​​residues that are present in the hinge region and that bind the heavy chains of antibodies. The light chains that make up Fab consist of the variable region and C L The heavy chain that constitutes Fab contains the variable region and the C H It may consist of one domain, a variable domain, C HIn addition to the variable region, a portion of the hinge region may be included. In this case, however, the hinge region is selected so as not to contain a cysteine ​​residue that bonds between 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 is composed of a variable region and a C H In addition to the above region, the heavy chain includes all or a part of the hinge region containing the cysteine ​​residue that binds the heavy chains together. F(ab')2 refers to a molecule in which two F(ab')s are bonded by disulfide bonds between the cysteine ​​residues present in the hinge regions of each other. The heavy chain that forms F(ab') or F(ab')2 is called a Fab' heavy chain. In addition, polymers such as dimers and trimers formed by bonding multiple antibodies directly or via a linker are also antibodies. Furthermore, not limited to these, any one that contains a part of an immunoglobulin molecule and has the property of specifically binding to an antigen is included in the "antibody" as used in the present invention. That is, when the immunoglobulin light chain is referred to in the present invention, it includes one that is derived from an immunoglobulin light chain and has all or a part of the amino acid sequence of its variable region. In addition, when the immunoglobulin heavy chain is referred to, it includes one that is derived from an immunoglobulin heavy chain and has all or a 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 one lacking, for example, the Fc region, is an immunoglobulin heavy chain.

[0031] In addition, Fc or Fc region refers to the C region in an antibody molecule. H This refers to a region that contains a fragment consisting of the CH2 region (part 2 of the heavy chain constant region) and the CH3 region (part 3 of the heavy chain constant region).

[0032] Furthermore, the antibody in one embodiment of the present invention is (8) Also included are scFab, scF(ab'), and scF(ab')2, which are single-chain antibodies formed by linking the light chain and heavy chain constituting the Fab, F(ab'), or F(ab')2 shown in (6) above via a linker sequence. Here, scFab, scF(ab'), and scF(ab')2 may be formed by linking a linker sequence to the C-terminus of the light chain and further linking a heavy chain to the C-terminus thereof, or may be formed by linking a linker to the C-terminus of the heavy chain and further linking a light chain to the C-terminus thereof. Furthermore, the antibody of the present invention also includes scFv, which is a single-chain antibody formed by linking the variable region of the light chain and the variable region of the heavy chain via a linker. In the case of scFv, it may be composed of a linker sequence attached to the C-terminus of the light chain variable region and a heavy chain variable region further attached to the C-terminus of that, or it may be composed of a linker sequence attached to the C-terminus of the heavy chain variable region and a light chain variable region further attached to the C-terminus of that.

[0033] Furthermore, the term "antibody" as used herein includes full-length antibodies, as well as those listed in (1) to (8) above, and antigen-binding fragments (antibody fragments) in which a portion of a full-length antibody is deleted, which is a broader concept including (1) to (8). Antigen-binding fragments include heavy chain antibodies, light chain antibodies, VHHs, VNARs, and those in which a portion of these antibodies is deleted.

[0034] The term "antigen-binding fragment" refers to a fragment of an antibody that retains at least a part of the specific binding activity with an antigen. Examples of binding fragments include, in addition to those shown in (4) and (5) above, Fab, Fab', F(ab')2, variable region (Fv), variable region of the heavy chain (V H ) and the light chain variable region (V L ) linked with an appropriate linker, and the variable region of the heavy chain (V H ) and the light chain variable region (V L Diabodies are dimers of polypeptides that contain a portion of the constant region (C) in the heavy chain (H chain) of scFv. H3) and other low molecular weight antibodies. However, the term is not limited to these molecules as long as they have the ability to bind to an antigen, and any molecule that can bind to a specific substance with specificity, even if it is made of an Fc region or a mutated Fc region, is included in the antigen-binding fragment. In addition, these antigen-binding fragments also include antibodies treated with an appropriate enzyme and proteins produced in an appropriate host cell using an antibody gene modified by genetic engineering. In this specification, when the term "antibody" is used simply, it is also intended to include antigen-binding fragments depending on the context.

[0035] In one embodiment of the present invention, the term "single-chain antibody" refers to a protein that is capable of specifically binding to a specific antigen, comprising an amino acid sequence that includes all or part of the variable region of an immunoglobulin light chain, to which a linker is attached at the C-terminus, and further to which an amino acid sequence that includes all or part of the variable region of an immunoglobulin heavy chain is attached at the C-terminus. In addition, a protein that is capable of specifically binding to a specific antigen, comprising an amino acid sequence that includes all or part of the variable region of an immunoglobulin heavy chain, to which a linker is attached at the C-terminus, and further to which an amino acid sequence that includes all or part of the variable region of an immunoglobulin light chain is attached at the C-terminus, is also a "single-chain antibody" in the present invention. For example, those shown in (2) and (3) above are included in single-chain antibodies. In a single-chain antibody in which an immunoglobulin light chain is attached to the C-terminus of an immunoglobulin heavy chain via a linker, the immunoglobulin heavy chain usually lacks the Fc region. The variable region of an 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, it is preferable that a single-chain antibody 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.

[0036] In a single-chain antibody, the linker disposed between the light and heavy chains of the 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 30 to 30 or 30 to 30, for example, 30 or 30 amino acid residues. There are no limitations on the amino acid sequence of such a linker, so long as the anti-hTfR antibody formed by linking both chains with the linker retains affinity for hTfR, but preferably the linker is composed of only glycine or glycine and serine, and for example, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence shown in SEQ ID NO: 4, or a sequence in which these amino acid sequences are repeated 2 to 10 times or 2 to 5 times. For example, when an ScFV is produced by linking the variable region of an immunoglobulin light chain to the C-terminus of an amino acid sequence consisting of the entire variable region of an immunoglobulin heavy chain via a linker, a linker having the amino acid sequence shown in SEQ ID NO: 4 is preferably used.

[0037] The antibody in one embodiment of the present invention is an antibody derived from a camelid (including an alpaca). Some camelid antibodies are composed of two heavy chains linked by a disulfide bond. An antibody composed of these two heavy chains is called a heavy chain antibody. A VHH is an antibody composed of one heavy chain including the variable region of the heavy chain constituting a heavy chain antibody, or an antibody composed of one heavy chain lacking the constant region (CH) constituting a heavy chain antibody. VHH is also one of the antibodies in the embodiment of the present invention. In order to reduce the antigenicity of an antibody (including VHH) derived from a camelid when administered to a human, an antibody in one embodiment of the present invention is also an antibody in which a mutation has been added to the amino acid sequence of an antibody of a camelid. When a mutation is added to the amino acid of an antibody of a camelid, a mutation similar to the mutation that can be added to the antibody described in this specification can be added. In addition, an antibody composed of two light chains linked by a disulfide bond is also one of the antibodies in the embodiment of the present invention. An antibody composed of these two light chains is called a light chain antibody.

[0038] The antibody in one embodiment of the present invention is an antibody derived from a shark. A shark antibody consists of two heavy chains linked by a disulfide bond. An antibody consisting of these two heavy chains is called a heavy chain antibody. A VNAR is an antibody consisting of one heavy chain including the variable region of the heavy chain constituting a heavy chain antibody, or an antibody consisting of one heavy chain lacking the constant region (CH) constituting a heavy chain antibody. VNAR is also one of the antibodies in the embodiment of the present invention. In order to reduce the antigenicity when an antibody derived from a shark (including VNAR) is administered to a human, an antibody in one embodiment of the present invention is also an antibody in which a mutation has been added to the amino acid sequence of the shark antibody. When a mutation is added to the amino acid of a shark antibody, the same mutation as that which can be added to the antibody described in this specification can be added. A humanized shark antibody is also one of the antibodies in the embodiment of the present invention.

[0039] In one embodiment of the present invention, a single domain antibody refers to an antibody that has the property of specifically binding to an antigen through a single variable region. Single domain antibodies include antibodies whose variable region consists only of the heavy chain variable region (heavy chain single domain antibodies) and antibodies whose variable region consists only of the light chain variable region (light chain single domain antibodies). VHH and VNAR are types of single domain antibodies.

[0040] In the present invention, the antigen that the antibody specifically recognizes is, for example, a molecule (surface antigen) present on the surface of vascular endothelial cells. Examples of such surface antigens include, but are not limited to, transferrin receptor (TfR), insulin receptor, leptin receptor, lipoprotein receptor, IGF receptor, organic anion transporters such as OATP-F, monocarboxylate transporters, and Fc receptors. The monocarboxylate transporter (MCT) may be any of 14 subtypes (MCT1 to MCT14), and is particularly preferably MCT1, MCT2, MCT4, or MCT8, for example MCT8. The antigen is preferably such a molecule (surface antigen) present on the surface of human vascular endothelial cells. 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:5. In one embodiment of the present invention, the anti-hTfR antibody specifically refers to an antibody that specifically binds to the portion from the 89th cysteine ​​residue from the N-terminus to the phenylalanine at the C-terminus in the amino acid sequence shown in SEQ ID NO:5 (the extracellular domain of hTfR), but is not limited to this.

[0041] Among the above surface antigens, transferrin receptor (TfR), insulin receptor, leptin receptor, lipoprotein receptor, IGF receptor, organic anion transporters such as OATP-F, and monocarboxylate transporters such as MCT-8 are antigens present on the surface of cells that form the blood-retinal barrier (BRB). The blood-retinal barrier (BRB) controls the movement of substances between the circulating blood and the retina. The BRB consists of an inner BRB composed of retinal capillary endothelial cells and an outer BRB composed of retinal pigment epithelial cells.

[0042] Antibodies that can recognize antigens present on the surface of cells that constitute the blood-retinal barrier can bind to the cells via the antigens. The antibodies that bind to the cells can then pass through the blood-retinal barrier and reach the retina. Therefore, by binding a desired substance to such an antibody to form a conjugate, it can be made to reach the retina. In this case, the conjugate may pass through either the inner BRB or the outer BRB, or may pass through both.

[0043] The retina is generally considered to be composed of three parts: the retinal visual part, the retinal ciliary part, and the retinal iris part. According to one embodiment of the present invention, the combination can reach at least one of these three parts.

[0044] Substances to be bound to antibodies include substances that have an activity that should exert a pharmacological effect in the retina. Such substances also include proteins, low molecular weight compounds, etc. Here, there is no particular limitation on the molecular weight of the protein, and peptides consisting of several amino acids are also included in the protein. Furthermore, proteins are not limited to monomers, and may be homomultimers or heteromultimers consisting of multiple peptide chains. Furthermore, the low molecular weight compound here refers to a compound that is hardly capable of crossing the blood-retinal barrier itself, and has a molecular weight of preferably 100 to 100,000 kD, for example, 100 to 10,000 kD, 100 to 5,000 kD, or 200 to 5,000 kD.

[0045] When the desired substance is a protein, the protein can be, for example, a lysosomal enzyme that is missing or dysfunctional in patients with lysosomal disease accompanied by retinal damage. Such lysosomal enzymes cannot reach the retina as is and have no medicinal effect on the patient's retinal damage, but by binding to these antibodies, they can pass through the blood-retinal barrier, thereby improving the retinal damage seen in patients with lysosomal disease.

[0046] In the present invention, the human lysosomal enzyme to be bound to the antibody is not particularly limited, but may be α-L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, etc. Lysosomal enzymes such as α-galactosidase A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase-N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1-6-glucosidase, sialidase, aspartylglucosaminidase, palmitoyl protein thioesterase-1 (PPT-1), tripeptidyl peptidase-1 (TPP-1), hyaluronidase-1, CLN1, CLN2, CLN3, CLN6, and CLN8, in particular iduronate-2-sulfatase and α-L-iduronidase.

[0047] Human iduronate-2-sulfatase (hI2S), a type of human lysosomal enzyme, is a type of lysosomal enzyme that has the activity of hydrolyzing sulfate ester bonds present in glycosaminoglycan (GAG) molecules such as heparan sulfate and dermatan sulfate. Mucopolysaccharidosis type II is a genetic disease caused by a mutation in the gene encoding this enzyme. Hunter syndrome is synonymous with mucopolysaccharidosis type II. In these patients, retinal degeneration (retinopathy) may occur as a result of accumulation of heparan sulfate and dermatan sulfate in the retinal tissue. The conjugate of the antibody and hI2S can degrade GAG ​​accumulated in the retina by passing through the BRB, so it can be used as a therapeutic agent to degrade GAG ​​accumulated in the retina by administering it to patients with Hunter syndrome who show retinal degeneration.

[0048] Human α-L-iduronidase (hIDUA), a type of human lysosomal enzyme, is a type of lysosomal enzyme that has the activity of hydrolyzing iduronic acid bonds present in glycosaminoglycan (GAG) molecules such as heparan sulfate and dermatan sulfate. Mucopolysaccharidosis type II is a genetic disease caused by mutations in the gene encoding this enzyme. Mucopolysaccharidosis type I is a genetic disease caused by mutations in the gene encoding this enzyme. Mucopolysaccharidosis type I is classified into Hurler syndrome, Hurler-Scheie syndrome, and Scheie syndrome, with Hurler syndrome being the severe type, Hurler-Scheie syndrome being the intermediate type, and Scheie syndrome being the mild type. In these patients, retinal degeneration (retinopathy) may occur as a result of the accumulation of heparan sulfate and dermatan sulfate in the retinal tissue. The conjugate of this antibody and hIDUA can degrade GAG ​​accumulated in the retina by passing through the BRB, and therefore can be used as a therapeutic agent for degrading GAG accumulated in the retina by administering it to patients with mucopolysaccharidosis type I who exhibit retinal degeneration.

[0049] In the present invention, the term "human iduronate-2-sulfatase" or "hI2S" particularly refers to hI2S having the same amino acid sequence as wild-type hI2S. Wild-type hI2S has an amino acid sequence consisting of 525 amino acids as shown in SEQ ID NO: 6. However, without being limited thereto, hI2S also includes those obtained by adding mutations such as substitution, deletion, and addition to the amino acid sequence of wild-type hI2S as long as they have I2S activity. When amino acids in the amino acid sequence of hI2S are replaced with other amino acids, the number of amino acids to be replaced 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 hI2S 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 combining these amino acid substitutions and deletions can also be added. When amino acids are added to hI2S, 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 hI2S 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 mutated hI2S preferably has 80% or more identity with the original amino acid sequence of hI2S, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 99% or more identity. The same applies to hIDUA.

[0050] In the present invention, when hI2S is said to have I2S activity, it means that when hI2S is bound to an antibody, it has 3% or more of the activity that natural hI2S originally has. However, the activity is preferably 10% or more of the activity that natural hI2S originally has, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more. The same applies when the hI2S bound to the antibody is one that has been mutated. The antibody is, for example, an anti-hTfR antibody. The same applies to hIDUA.

[0051] In the present invention, the antibody and the desired substance can be linked directly or via a non-peptide linker or peptide linker. Examples of non-peptide linkers that can be used include polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitins, and hyaluronic acid, or derivatives thereof, or combinations thereof. The peptide linker is a peptide chain or derivative thereof consisting of 1 to 100 amino acids bonded by peptide bonds, and its N-terminus and C-terminus form covalent bonds with either the antibody or the desired substance, respectively, thereby linking the antibody and the desired substance.

[0052] When biotin-streptavidin is used as the non-peptide linker, the antibody may be bound to biotin and the desired substance may be bound to streptavidin, and the antibody and the desired substance may be bound via the bond between this biotin and streptavidin, or conversely, the antibody may be bound to streptavidin and the desired substance may be bound to biotin, and the antibody and the desired substance may be bound via the bond between this biotin and streptavidin.

[0053] The antibody of the present invention is linked to a desired substance using PEG as a non-peptide linker, and is particularly referred to as an antibody-PEG substance. The antibody-PEG substance can be produced by linking an antibody to PEG to prepare an antibody-PEG, and then linking the antibody-PEG to the desired substance. Alternatively, the antibody-PEG substance can be produced by linking a desired substance to PEG to prepare a substance-PEG, and then linking the substance-PEG to an antibody. When linking PEG to an antibody and a desired substance, 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. These functional groups introduced into PEG react mainly with amino groups in the antibody and desired substance molecules, thereby covalently linking PEG to the antibody and the desired substance. Although there is no particular limitation on the molecular weight and shape of the PEG used in this case, its average molecular weight (MW) is preferably MW=300 to 60000, and more preferably MW=500 to 20000. For example, PEGs with average molecular weights of about 300, about 500, about 1000, about 2000, about 4000, about 10000, about 20000, etc. can be suitably used as non-peptide linkers.

[0054] For example, an antibody-PEG can be obtained by mixing an antibody with polyethylene glycol having an aldehyde group as a functional group (ALD-PEG-ALD) so that the molar ratio of ALD-PEG-ALD to the antibody is 1:11, 1:12.5, 1:15, 1:110, 1:120, etc., and then adding a reducing agent such as NaCNBH3 to the mixture to cause a reaction. The antibody-PEG can then be reacted with a desired substance in the presence of a reducing agent such as NaCNBH3 to obtain an antibody-PEG-substance. Conversely, an antibody-PEG-substance can also be obtained by first binding the desired substance to ALD-PEG-ALD to prepare a substance-PEG, and then binding the substance-PEG to an antibody.

[0055] A method for binding an antibody to a protein will be explained below using hI2S as an example. The antibody and hI2S can be bound to the C-terminus or N-terminus of the heavy or light chain of the antibody by a peptide bond at the N-terminus or C-terminus of hI2S, respectively, via a linker or directly. A conjugate formed by binding an antibody to hI2S in this way can be obtained by incorporating a DNA fragment in which a cDNA encoding hI2S is arranged in frame at the 3'-terminus or 5'-terminus of the cDNA encoding the heavy or light chain of the antibody, either directly or with a DNA fragment encoding a linker between them, into an expression vector for eukaryotes such as mammalian cells or yeast, and culturing mammalian cells into which this expression vector has been introduced. In the case where a DNA fragment encoding hI2S is bound to a heavy chain, an expression vector for mammalian cells incorporating a cDNA fragment encoding the light chain of the antibody is also introduced into the same host cell, and in the case where a DNA fragment encoding hI2S is bound to a light chain, an expression vector for mammalian cells incorporating a cDNA fragment encoding the heavy chain of the antibody is also introduced into the same host cell. When the antibody is a single-chain antibody, the conjugate of the antibody and hI2S can be obtained by incorporating a DNA fragment in which a cDNA encoding a single-chain antibody is linked to the 5'-end or 3'-end of the cDNA encoding hI2S directly or via a DNA fragment encoding a linker into an expression vector for eukaryotes such as mammalian cells or yeast, and expressing the antibody in the cells into which the expression vector has been introduced.

[0056] The type of conjugate in which hI2S is bound to the C-terminus of the light chain of an antibody is one in which the antibody contains an amino acid sequence including all or part of the variable region of the light chain and an amino acid sequence including all or part of the variable region of the heavy chain, and hI2S is bound to the C-terminus of the light chain of the antibody. Here, the light chain of the antibody and hI2S may be bound directly or via a linker.

[0057] The type of conjugate in which hI2S is bound to the C-terminus of the heavy chain of an antibody is one in which the antibody contains an amino acid sequence including all or part of the variable region of the light chain and an amino acid sequence including all or part of the variable region of the heavy chain, and hI2S is bound to the C-terminus of the heavy chain of this antibody. Here, the antibody heavy chain and hI2S may be bound directly or via a linker.

[0058] The type of conjugate in which hI2S is bound to the N-terminus of the light chain of an antibody is one in which the antibody contains an amino acid sequence including all or part of the variable region of the light chain and an amino acid sequence including all or part of the variable region of the heavy chain, and hI2S is bound to the N-terminus of the light chain of the antibody. Here, the light chain of the antibody and hI2S may be bound directly or via a linker.

[0059] The type of conjugate in which hI2S is bound to the N-terminus of the heavy chain of an antibody is one in which the antibody contains an amino acid sequence including all or part of the variable region of the light chain and an amino acid sequence including all or part of the variable region of the heavy chain, and hI2S is bound to the N-terminus of the heavy chain of the antibody. Here, the heavy chain of the antibody and hI2S may be bound directly or via a linker.

[0060] In this case, when a linker is placed between the antibody and the hI2S, the sequence is preferably composed of 1 to 50, more preferably 1 to 17, even more preferably 1 to 10, and even more preferably 1 to 5 amino acids, but depending on the hI2S to be bound to the antibody, the number of amino acids constituting the linker 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 is not limited in its amino acid sequence as long as the antibody linked thereto retains the affinity with hTfR and the hI2S linked by the linker can exert the physiological activity of the protein under physiological conditions, but is preferably composed of glycine and serine, and for example, those consisting of one amino acid of either glycine or serine, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence shown in SEQ ID NO: 4, or a sequence consisting of 1 to 50 amino acids consisting of 1 to 10 or 2 to 5 consecutive amino acids of these amino acid sequences, a sequence consisting of 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, or 25 to 29 amino acids, etc. For example, those consisting of the amino acid sequence Gly-Ser and those having the amino acid sequence shown in SEQ ID NO: 4 can be suitably used as linkers. The same applies to the antibody being a single-chain antibody.

[0061] When one peptide chain contains multiple linkers, for convenience, each linker is named, starting from the N-terminus, as the first linker, the second linker, and so on.

[0062] In a preferred embodiment of the antibody, when the antibody is an anti-human transferrin receptor antibody, In the light chain variable region: (a) CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7 or 8, (b) CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9 or 10, and (c) CDR3 comprises the amino acid sequence set forth in SEQ ID NO:11; In the heavy chain variable region: (d) CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 12 or 13, (e) CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14 or 15, and (f) An example is an antibody whose CDR3 comprises the amino acid sequence shown in SEQ ID NO: 16 or 17. The antibody herein is preferably a human antibody or a humanized antibody.

[0063] The combination of amino acid sequences of each CDR shown in (a) to (f) above may be any combination.

[0064] In a further preferred embodiment of the antibody, when the antibody is an anti-human transferrin receptor antibody, (x) An example of an antibody is one in which the variable region of the light chain has the amino acid sequence shown in SEQ ID NO: 18 and the variable region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 19. The antibody is preferably a human antibody or a humanized antibody. Here, the amino acid sequence of the light chain variable region shown in SEQ ID NO:18 comprises the amino acid sequences shown in SEQ ID NO:7 and SEQ ID NO:8 as CDR1, the amino acid sequences shown in SEQ ID NO:9 and SEQ ID NO:10 as CDR2, and the amino acid sequence shown in SEQ ID NO:11 as CDR3. Also, the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:19 comprises the amino acid sequences shown in SEQ ID NO:12 and SEQ ID NO:13 as CDR1, the amino acid sequences shown in SEQ ID NO:14 and SEQ ID NO:15 as CDR2, and the amino acid sequences shown in SEQ ID NO:16 and SEQ ID NO:17 as CDR3.

[0065] However, when the antibody is a humanized antibody and an anti-human transferrin receptor antibody, the preferred embodiment of the antibody is not limited to the above (x). For example, an antibody whose light chain variable region amino acid sequence has 80% or more identity with the light chain variable region amino acid sequence in the above (x) and whose heavy chain variable region amino acid sequence has 80% or more identity with the heavy chain variable region amino acid sequence in the above (x) can also be used in the present invention as long as it has affinity for hTfR.

[0066] Furthermore, an antibody in which the amino acid sequence of the light chain variable region has 85% or more identity with the amino acid sequence of the light chain variable region in (x) above, and the amino acid sequence of the heavy chain variable region has 85% or more identity with the amino acid sequence of the heavy chain variable region in (x) above, An antibody having an amino acid sequence of a light chain variable region that is 90% or more identical to the amino acid sequence of the light chain variable region in (x) above, and an amino acid sequence of a heavy chain variable region that is 90% or more identical to the amino acid sequence of the heavy chain variable region in (x) above, and Antibodies in which the amino acid sequence of the light chain variable region has 95% or more identity with the amino acid sequence of the light chain variable region in (x) above and the amino acid sequence of the heavy chain variable region has 95% or more identity with the amino acid sequence of the heavy chain variable region in (x) above can also be used in the present invention, so long as they have affinity for hTfR.

[0067] An antibody having an amino acid sequence of a light chain variable region that is identical to the amino acid sequence of the light chain variable region in (x) above, and an amino acid sequence of a heavy chain variable region that is identical to the amino acid sequence of the heavy chain variable region in (x) above, (xa) a light chain variable region comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18 and comprising the amino acid sequence of SEQ ID NO: 7 or 8 as CDR1, the amino acid sequence of SEQ ID NO: 9 or 10 as CDR2, and the amino acid sequence of SEQ ID NO: 11 as CDR3, respectively; and a heavy chain variable region comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19 and comprising the amino acid sequence of SEQ ID NO: 12 or 13 as CDR1, the amino acid sequence of SEQ ID NO: 14 or 15 as CDR2, and the amino acid sequence of SEQ ID NO: 16 or 17 as CDR3, respectively; (xb) a light chain variable region comprising an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18 and comprising the amino acid sequence of SEQ ID NO: 7 or 8 as CDR1, the amino acid sequence of SEQ ID NO: 9 or 10 as CDR2, and the amino acid sequence of SEQ ID NO: 11 as CDR3, respectively; and a heavy chain variable region comprising an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19 and comprising the amino acid sequence of SEQ ID NO: 12 or 13 as CDR1, the amino acid sequence of SEQ ID NO: 14 or 15 as CDR2, and the amino acid sequence of SEQ ID NO: 16 or 17 as CDR3, respectively; (xc) a light chain variable region comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18 and comprising the amino acid sequence of SEQ ID NO: 7 or 8 as CDR1, the amino acid sequence of SEQ ID NO: 9 or 10 as CDR2, and the amino acid sequence of SEQ ID NO: 11 as CDR3, respectively; and a heavy chain variable region comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19 and comprising the amino acid sequence of SEQ ID NO: 12 or 13 as CDR1, the amino acid sequence of SEQ ID NO: 14 or 15 as CDR2, and the amino acid sequence of SEQ ID NO: 16 or 17 as CDR3, respectively; (xd) a light chain variable region comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, and comprising the amino acid sequence of SEQ ID NO: 7 or 8 as CDR1, the amino acid sequence of SEQ ID NO: 9 or 10 as CDR2, and the amino acid sequence of SEQ ID NO: 11 as CDR3, respectively; and a heavy chain variable region comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, and comprising the amino acid sequence of SEQ ID NO: 12 or 13 as CDR1, the amino acid sequence of SEQ ID NO: 14 or 15 as CDR2, and the amino acid sequence of SEQ ID NO: 16 or 17 as CDR3, respectively; Examples include:

[0068] Further, when the antibody is an anti-human transferrin receptor antibody, a preferred embodiment of the antibody is The amino acid sequence of the light chain variable region in the above (x) has 1 to 5 amino acids substituted, deleted or added in the amino acid sequence constituting the light chain variable region, and the amino acid sequence of the heavy chain variable region in the above (x) has 1 to 5 amino acids substituted, deleted or added in the amino acid sequence constituting the heavy chain variable region. The amino acid sequence of the light chain variable region in the above (x) in which 1 to 3 amino acids are substituted, deleted or added, and the amino acid sequence of the heavy chain variable region in the above (x) in which 1 to 3 amino acids are substituted, deleted or added, and In the above (x) the amino acid sequence of the light chain variable region in which one or two amino acids are substituted, deleted or added, and in the above (x) the amino acid sequence of the heavy chain variable region in which one or two amino acids are substituted, deleted or added, can also be used in the present invention, so long as they have affinity for hTfR. Here, the antibody is preferably a human antibody or a humanized antibody.

[0069] An antibody in which the amino acid sequence of the light chain variable region of the antibody is the amino acid sequence of the light chain variable region in the above (x) in which amino acids in the amino acid sequence constituting the light chain variable region have been substituted, deleted or added, and the amino acid sequence of the heavy chain variable region in the above (x) in which amino acids in the amino acid sequence constituting the light chain variable region have been substituted, deleted or added, (xe) A light chain variable region amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and which comprises the amino acid sequence of SEQ ID NO: 7 or 8 as CDR1, the amino acid sequence of SEQ ID NO: 9 or 10 as CDR2, and the amino acid sequence of SEQ ID NO: 11 as CDR3, respectively; a heavy chain variable region amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and which comprises the amino acid sequence of SEQ ID NO: 12 or 13 as CDR1, the amino acid sequence of SEQ ID NO: 14 or 15 as CDR2, and the amino acid sequence of SEQ ID NO: 16 or 17 as CDR3, respectively; (xf) A light chain variable region amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added, and which comprises the amino acid sequence of SEQ ID NO: 7 or 8 as CDR1, the amino acid sequence of SEQ ID NO: 9 or 10 as CDR2, and the amino acid sequence of SEQ ID NO: 11 as CDR3, respectively; a heavy chain variable region amino acid sequence in which 1 to 3 amino acids are substituted, deleted or added, and which comprises the amino acid sequence of SEQ ID NO: 12 or 13 as CDR1, the amino acid sequence of SEQ ID NO: 14 or 15 as CDR2, and the amino acid sequence of SEQ ID NO: 16 or 17 as CDR3, respectively; (xg) A light chain variable region amino acid sequence in which one or two amino acids are substituted, deleted or added, and which comprises the amino acid sequence of SEQ ID NO: 7 or 8 as CDR1, the amino acid sequence of SEQ ID NO: 9 or 10 as CDR2, and the amino acid sequence of SEQ ID NO: 11 as CDR3, respectively; a heavy chain variable region amino acid sequence in which one or two amino acids are substituted, deleted or added, and which comprises the amino acid sequence of SEQ ID NO: 12 or 13 as CDR1, the amino acid sequence of SEQ ID NO: 14 or 15 as CDR2, and the amino acid sequence of SEQ ID NO: 16 or 17 as CDR3, respectively; Examples include:

[0070] In the above (xa) to (xg), the combination of amino acid sequences of each CDR may be any combination.

[0071] When the antibody is a humanized anti-human transferrin receptor antibody and the human lysosomal enzyme is hI2S, the preferred form of the conjugate is as follows: A conjugate consisting of a light chain of a humanized anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO: 20 and hI2S shown in SEQ ID NO: 6 linked to the C-terminus of a humanized anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO: 21 via a linker shown by Gly-Ser.

[0072] Furthermore, when the antibody is a humanized anti-human transferrin receptor antibody and the human lysosomal enzyme is hI2S, the preferred form of the fusion protein is as follows: the light chain of the humanized anti-hTfR antibody has the amino acid sequence shown in SEQ ID NO: 22; A conjugate comprising the humanized anti-hTfR antibody and a fusion protein having the amino acid sequence shown in SEQ ID NO: 22, in which the humanized anti-hTfR antibody is linked at its C-terminus to hI2S shown in SEQ ID NO: 6 via a linker having the amino acid sequence shown in Gly-Ser.

[0073] In the present invention, a physiologically active protein can be produced, for example, by culturing mammalian cells that have been artificially manipulated to produce the protein by expressing or strongly expressing a gene encoding the protein. In this case, the gene to be strongly expressed in the mammalian cell that produces the protein is generally introduced into the mammalian cell by transforming the mammalian cell with an expression vector in which the gene is incorporated. There is no particular limitation on the mammalian cell used in this case, but cells derived from humans, mice, or Chinese hamsters are preferred, and CHO cells derived from Chinese hamster ovary cells are particularly preferred. In the present invention, the term "protein" refers specifically to a recombinant protein secreted into a medium when mammalian cells that produce such a protein are cultured.

[0074] A conjugate obtained by combining an antibody or a fragment thereof that specifically binds to a protein present on cells forming the blood-retinal barrier with a substance having an activity that is to exert a pharmacological effect in the retina can be used as an active ingredient of a therapeutic agent for diseases accompanied by retinal abnormalities. A pharmaceutical composition containing such a conjugate as an active ingredient is administered parenterally, for example, by intravenous injection, intravenous drip infusion, etc.

[0075] The conjugate injected intravenously passes through the BRB to reach the retina, where it exerts its physiological activity to ameliorate symptoms of retinal diseases or prevent the onset or progression of such symptoms.

[0076] In one embodiment of the present invention, the pharmaceutical composition is prepared as a freeze-dried preparation or an aqueous pharmaceutical composition, and is provided to a medical institution filled and sealed in a container or injector such as a syringe or vial. EXAMPLES

[0077] The present invention will be described in more detail below with reference to examples, but it is not intended that the present invention be limited to the examples.

[0078] [Example 1] Preparation of a conjugate of hI2S and humanized anti-hTfR antibody The conjugate of hI2S and humanized anti-hTfR antibody (hI2S-anti-hTfR antibody) was prepared according to the method described in WO2018 / 124277. The hI2S-humanized anti-hTfR antibody is the same protein as the fusion protein of hI2S and humanized anti-hTfR antibody described as I2S-anti-hTfR antibody 3 in WO2018 / 124277. That is, the hI2S-anti-hTfR antibody is a product in which hI2S is fused by peptide bond to a light chain of a humanized anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO: 20 and a heavy chain of a humanized anti-hTfR antibody having the amino acid sequence shown in SEQ ID NO: 22, via a linker having an amino acid sequence shown by Gly-Ser at the C-terminus of the heavy chain. Here, the amino acid sequence of the heavy chain and hI2S fused via the linker is that shown in SEQ ID NO: 22.

[0079] Example 2: Generation of I2S-KO / hTfR-KI mice hTfR knock-in mice (hTfR-KI mice), in which the gene encoding the extracellular domain of mouse transferrin receptor was replaced with a gene encoding the extracellular domain of the human transferrin receptor gene, were generated generally by the following method. A DNA fragment having the base sequence shown in SEQ ID NO: 23, in which a neomycin resistance gene flanked by loxP sequences was placed on the 3' side of cDNA encoding a chimeric hTfR, whose intracellular domain is the amino acid sequence of mouse hTfR and whose extracellular domain is the amino acid sequence of human hTfR, was chemically synthesized. This DNA fragment was incorporated into a targeting vector having the base sequence shown in SEQ ID NO: 24 as the 5' arm sequence and the base sequence shown in SEQ ID NO: 25 as the 3' arm sequence by a standard method, and this was introduced into mouse ES cells by electroporation. Mouse ES cells after gene introduction were selectively cultured in the presence of neomycin, and mouse ES cells in which the targeting vector had been incorporated into the chromosome by homologous recombination were selected. The resulting recombinant mouse ES cells were injected into 8-cell stage embryos (host embryos) of ICR mice and transplanted into pseudopregnant mice (recipient mice) obtained by mating with vasectomized mice. The resulting offspring (chimeric mice) were judged for coat color, and individuals in which the ES cells contributed highly efficiently to the formation of the organism, i.e., individuals with a high ratio of white hair to total hair, were selected. These chimeric mice were crossed with ICR mice to obtain F1 mice. White F1 mice were selected, and DNA extracted from the tail tissue was analyzed. Mice in which the mouse transferrin receptor gene was replaced on the chromosome with the chimeric hTfR were designated hTfR-KI mice. These hTfR-KI mice were crossed with I2S gene knockout mice to obtain I2S-KO / hTfR-KI mice.

[0080] [Example 3] Retinal distribution test 1 of hI2S-anti-hTfR antibody A retinal distribution test of the hI2S-anti-hTfR antibody was carried out using I2S-KO / hTfR-KI mice (male, 26 weeks old, 4 mice) prepared in Example 2. The hI2S-anti-hTfR antibody or hI2S was administered once at a dose of 2 mg / kg via the tail vein to two I2S-KO / hTfR-KI mice.

[0081] Immunohistochemical staining was used to detect hI2S in the retina, and was generally performed as follows. 17 hours after administration, the eyeballs were removed from the anesthetized mice. Frozen tissue sections of 7 μm thickness were prepared from the collected ocular tissue using a LEICA CM1860 UV (Leica), and these were attached to slides. The slides were immersed in 4% paraformaldehyde for 10 minutes to fix the tissue sections on the slides, and then washed three times with phosphate buffered saline (PBST) containing 0.05% Tween 20.

[0082] Next, appropriate amounts of Streptavidin Block and Biotin Block (Vector) were dropped onto the tissue sections, and each was shaken for 15 minutes in a humid box. Next, 150 μL of SuperBlock Blocking Buffer in PBS (Thermo Fisher Scientific) was dropped onto the tissue sections, and the sections were shaken for 15 minutes in a humid box, and then lightly washed with PBST.

[0083] A rabbit anti-hI2S polyclonal antibody diluted with Can get signal solution A (Toyobo) was dropped onto the tissue section fixed on the slide as the primary antibody and left to stand in a humid box at 4°C. The slide was then immersed in 0.1 M Tris-HCl buffer (TNT wash buffer) containing 0.05% Tween 20 and 0.15 M NaCl for 5 minutes. This was repeated three times to remove excess rabbit anti-hI2S polyclonal antibody. Next, 150 μL of Goat Anti-Rabbit IgG H&L (HRP-polymer) (Abcam) was dropped onto the tissue section as the secondary antibody and shaken in a humid box for 30 minutes. The slide was then washed in the same manner as for the primary antibody.

[0084] Next, 100 μL of Tyramide Working Reagent (Thermo Fisher Scientific) was dropped onto the tissue section and left to stand in a humid box for 8 minutes, after which the slide glass was washed in the same manner as for the primary antibody above. Next, 150 μL of Streptavidin-Peroxidase Polymer, Ultrasensitive (Sigma-Aldrich) diluted 1000-fold with phosphate-buffered saline (PBS, Sigma-Aldrich) was dropped onto the tissue section and shaken in a humid box for 30 minutes, after which the slide glass was washed in the same manner as for the primary antibody above.

[0085] Next, the tissue sections were stained by dropping ImmPACT NovaRED (Vector, registered trademark), a chromogenic substrate for peroxidase, and the slides were washed with tap water. The slides were then immersed in a chamber filled with hematoxylin (Merck) for 10 seconds, and then washed with ion-exchanged water for 3 minutes. The slides were dehydrated, cleared, and mounted, and then observed under an optical microscope.

[0086] In mice administered hI2S-anti-hTfR antibody, staining with a chromogenic substrate was detected in tissue sections, but in mice administered hI2S, no such staining was detected (data not shown). These results indicate that hI2S by itself cannot pass through the BRB to reach retinal tissue, but that fusion of hI2S with anti-hTfR can enable it to pass through the BRB and reach retinal tissue.

[0087] [Example 4] Retinal distribution test 2 of hI2S-anti-hTfR antibody The concentration of hI2S-anti-hTfR antibody in the retina was quantified by an electrochemiluminescence method based on the immunoreaction. The procedure is outlined below.

[0088] hI2S-anti-hTfR antibody or hI2S was administered once at a dose of 2 mg / kg via the tail vein to three I2S-KO / hTfR-KI mice. 17 hours after administration, the right eye was removed from the anesthetized mouse and placed in a Petri dish filled with PBS. Excess tissue attached around the eyeball was removed using ophthalmic scissors and tweezers under a stereomicroscope. The lens and cornea / iris were removed by cutting along the ora serrata through the hole made with a needle, and then the retina was isolated by removing the retinal pigment epithelium / choroid / sclera complex. 200 μL of RIPA buffer (Fujifilm Wako Pure Chemical Industries, Ltd.) containing Protease Inhibitor Cocktail (Sigma-Aldrich) was added to the collected retina and homogenized. The homogenate was centrifuged to obtain the supernatant. This supernatant was used as the sample material for the following experiments.

[0089] 25 μL of mouse anti-hI2S monoclonal antibody diluted to 3 μg / mL in PBS was added as the primary antibody to each well of a 96-well plate washed with PBS, and the plate was shaken for approximately 60 minutes on a plate shaker to immobilize the antibody. The solution in each well was removed, and the wells were washed three times with PBST. 150 μL of SuperBlock (Thermo Fisher Scientific) was added to each well, and the plate was shaken for approximately 60 minutes on a plate shaker, after which the solution was removed and the wells were washed three times with PBST. Next, 25 μL of the specimen sample and the calibration curve sample containing hI2S-anti-hTfR antibody or hI2S of known concentration were added to each well, and the plate was shaken for approximately 60 minutes on a plate shaker, after which the solution was removed and the wells were washed three times with PBST.

[0090] The rabbit anti-hI2S polyclonal antibody was used as the secondary antibody, diluted to 0.5 μg / mL with SuperBlock, and 25 μL of the antibody was added to each well and shaken on a plate shaker for about 60 minutes, after which the solution was removed and the wells were washed three times with PBST. Next, 25 μL of SULFO-Tag Goat Anti Rabbit IgG (Mesoscale) diluted to 0.5 μg / mL with SuperBlock was added to each well and shaken on a plate shaker for about 60 minutes, after which the solution was removed and the wells were washed three times with PBST. 150 μL of MSD Read Buffer T (4X) with Surfactant (Mesoscale) mixed with an equal amount of water for injection was added to each well, and the results were immediately measured using a Sector Imager S600 (Mesoscale). The concentration of protein (tissue protein) contained in the sample was measured using a Micro BCA Protein Assay Kit (Thermo Scientific) according to the method attached to the instructions.

[0091] In mice administered hI2S-anti-hTfR antibody, hI2S-anti-hTfR antibody was detected in tissue sections at a concentration of approximately 0.6 μg / g tissue protein. On the other hand, in mice administered hI2S, hI2S was not detected in tissue sections (Figure 1). These results also indicate that hI2S by itself cannot pass through the BRB to reach retinal tissue, but that by fusing hI2S with anti-hTfR, it can pass through the BRB and reach retinal tissue.

[0092] [Example 5] Evaluation of efficacy of hI2S-anti-hTfR antibody 1 The efficacy of hI2S-anti-hTfR antibody on the retina was evaluated by measuring the concentration of heparan sulfate (HS) accumulated in the retina of I2S-KO / hTfR-KI mice (pathological control mice), a model animal for mucopolysaccharidosis type II. The fusion protein 0.5 mg / kg and 2 mg / kg or hI2S 0.5 mg / kg were repeatedly administered into the tail vein of 10-week-old male pathological control mice once a week for 40 weeks. HS concentration was measured according to the standard method described in a previous paper (Tanaka et al., Mol Genet Metab, 125, 53-58, 2018).

[0093] The experimental results are shown in Figure 2. In wild-type mice, HS is hardly present in the retina, but in pathological control mice, which are model animals for mucopolysaccharidosis type II, abnormal HS concentrations are found to have accumulated in the retina. The retinal HS concentrations in mice administered hI2S were not significantly different from those in the model animals, but a dose-dependent decrease in HS concentrations was observed with statistical significance in mice administered hI2S-anti-hTfR antibody. These results indicate that the hI2S-anti-hTfR antibody, which passes through the BRB and reaches the retinal tissue, exerts enzymatic activity as hI2S in the retina and breaks down HS accumulated in the retina.

[0094] [Example 6] Evaluation of efficacy of hI2S-anti-hTfR antibody 2 The efficacy of hI2S-anti-hTfR antibody on the retina was evaluated by measuring the retinal function using electroretinograms (ERG) in I2S-KO / hTfR-KI mice (pathological control mice), an animal model of mucopolysaccharidosis type II in which HS accumulates in the retina. hI2S-anti-hTfR antibody 0.5 mg / kg and 2 mg / kg or hI2S 0.5 mg / kg were repeatedly administered into the tail vein of 10-week-old pathological control mice once a week for 38 weeks. ERG measurements were performed before the start of administration and after the final administration. The number of mice per group was n=12-14.

[0095] ERG was measured using a PuRec recording system (Mayo) as follows: On the day before ERG measurement, mice were moved to a dark room and dark-adapted. All subsequent procedures were performed under dim red light. Mice were anesthetized with a triple-anesthesia mixture (medetomidine / midazolam / butorphanol: 0.75 / 4.0 / 5.0 mg / kg) and dilated with 1% atropine (Japan Eye Drops Research Institute). The mice were then placed in a Ganzfelt dome (Mayo) covered with a heating pad (Bio Research Center). A reference electrode was placed in the mouth, a ground electrode was attached to the tail, and a contact lens-type electrode was placed at the center of the cornea. Dark-adapted ERG responses were measured using a single white light of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, and 10 cd·s / cm, respectively. 2 The mean values ​​for the left and right eyes were calculated.

[0096] The results are shown in Figures 3 and 4. In the pathological control mice, at 10 weeks of age (before the start of treatment), the ERG b-wave (signal derived from amacrine cells / bipolar cells) was almost equivalent to that of the wild-type mice (Figure 3(b)), but the ERG a-wave (signal derived from photoreceptor cells) was statistically significantly attenuated (Figure 3(a)), indicating that damage had occurred to the photoreceptor cells. After the 38-week treatment period, in the pathological control mice, both the a-wave and b-wave were significantly attenuated compared to the wild-type mice before the start of treatment (lanes (1) and (2) in Figure 4(a) and lanes (1) and (2) in Figure 4(b), respectively), indicating that damage to the photoreceptor cells and therefore damage to the retina had progressed during this period.

[0097] In pathological control mice administered with hI2S-anti-hTfR antibody, the attenuation of both the a-wave and the b-wave of the ERG was dose-dependently suppressed compared to pathological control mice not administered with hI2S-anti-hTfR antibody (Fig. 4(a) and (b)). In particular, in mice administered with 2 mg / kg of hI2S-anti-hTfR antibody, both the a-wave and the b-wave of the ERG were restored to the level of wild-type mice (Fig. 4(a) lane (4) and Fig. 4(b) lane (4), respectively). On the other hand, in pathological control mice administered with hI2S, the attenuation of the a-wave and the b-wave was not suppressed to a significant level (Fig. 4(a) lane (5) and Fig. 4(b) lane (5), respectively). These results indicate that the hI2S-anti-hTfR antibody, which passes through the BRB and reaches retinal tissue, exerts its enzymatic activity as hI2S in the retina, breaking down HS accumulated in the retina, thereby suppressing the progression of functional damage to photoreceptors and ultimately the retina, or restoring such function. [Industrial Applicability]

[0098] According to the present invention, it is possible to provide a pharmaceutical composition containing as an active ingredient a substance having an activity that should exert a medicinal effect in the retina, which is a substance that is barely able to pass through the blood-retinal barrier, but which has been improved so that it can pass through the blood-retinal barrier. [Sequence List Free Text]

[0099] SEQ ID NO: 1: Amino acid sequence of linker example 1 SEQ ID NO:2: Amino acid sequence of linker example 2 SEQ ID NO: 3: Amino acid sequence of linker example 3 SEQ ID NO: 4: Amino acid sequence of linker example 4 SEQ ID NO: 5: Amino acid sequence of the human transferrin receptor SEQ ID NO: 6: Amino acid sequence of human I2S SEQ ID NO: 7: Amino acid sequence of the light chain CDR1 of anti-hTfR antibody 1 SEQ ID NO: 8: Amino acid sequence 2 of the light chain CDR1 of the anti-hTfR antibody SEQ ID NO: 9: Amino acid sequence of the light chain CDR2 of anti-hTfR antibody 1 SEQ ID NO: 10: Amino acid sequence of the light chain CDR2 of anti-hTfR antibody 2 SEQ ID NO: 11: Amino acid sequence of the light chain CDR3 of anti-hTfR antibody 1 SEQ ID NO: 12: Amino acid sequence of heavy chain CDR1 of anti-hTfR antibody 1 SEQ ID NO: 13: Amino acid sequence 2 of heavy chain CDR1 of anti-hTfR antibody SEQ ID NO: 14: Amino acid sequence of the heavy chain CDR2 of anti-hTfR antibody 1 SEQ ID NO: 15: Amino acid sequence 2 of the heavy chain CDR2 of the anti-hTfR antibody SEQ ID NO: 16: Amino acid sequence of the heavy chain CDR3 of anti-hTfR antibody 1 SEQ ID NO: 17: Amino acid sequence 2 of the heavy chain CDR3 of anti-hTfR antibody SEQ ID NO: 18: Amino acid sequence of the variable region of the light chain of an anti-hTfR antibody SEQ ID NO: 19: Amino acid sequence of the variable region of the heavy chain of an anti-hTfR antibody SEQ ID NO: 20: Amino acid sequence of the light chain of the anti-hTfR antibody SEQ ID NO: 21: Amino acid sequence of anti-hTfR antibody SEQ ID NO: 22: Amino acid sequence of the fusion protein of humanized anti-hTfR antibody and human I2S SEQ ID NO: 23: DNA sequence in which a neomycin resistance gene is placed between loxP sequences on the 3' side of the cDNA encoding chimeric hTfR; synthetic sequence SEQ ID NO: 24: 5' arm sequence of the targeting vector SEQ ID NO: 25: 3' arm sequence of the targeting vector

Claims

1. A pharmaceutical composition for treating or preventing retinal diseases, comprising as an active ingredient a conjugate of an antibody or a fragment thereof that specifically binds to a protein present on cells that form the blood-retinal barrier and a substance having an activity that is to exert a medicinal effect in the retina.

2. The pharmaceutical composition according to claim 1, wherein the protein present on cells forming the blood-retinal barrier is selected from the group consisting of transferrin receptor (TfR), insulin receptor, leptin receptor, lipoprotein receptor, IGF receptor, organic anion transporters such as OATP-F, monocarboxylate transporters, and Fc receptors.

3. The pharmaceutical composition according to claim 1, wherein the antibody is an anti-human transferrin receptor antibody or an anti-human insulin receptor antibody.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the substance having an activity to exert a pharmacological effect in the retina is a lysosomal enzyme.

5. The pharmaceutical composition according to claim 4, wherein the lysosomal enzyme is human iduronate-2-sulfatase (hI2S) or human α-L-iduronidase.

6. A pharmaceutical composition for treating or preventing a disease accompanied by accumulation of glycosaminoglycan (GAG) in the retina, comprising as an active ingredient a conjugate formed by binding an antibody or a fragment thereof having affinity for the human transferrin receptor to hI2S.

7. Use of an antibody or a fragment thereof that specifically binds to a protein present on cells that form the blood-retinal barrier, in order to allow a substance having an activity to exert a pharmacological effect in the retina to pass through the blood-retinal barrier.

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