阅读说明：本技术 抗cldn-5抗体及含有该抗体的药物 (anti-CLDN-5 antibody and medicine containing the same ) 是由 近藤昌夫 八木清仁 土井健史 冈田欣晃 桥本洋佑 泽崎达也 竹田浩之 远藤幸喜 田村 于 2018-04-26 设计创作，主要内容包括：本发明通过特异性识别Claudin-5蛋白的胞外区的三维结构或一级结构的抗体,解决提供一种CLDN-5特异性高、识别CLDN-5的胞外区的分子这一技术问题。(The invention solves the technical problem of providing a molecule which has high CLDN-5 specificity and can recognize the extracellular region of CLDN-5 through an antibody which can specifically recognize the three-dimensional structure or the primary structure of the extracellular region of the Claudin-5 protein.)

1. An antibody that specifically recognizes the three-dimensional structure or the primary structure of the extracellular region of the Claudin-5 protein.

2. The antibody of claim 1, which does not bind to the extracellular domain of any of the Claudin-1 protein, Claudin-2 protein, Claudin-3 protein, Claudin-4 protein, Claudin-6 protein and Claudin-7 protein.

3. The antibody according to claim 1 or 2, which specifically recognizes a region of amino acids (proline) at position 28 to amino acid (alanine) at position 80 of the N-terminus of the human CLDN-5 protein formed from the amino acid sequence represented by SEQ ID NO. 27.

4. The antibody according to any one of claims 1 to 3, which has a binding property to the protein 1-1-5 comprising the amino acid sequence represented by SEQ ID NO. 45 of 1/5 or less to the human Claudin-5 protein comprising the amino acid sequence represented by SEQ ID NO. 27, wherein the first extracellular loop of the human Claudin-5 protein is replaced with the first loop of the human Claudin-1 protein in the protein 1-1-5.

5. The antibody according to claim 4, which has a binding ability to human Claudin-5 protein site mutant D68E having the amino acid sequence represented by SEQ ID NO. 41 of 1/5 or less of the binding ability to human Claudin-5 protein having the amino acid sequence represented by SEQ ID NO. 27.

6. The antibody according to claim 1 or 2, which specifically recognizes a region of amino acids 147 (phenylalanine) to 163 (alanine) at the N-terminus in the human CLDN-5 protein formed from the amino acid sequence represented by SEQ ID NO. 27.

7. The antibody according to claims 1, 2 and 6, which has a binding property to the protein 5-5-1 formed from the amino acid sequence represented by SEQ ID NO. 48 of 1/5 or less of the binding property to the human Claudin-5 protein formed from the amino acid sequence represented by SEQ ID NO. 27, wherein the second extracellular loop of the human Claudin-5 protein in the protein 5-5-1 is replaced with the second loop of the human Claudin-1 protein.

8. The antibody according to claim 7, which has a binding ability to human Claudin-5 protein site mutant S151T having the amino acid sequence represented by SEQ ID NO. 43 of 1/5 or less of the binding ability to human Claudin-5 protein having the amino acid sequence represented by SEQ ID NO. 27.

9. The antibody according to claim 1 or 2, which specifically recognizes a three-dimensional structure formed by a region of N-terminal amino acids 28 (proline) to 80 (alanine) and a region of N-terminal amino acids 147 (phenylalanine) to 163 (alanine) in the human CLDN-5 protein having the amino acid sequence represented by SEQ ID NO. 27.

10. The antibody according to claims 1, 2 and 9, which has a binding property to a mutant TM formed of the amino acid sequence represented by seq id No. 44 in which the region other than the extracellular region of the human Claudin-5 protein is replaced with the mouse Claudin-5 protein, which has a binding property to the human Claudin-5 protein formed of the amino acid sequence represented by seq id No. 27 of 1/5 or less.

11. The antibody according to any one of claims 1 to 10, which is any one selected from the group consisting of:

(I) antibody I comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 106 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 108 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 110 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID No. 197 or an amino acid sequence having 95% or more identity to said amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID No. 199 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 201 or an amino acid sequence having 95% or more identity to the amino acid sequence;

(E) antibody E comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 78 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 80 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 82 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID No. 169 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 171 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID No. 173 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(B) antibody B comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 57 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 59 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 61 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 148 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 150 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 152 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(F) an antibody F comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 85 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 87 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 89 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 176 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 178 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 180 or an amino acid sequence having 95% or more identity to the amino acid sequence;

(C) antibody C comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 64 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 66 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 68 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 155 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 157 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 159 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(A) antibody A comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 50 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 52 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 54 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 141 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 143 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 145 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(K) antibody K comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 120 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 122 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 124 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 211 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 213 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 215 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(D) an antibody D comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 71 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 73 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 75 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 162 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 164 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 166 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(G) an antibody G comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 92 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 94 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 96 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 183 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID No. 185 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID No. 187 or an amino acid sequence having 95% or more identity to the amino acid sequence;

(H) antibody H comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 99 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 101 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 103 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 190 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 192 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 194 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(J) an antibody J comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 113 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 115 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 117 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 204 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 206 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 208 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(L) an antibody L comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 127 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 129 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 131 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 218 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 220 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 222 or an amino acid sequence having 95% or more identity to said amino acid sequence; and

(M) an antibody M comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 134 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 136 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 138 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 225 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 227 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 229 or an amino acid sequence having 95% or more identity to said amino acid sequence.

12. The antibody according to any one of claims 1 to 11, wherein the sequence of VH in the variable region of the antibody is the sequence of seq id No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 or an amino acid sequence having 90% or more identity to any one of these amino acid sequences, and the sequence of VL is the sequence of seq id No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 or an amino acid sequence having 90% or more identity to any one of these amino acid sequences.

13. A cell comprising a polynucleotide encoding the antibody of any one of claims 1-12.

Technical Field

The present invention relates to an anti-CLDN-5 antibody and a medicament containing the same. More specifically, the present invention relates to an antibody recognizing the extracellular domain of CLDN-5, a blood brain barrier controlling drug, and the like.

Background

The blood brain barrier is a mechanism for limiting the exchange of substances between blood and brain, and plays an important role in protecting the brain from foreign matter invasion. On the other hand, the blood-brain barrier hinders the transfer of an intravenously administered drug to the brain, and therefore, has been a major obstacle to the development of a therapeutic drug for brain diseases. The blood brain barrier function is caused by the high level of tight junctions formed between brain capillary endothelial cells, which is different from the large gap between capillary endothelial cells of other organs that allow substances to pass through. Heretofore, as a theoretical method for regulating the tight junction, intercellular space delivery of drugs that limit the delivery of this substance, a method of administering a hypertonic solution of mannitol via the carotid artery has been proposed. However, this method involves physical changes in cell morphology and disruption of tight junctions due to cell dehydration, and thus has a large side effect. Therefore, a new technology for specifically controlling only tight junctions has been developed.

The CLDN family molecules play an important role in the formation of tight junctions between cells present in epithelial cells or vascular endothelial cells. The CLDN family consists of members of 27 tetraspanin proteins with 2 loops (loops) outside the cell (the first and second extracellular loops on the N-terminal side), and interactions between these cells contribute to the formation of tight junctions. The CLDN family molecules expressed in each tissue differ in kind (make-up ratio) or amount, which results in tissue-specific tight junctions, barrier functions. Particularly, in the tight connection between cerebrovascular endothelial cells, CLDN-5 is highly expressed, which has a great influence on the function of the blood brain barrier. Indeed, CLDN-5 deficient mice have impaired function of the blood-brain barrier, allowing substances with molecular weights below 1000 to permeate. Furthermore, it has been reported that in an experiment in which CLDN-5siRNA was intravenously administered to mice, expression of CLDN-5 by cerebral vascular endothelial cells was decreased, which resulted in the induction of delivery of substances having a molecular weight of 1000 or less into the brain without serious side effects (non-patent document 1). Based on these findings, the functional regulation of CLDN-5 is expected to become a novel intracerebral drug delivery strategy via the intercellular space.

To date, a number of CLDN-5 interacting molecules have been made with the aim of inhibiting barrier function based on CLDN-5. For example, it has been reported that the barrier of mouse brain capillary endothelial cells can be controlled by inhibiting the interaction of CLDN-1 and CLDN-5 using a peptide (about 20 amino acids) derived from a partial sequence of the first extracellular loop of CLDN-1 (non-patent document 2). In addition, an example of creating a CLDN-5 binding molecule by introducing a mutation into the C-terminus of Clostridium perfringens enterotoxin (Clostridium perfringens enterotoxin) known as a molecule having high barrier controlling activity by binding CLDN-3 to CLDN-4 has been reported (non-patent document 3). However, these molecules have problems that binding specificity to CLDN-5 is low and they bind to other CLDN molecules, and therefore it is considered that the development of a blood brain barrier regulation technique using these molecules is very difficult from the viewpoint of enhancing barrier regulation activity and reducing side effects. As another example, there are reports concerning peptides and antibodies that regulate cell adhesion of CLDN family molecules, but in the report, no data concerning the confirmation of regulation of the blood brain barrier by an anti-CLDN-5 antibody is disclosed (patent document 1). Thus, molecules that specifically interact with CLDN-5 to modulate its function are not yet available.

Disclosure of Invention

Technical problem to be solved by the invention

The technical problem of the present invention is to provide a novel molecule having high specificity for CLDN-5 and recognizing the extracellular region of CLDN-5. Further, the technical problem of the present invention is to provide a technique for detecting CLDN-5-expressing cells and a technique for regulating the blood-brain barrier using the molecules.

Means for solving the problems

There is no example of making a binding molecule with high specificity for CLDN-5 and demonstrating its useful activity, preferably the activity of modulating the barrier of the blood brain barrier. One reason for this is that CLDN-5 is a membrane protein, and therefore, it is difficult to purify the protein from the viewpoint of solubility and agglutination, and sufficient quality and amount cannot be obtained as a screening material or immunogen for phage display. In view of the above-mentioned technical problems, the inventors of the present application have conducted earnest studies and, as a result, succeeded in producing an antibody recognizing a region within the extracellular region of CLDN-5 protein (also referred to as "antibody of the present invention", "CLDN-5 extracellular region antibody", and the like in the present specification). And unexpectedly, the antibody has the function of opening the connection between the endothelial cells of blood vessels in the brain, namely has blood brain barrier regulation activity. The inventors of the present application have further studied based on this finding and, as a result, have completed the present invention.

The present invention includes the following embodiments as one embodiment.

Item 1.

An antibody that specifically recognizes the three-dimensional structure or the primary structure of the extracellular region of the Claudin-5 protein (Claudin-5 protein).

Item 2.

The antibody of claim 1, which does not bind to the extracellular domain of any of the Claudin-1 protein, Claudin-2 protein, Claudin-3 protein, Claudin-4 protein, Claudin-6 protein and Claudin-7 protein.

Item 3.

The antibody according to item 1 or 2, which specifically recognizes a region from amino acid (proline) at position 28 to amino acid (alanine) at position 80 of the N-terminus in the human CLDN-5 protein formed from the amino acid sequence represented by SEQ ID NO. 27.

Item 4.

The antibody according to any one of items 1 to 3, which has a binding property to protein 1-1-5 having an amino acid sequence represented by SEQ ID NO. 45 of 1/5 or less, wherein the first extracellular loop of the human Claudin-5 protein in the protein 1-1-5 is replaced with the first loop of the human Claudin-1 protein, and the binding property to the human Claudin-5 protein having an amino acid sequence represented by SEQ ID NO. 27 is.

Item 5.

The antibody according to item 4, which has a binding ability to human Claudin-5 protein site mutant D68E having an amino acid sequence represented by SEQ ID NO. 41 of 1/5 or less of the binding ability to human Claudin-5 protein having an amino acid sequence represented by SEQ ID NO. 27.

Item 6.

The antibody according to item 1 or 2, which specifically recognizes a region of amino acids 147 (phenylalanine) to 163 (alanine) at the N-terminus in the human CLDN-5 protein formed from the amino acid sequence represented by SEQ ID NO. 27.

Item 7.

The antibody according to items 1, 2 and 6, which has a binding property to protein 5-5-1 formed from the amino acid sequence represented by SEQ ID NO. 48 of 1/5 or less which is a binding property to human Claudin-5 protein formed from the amino acid sequence represented by SEQ ID NO. 27, wherein the second extracellular loop of the human Claudin-5 protein in the protein 5-5-1 is replaced with the second loop of the human Claudin-1 protein.

Item 8.

The antibody according to item 7, which has a binding ability to human Claudin-5 protein site mutant S151T having the amino acid sequence represented by SEQ ID NO. 43 of 1/5 or less of the binding ability to human Claudin-5 protein having the amino acid sequence represented by SEQ ID NO. 27.

Item 9.

The antibody according to item 1 or 2, which specifically recognizes a three-dimensional structure formed by a region of N-terminal amino acids 28 (proline) to 80 (alanine) and a region of N-terminal amino acids 147 (phenylalanine) to 163 (alanine) in the human CLDN-5 protein having the amino acid sequence represented by SEQ ID NO. 27.

Item 10.

The antibody according to items 1, 2 and 9, which has a binding property to a mutant TM having a mouse Claudin-5 protein substituted for a region other than the extracellular region of the human Claudin-5 protein, said mutant TM having a binding property to the human Claudin-5 protein having an amino acid sequence represented by SEQ ID NO. 27 of 1/5 or less.

Item 11.

The antibody according to any one of claims 1 to 10, which is any one selected from the group consisting of:

(I) antibody I comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 106 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 108 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 110 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID No. 197 or an amino acid sequence having 95% or more identity to said amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID No. 199 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 201 or an amino acid sequence having 95% or more identity to the amino acid sequence;

(E) antibody E comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 78 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 80 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 82 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID No. 169 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 171 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID No. 173 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(B) antibody B comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 57 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 59 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 61 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 148 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 150 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 152 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(F) an antibody F comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 85 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 87 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 89 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 176 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 178 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 180 or an amino acid sequence having 95% or more identity to the amino acid sequence;

(C) antibody C comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 64 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 66 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 68 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 155 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 157 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 159 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(A) antibody A comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 50 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 52 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 54 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 141 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 143 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 145 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(K) antibody K comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 120 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 122 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 124 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 211 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 213 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 215 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(D) an antibody D comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 71 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 73 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 75 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 162 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 164 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 166 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(G) an antibody G comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 92 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 94 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 96 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 183 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID No. 185 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID No. 187 or an amino acid sequence having 95% or more identity to the amino acid sequence;

(H) antibody H comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 99 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 101 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 103 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 190 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 192 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 194 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(J) an antibody J comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 113 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 115 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 117 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 204 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 206 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 208 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(L) an antibody L comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 127 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 129 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 131 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 218 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 220 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 222 or an amino acid sequence having 95% or more identity to said amino acid sequence; and

(M) an antibody M comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 134 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 136 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 138 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 225 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 227 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 229 or an amino acid sequence having 95% or more identity to said amino acid sequence.

Item 12.

The antibody according to any one of claims 1 to 11, wherein the sequence of VH in the variable region of the antibody is the sequence of seq id No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or an amino acid sequence having 90% or more identity to any one of these amino acid sequences, and the sequence of VL is the sequence of seq id No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, or an amino acid sequence having 90% or more identity to any one of these amino acid sequences.

Item 13.

A cell comprising a polynucleotide encoding the antibody of any one of claims 1-12.

Further, as another embodiment, the present invention includes the following modes:

item 1A.

An antibody that specifically recognizes the three-dimensional structure or the primary structure of the extracellular region of the Claudin-5 protein.

Item 2A.

The antibody of item 1A, which does not bind to the extracellular domain of Claudin-1 protein, Claudin-2 protein, Claudin-3 protein, Claudin-4 protein, Claudin-6 protein and Claudin-7 protein.

Item 3A.

The antibody according to item 1A or 2A, which specifically recognizes a region from amino acid (proline) at position 28 to amino acid (alanine) at position 80 of the N-terminus in the human CLDN-5 protein formed from the amino acid sequence represented by SEQ ID NO. 27.

Item 4A.

The antibody according to any one of items 1A to 3A, which has a binding property to protein 1-1-5 formed from the amino acid sequence represented by SEQ ID NO. 45 of 1/5 or less of a binding property to human Claudin-5 protein formed from the amino acid sequence represented by SEQ ID NO. 27, wherein the first extracellular loop of human Claudin-5 protein is replaced with the first loop of human Claudin-1 protein in the protein 1-1-5.

Item 5A.

The antibody according to item 4A, which has a binding ability to human Claudin-5 protein site mutant D68E having the amino acid sequence represented by SEQ ID NO. 41 of 1/5 or less of the binding ability to human Claudin-5 protein having the amino acid sequence represented by SEQ ID NO. 27.

Item 6A.

The antibody according to item 1A or 2A, which specifically recognizes a region of N-terminal amino acids 147 (phenylalanine) to 163 (alanine) of the human CLDN-5 protein formed from the amino acid sequence represented by SEQ ID NO. 27.

Item 7A.

The antibody according to items 1A, 2A and 6A, which has a binding property to protein 5-5-1 formed from the amino acid sequence represented by SEQ ID NO. 48 of 1/5 or less of a binding property to human Claudin-5 protein formed from the amino acid sequence represented by SEQ ID NO. 27, wherein the second extracellular loop of human Claudin-5 protein is replaced with the second loop of human Claudin-1 protein in the protein 5-5.

Item 8A.

The antibody according to item 7A, which has a binding ability to human Claudin-5 protein site mutant S151T having the amino acid sequence represented by SEQ ID NO. 43 of 1/5 or less of the binding ability to human Claudin-5 protein having the amino acid sequence represented by SEQ ID NO. 27.

Item 9A.

The antibody according to item 1A or 2A, which specifically recognizes a three-dimensional structure formed by a region of N-terminal amino acids (proline) at positions 28 to 80 (alanine) and a region of N-terminal amino acids (phenylalanine) to 163 (alanine) in the human CLDN-5 protein having the amino acid sequence represented by SEQ ID NO. 27.

Item 10A.

The antibody according to items 1A, 2A and 9A, which has a binding property to a mutant TM having a mouse Claudin-5 protein in addition to the extracellular region of the human Claudin-5 protein, wherein the binding property to the mutant TM has a binding property to the human Claudin-5 protein having an amino acid sequence represented by SEQ ID NO. 27 of 1/5 or less.

Item 11A.

The antibody according to any one of claims 1A to 10A, selected from the group consisting of any one of antibodies a to M:

(A) antibody A comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 50 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 52 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 54 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 141 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 143 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 145 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(B) antibody B comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 57 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 59 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 61 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 148 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 150 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 152 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(C) antibody C comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 64 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 66 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 68 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 155 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 157 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 159 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(D) an antibody D comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 71 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 73 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 75 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 162 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 164 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 166 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(E) antibody E comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 78 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 80 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 82 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID No. 169 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 171 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID No. 173 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(F) an antibody F comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 85 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 87 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 89 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 176 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 178 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 180 or an amino acid sequence having 95% or more identity to the amino acid sequence;

(G) an antibody G comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 92 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 94 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 96 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 183 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID No. 185 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID No. 187 or an amino acid sequence having 95% or more identity to the amino acid sequence;

(H) antibody H comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 99 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 101 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 103 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 190 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 192 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 194 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(I) antibody I comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 106 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 108 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 110 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID No. 197 or an amino acid sequence having 95% or more identity to said amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID No. 199 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 201 or an amino acid sequence having 95% or more identity to the amino acid sequence;

(J) an antibody J comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 113 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 115 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 117 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 204 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 206 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 208 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(K) antibody K comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 120 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 122 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 124 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 211 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 213 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 215 or an amino acid sequence having 95% or more identity to said amino acid sequence;

(L) an antibody L comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 127 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 129 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 131 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 218 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 220 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 222 or an amino acid sequence having 95% or more identity to said amino acid sequence; and

(M) an antibody M comprising a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 134 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 136 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 138 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 225 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 227 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 229 or an amino acid sequence having 95% or more identity to said amino acid sequence.

Item 12A.

The antibody according to any one of claims 1A to 11A, wherein the sequence of VH of the variable region of the antibody is the sequence of seq id No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or an amino acid sequence having 90% or more identity to any one of these amino acid sequences, and the sequence of VL is the sequence of seq id No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, or an amino acid sequence having 90% or more identity to any one of these amino acid sequences.

Item 13A.

A human IgG1 chimeric antibody, a human IgG4 chimeric antibody, a human IgG1 mutant chimeric antibody, or a human IgG4 mutant chimeric antibody having a variable region of the antibody of item 11A or 12A, or a modified chimeric antibody of these antibodies.

Item 14A.

A humanized antibody or a modified humanized antibody having complementarity determining regions of the antibody according to item 11A or 12A.

Item 15A.

A Fab fragment, F (ab') 2 fragment, Fv fragment, minibody (minibody), scFv-Fc, scFv, diabody, triabody, or tetrabody having the variable region of the antibody of item 11A or 12A.

Item 16A.

A cell comprising a polynucleotide encoding the antibody or fragment of any one of claims 1A-15A.

Item 17A.

A hybridoma cell comprising a polynucleotide encoding the antibody or fragment of any one of claims 1A-15A.

Item 18A.

A complex of the antibody or fragment according to any one of claims 1A to 15A and a drug.

Item 19A.

An agent comprising at least one selected from the group consisting of the antibody or fragment thereof according to any one of items 1A to 15A, and the complex according to item 18.

Item 20A.

A pharmaceutical agent comprising at least one selected from the group consisting of the antibody or fragment thereof according to any one of items 1A to 15A, and the complex according to item 18.

Item 21A.

The medicament of item 20A, for blood brain barrier modulation.

Item 22A.

The medicament of item 20A, for use in the localized alteration of the Claudin-5 protein.

Effects of the invention

The monoclonal antibody recognizing the extracellular region of CLDN-5 obtained by the present invention has high specificity for CLDN-5, unlike the existing molecules bound to the extracellular region of CLDN-5. Therefore, minicells among cell populations expressing CLDN-5 can be detected and isolated without cell immobilization and permeabilization. Furthermore, the antibody obtained by the present invention can also be used academically. Although it is known that vascular endothelial cells including brain vascular endothelial cells are heterogeneous cell groups, the use of the antibody obtained in the present invention makes it possible to prepare a useful reagent for understanding the mechanism of the blood-brain barrier and key clues of disease failure, such as grouping and analyzing these cells by the difference in expression level of CLDN-5.

The antibody developed in the present invention has sufficient activity to regulate the barrier formed by tight junctions produced by intracerebral vascular endothelial cells, and therefore, for example, by administering the antibody in combination with a drug or by administering a complex of a drug and an antibody, it is possible to promote the passage of the drug through the blood-brain barrier, and to further improve the effect of the drug in preventing or treating diseases such as central nervous diseases.

The present invention can provide an antibody against the extracellular domain of CLDN-5 as a novel antibody, and is useful for studies involving CLDN-5, the blood-brain barrier, and the like.

Drawings

FIG. 1 shows the results of CLDN specificity of the anti-CLDN-5 antibody obtained in example 3-1). The vertical axis is a value obtained by dividing the average fluorescence intensity obtained when each antibody is reacted with each CLDN-expressing cell by the average fluorescence intensity obtained when a solvent is reacted with each CLDN-expressing cell; the horizontal axis represents each antibody, and the columns represent the reaction of each CLDN or mock (mock is a cell not expressing a CLDN protein) expressing cell with the antibody; fig. 1A shows binding to human CLDN and fig. 1B shows binding to mouse CLDN.

FIG. 2 is a FACS histogram showing the binding of the anti-CLDN-5 antibody obtained in example 3-2) to CLDN-5 of each animal species. The names of the various animal species CLDN-5 expressed in the cells using retroviruses are shown at the top of the histogram (h denotes human abbreviation, c denotes cynomolgus monkey abbreviation, m denotes mouse abbreviation, mock denotes retrovirus-infected cells that do not express CLDN protein); the antibody used as the first antibody is shown on the far left of the histogram; in each histogram, the horizontal axis represents a fluorescence signal, the vertical axis represents the number of cells, and Vehicle represents a case where the first antibody is not allowed to act.

FIG. 3 is a FACS histogram suggesting to which of the first or second extracellular region of CLDN-5 the anti-CLDN-5 antibody obtained in example 3-4) binds; the name of a CLDN-5 mutant replacing the extracellular region of CLDN-5 expressed in cells using a retrovirus with the extracellular region of CLDN-1 is shown at the top of the histogram, and the summary of the CLDN-5 mutant is shown at the upper part thereof; the antibody used as the first antibody is shown on the far left of the histogram; in each histogram, the horizontal axis represents a fluorescence signal, and the vertical axis represents the number of cells; vehicle indicates that the primary antibody is not allowed to act.

FIG. 4 is a FACS histogram suggesting which site of the anti-CLDN-5 antibody obtained in example 3-5) is necessary for the recognition of CLDN-5. The name of a CLDN-5 mutant replacing the extracellular region of CLDN-5 expressed in cells using a retrovirus with the extracellular region of CLDN-1 is shown at the top of the histogram, and the summary of the CLDN-5 mutant is shown at the upper part thereof; the antibody used as the first antibody is shown on the far left of the histogram; in each histogram, the horizontal axis represents a fluorescence signal, and the vertical axis represents the number of cells; vehicle indicates that the primary antibody is not allowed to act.

FIG. 5 is a Western blot photograph showing the recognition of the primary structure of CLDN-5 by the anti-CLDN-5 antibody obtained in example 3-6); the first antibody used is shown at the top of the photograph, and the cells used are shown below; the protein names represented by the bands are shown on the left side of the photograph.

FIG. 6 shows the results of daily changes in TEER of cells prepared by forced expression of CLDN-5 of each animal species using retrovirus in MDCKII obtained in example 4-2). The vertical axis represents TEER and the horizontal axis represents the number of days from the day that cells were confluent.

FIG. 7 shows the results of the temporal changes in TEER when cells prepared by forced expression of CLDN-5 of each animal species with retrovirus were treated with anti-CLDN-5 antibody in MDCKII obtained in example 4-3); the horizontal axis represents the time from the addition of the test substance, and the vertical axis represents the value obtained by dividing the TEER value measured at each time by the TEER value at the start of the test and recording the divided value as a percentage; the concentrations shown in the graph indicate the concentrations of the respective substances to be detected in the medium, while the Vehicle indicates the case where the solution for diluting the antibody is added; each point (plot) is the mean (n ═ 3), and the error bars represent the standard deviation; the results of MDCKII/mock are shown in FIG. 7A, the results of MDCKII/hCLDN-5 are shown in FIG. 7B, the results of MDCKII/cCLDN-5 are shown in FIG. 7C, the results of MDCKII/mCLDN-5 are shown in FIG. 7D, the results of treatment with a mouse antibody are shown on the left, and the results of treatment with a rat antibody are shown on the right; furthermore, the results for the Vehicle processing group and the C-CPEmt processing group of the left and right graphs are common values.

FIG. 8 shows the results of measuring TEER after 12 hours of culture medium replacement and 24 hours of culture in MDCKII obtained in example 4-3) by treating cells prepared by forced expression of CLDN-5 of each animal species using retrovirus with an anti-CLDN-5 antibody; the vertical axis represents the value obtained by dividing the TEER value measured at each time by the TEER value at the start of the test and recording the divided value as a percentage; the horizontal axis represents the name and concentration of each test substance, and the columns represent the results of TEER measurement at the start of the test, before medium replacement, and 24 hours after medium replacement; vehicle indicates the case where a solution for diluting the antibody was added; each value is the mean (n-3) and the error bars represent the standard deviation; FIG. 8A shows the results of MDCKII/mock, FIG. 8B shows the results of MDCKII/hLDN-5, FIG. 8C shows the results of MDCKII/cCLDN-5, FIG. 8D shows the results of MDCKII/mLDN-5, the left side shows the results of treatment with a mouse antibody, and the right side shows the results of treatment with a rat antibody; furthermore, the results for the Vehicle processing group and the C-CPEmt processing group of the left and right graphs are common values.

FIG. 9 shows the results of evaluating the permeability of fluorescein sodium after 12 hours from the treatment of cells prepared by forced expression of CLDN-5 of each animal species by retrovirus with anti-CLDN-5 antibody in MDCKII obtained in example 4-4); the vertical axis represents the apparent permeability coefficient, and the horizontal axis represents the name and concentration of each substance to be detected; vehicle indicates the case where a solution for diluting the antibody was added; each value is the mean (n-3) and the error bars represent the standard deviation; in the graph, the results of MDCKII/mock are shown in row 1, the results of MDCKII/hLDN-5 are shown in row 2, the results of MDCKII/cCLDN-5 are shown in row 3, the results of MDCKII/mLDN-5 are shown in row 4, the results of treatment with a mouse antibody are shown on the left, and the results of treatment with a rat antibody are shown on the right; furthermore, the results of the Vehicle processing group and the C-CPEmt processing group of the graphs on the left and right sides of the same row are common values.

FIG. 10 shows the results of evaluating the permeability of 4kDa FITC-labeled dextran after 12 hours for cells prepared by forcedly expressing CLDN-5 of each animal species by retrovirus using an anti-CLDN-5 antibody in MDCKII obtained in example 4-4); the vertical axis represents the apparent permeability coefficient, and the horizontal axis represents the name and concentration of each substance to be detected; vehicle indicates the case where a solution for diluting the antibody was added; each value is the mean (n-3) and the error bars represent the standard deviation; in the graph, the results of MDCKII/mock are shown in row 1, the results of MDCKII/hLDN-5 are shown in row 2, the results of MDCKII/cCLDN-5 are shown in row 3, the results of MDCKII/mLDN-5 are shown in row 4, the results of treatment with a mouse antibody are shown on the left, and the results of treatment with a rat antibody are shown on the right; furthermore, the results of the Vehicle processing group and the C-CPEmt processing group of the graphs on the left and right sides of the same row are common values.

FIG. 11 shows the results of the temporal change in TEER upon treatment of the blood brain barrier mimetic system obtained in example 5-1) with an anti-CLDN-5 antibody; the horizontal axis represents the time from the addition of the test substance, and the vertical axis represents the value obtained by dividing the TEER value measured at each time by the TEER value at the start of the test and recording the divided value as a percentage; the concentrations shown in the graph indicate the concentrations of the respective substances to be detected in the medium, while the Vehicle indicates the case where the solution for diluting the antibody is added; each point is the mean (n is 3), and the error bars represent the standard deviation; the left side shows the results when treated with mouse antibody, and the right side shows the results when treated with rat antibody; furthermore, the results for the Vehicle processing group and the C-CPEmt processing group of the left and right graphs are common values.

FIG. 12 shows the results of evaluating the permeability of sodium fluorescein after 12 hours by treating the blood-brain barrier mimetic system obtained in example 5-2) with an anti-CLDN-5 antibody; the vertical axis represents the apparent permeability coefficient, and the horizontal axis represents the name and concentration of each substance to be detected; vehicle indicates the case where a solution for diluting the antibody was added; each value is the mean (n-3) and the error bars represent the standard deviation; the left side shows the results when treated with mouse antibody, and the right side shows the results when treated with rat antibody; furthermore, the results for the Vehicle processing group and the C-CPEmt processing group of the left and right graphs are common values.

FIG. 13 shows the results of evaluating the permeability of 4kDa FITC-labeled dextran after 12 hours by treating the blood-brain barrier mimetic system obtained in example 5-2) with an anti-CLDN-5 antibody; the vertical axis represents the apparent permeability coefficient, and the horizontal axis represents the name and concentration of each substance to be detected; vehicle indicates the case where a solution for diluting the antibody was added; each value is the mean (n-3) and the error bars represent the standard deviation; the left side shows the results when treated with mouse antibody, and the right side shows the results when treated with rat antibody; furthermore, the results for the Vehicle processing group and the C-CPEmt processing group of the left and right graphs are common values.

FIG. 14 shows the results of evaluation of the localization of ZO-1 and CLDN-5 by immunostaining after 12 hours of treatment of the blood-brain barrier mimetic system obtained in example 5-3) with an anti-CLDN-5 antibody; the first antibody used is shown at the top of the image, the staining image of ZO-1 is shown on the left side, and the staining image of CLDN-5 is shown on the right side; the name of the detected substance subjected to the processing is shown in the upper left of each image; vehicle shows the case where a solution for diluting the antibody was added.

Detailed Description

1. Definition of

In this specification, expressions such as "including" and "including" include concepts of "including", "formed substantially of …", and "formed of … only".

"identity" of amino acid sequences means the degree of amino acid sequence identity of 2 or more amino acid sequences that can be compared with each other. Thus, the higher the identity of two amino acid sequences, the higher the identity or similarity of the two sequences. The degree of identity of amino acid sequences can be determined, for example, by using FASTA as a tool for sequence analysis and using default parameters (default parameters). Alternatively, it can be determined using the algorithm BLAST of Karlin and Altschul (Karlins, Altschul SF. "method for assessing the statistical design of reactions by using general strategies" Proc Natl Acad Sci USA.87: 2264. sup.2268 (1990), Karlins, Altschul SF. "Applications and statistics for multiple-sequencing sequences in molecular sequences" Proc Natl Acad Sci USA.90:5873-7 (1993)). A program called BLASTX based on the BLAST algorithm was developed. Specific methods for these analysis methods are known, and reference may be made to the website of National Center of Biotechnology Information (NCBI) (http:// www.ncbi.nlm.nih.gov /). The "identity" of the nucleotide sequence is also defined as follows.

In the present specification, "conservative substitution" means that an amino acid residue is substituted with an amino acid residue having a similar side chain. For example, a substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine corresponds to a conservative substitution. In addition, amino acid residues having acidic side chains such as aspartic acid and glutamic acid; glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, amino acid residues having uncharged polar side chains; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; threonine, valine, isoleucine, or an amino acid residue having a β -branched side chain; substitutions between amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan and histidine correspond to conservative substitutions. .

In this specification, "CDR" isComplementarity Determining RThe abbreviation of egon, also known as complementarity determining region. CDR refers to a region present in the variable region of an immunoglobulin, which is a region deeply involved in the specific binding of an antibody to an antigen. Also, "light chain CDR" refers to the CDR present in the variable region of the light chain of an immunoglobulin, and "heavy chain CDR" refers to the CDR present in the variable region of the heavy chain of an immunoglobulin.

In the present specification, the term "variable region" refers to a region including CDRs1 to 3 (hereinafter, abbreviated as "CDRs 1-3"). The arrangement order of these CDRs1-3 is not particularly limited, but preferably refers to a region in which the CDRs1, CDR2 and CDR3 are arranged in the order of the CDRs or in the reverse order, continuously or via another amino acid sequence referred to as a Framework Region (FR) described later, in the direction from the N-terminal side to the C-terminal side. The "heavy chain variable region" is a region in which the heavy chain CDRs1-3 are arranged, and the "light chain variable region" is a region in which the light chain CDRs1-3 are arranged.

As described above, the region of each variable region other than the above-mentioned CDR1-3 is referred to as a Framework Region (FR). In particular, the region between the N-terminus of the variable region and the above-mentioned CDR1 is defined as FR1, the region between the CDR1 and the CDR2 is defined as FR2, the region between the CDR2 and the CDR3 is defined as FR3, and the region between the CDR3 and the C-terminus of the variable region is defined as FR 4.

The FR also functions as a linker sequence linking the CDRs1-3, which is a particularly important antigen recognition sequence, and is a region contributing to the formation of the three-dimensional structure of the entire variable region.

In the present specification, various Claudin proteins are also sometimes referred to as "X CLDN-Y" or "X CLDN-Y protein". X represents the species of organism from which the protein is derived (h: human, c: cynomolgus monkey, m: mouse), and Y represents the number of the Claudin protein. For example, the human Claudin-5 protein is designated as "hLDN-5" or "hLDN-5 protein".

2. Antibodies

In one embodiment, the present invention relates to an antibody specifically recognizing the three-dimensional structure or the primary structure of the extracellular region of CLDN-5 protein (in the present specification, sometimes referred to as "antibody of the present invention", "CLDN-5 extracellular region antibody", or simply "anti-CLDN-5 antibody", etc.).

This will be explained below.

The Claudin-5 protein is an expression product of the Claudin-5 (sometimes also referred to as CLDN-5, Cldn-5, CLDN5, Cldn5, etc.) gene, and is a CLDN-5 protein expressed in an organism. The type of organism from which the CLDN-5 protein is derived is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. Among them, human, monkey (especially cynomolgus monkey) and the like are preferable.

The amino acid sequences of CLDN-5 proteins from various biological species are well known. Specifically, for example, a human CLDN-5 protein includes a protein having an amino acid sequence represented by sequence number 27, a cynomolgus monkey CLDN-5 protein includes a protein having an amino acid sequence represented by sequence number 28, and a mouse CLDN-5 protein includes a protein having an amino acid sequence represented by sequence number 29.

The CLDN-5 protein may have amino acid mutations such as substitutions, deletions, additions, and insertions as long as it retains its original activity and the CLDN-5 protein can interact with each other through its extracellular loop to form a tight junction. From the viewpoint of less impairing activity, the mutation is preferably a substitution, and conservative substitutions can be more preferably enumerated.

Preferred specific examples of the CLDN-5 protein include at least one selected from the group consisting of the protein described in the following (a) and the protein described in the following (b):

(a) a protein comprising the amino acid sequence represented by any one of SEQ ID Nos. 27, 28 and 29, and

(b) a protein which is composed of an amino acid sequence having 85% or more identity to the amino acid sequence represented by any one of SEQ ID Nos. 27, 28 and 29 and which has a tight junction-forming ability.

In the above (a) and (b), the amino acid sequences are preferably SEQ ID Nos. 27 and 28.

In the above (b), the identity is more preferably 90% or more, still more preferably 95% or more, and still more preferably 98% or more.

Examples of the protein described in (b) above include, for example, (b') a protein having an inositol phosphate bond-hydrolyzing activity, which is formed by substituting, deleting, adding, or inserting 1 or more amino acids in the amino acid sequence represented by any one of SEQ ID Nos. 27, 28, and 29. In the above (b'), the number of the plurality is, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5, and still more preferably 2 or 3.

The extracellular region of the CLDN-5 protein is a region exposed outside the cell in a state where the CLDN-5 protein is arranged on a cell membrane (preferably an endothelial cell membrane, preferably a vascular endothelial cell membrane, more preferably a brain capillary endothelial cell membrane) across the membrane 4 times, and thus is not particularly limited. In addition, the extracellular region of the CLDN-5 protein is formed of a first extracellular loop present on the N-terminal side and a second extracellular loop present on the C-terminal side. The extracellular region, as well as the first extracellular loop and the second extracellular loop, of each CLDN-5 protein are known or can be readily determined using various transmembrane region prediction tools (e.g., SOSUI: http:// harrier. nagahama-i-bio. ac. jp/SOSUI/etc.), and the like.

Specific examples of the extracellular domain include a region from amino acid (proline) at position 28 to amino acid (alanine) at position 80 at the N-terminus (first extracellular loop) and a region from amino acid (phenylalanine) at position 147 to amino acid (alanine) at position 163 at the N-terminus (second extracellular loop) in the human CLDN-5 protein having the amino acid sequence represented by seq id No. 27. Specific examples of the extracellular region of another CLDN-5 protein include regions corresponding to the above-mentioned regions. In the present specification, the term "corresponding region" refers to a region corresponding to the amino acid sequence of a human CLDN-5 protein when aligned with other CLDN-5 amino acid sequences using a tool for sequence analysis (FASTA, BLAST, etc.).

The antibody of the present invention specifically recognizes the three-dimensional structure or the primary structure of the extracellular region of CLDN-5 protein. In other words, the antibody of the present invention binds or has binding to the three-dimensional structure or primary structure of the extracellular region of CLDN-5 protein.

When the recognition region of the antibody of the present invention is a primary structure, the recognition region is a continuous amino acid sequence. In addition, when the recognition region of the antibody of the present invention has a three-dimensional structure, the recognition region may be a continuous amino acid sequence or a plurality of discontinuous amino acid sequences.

The number of amino acid residues constituting the recognition region of the antibody of the present invention is not particularly limited, and is, for example, 40 or less, 35 or less, 6 to 30, 6 to 25, 6 to 20, 6 to 15, or 6 to 10.

Preferred specific examples of the recognition region of the antibody of the present invention include a region within the first extracellular loop (primary structure or three-dimensional structure), a region within the second extracellular loop (primary structure or three-dimensional structure), a three-dimensional structure formed by the first extracellular loop and/or the second extracellular loop, and the like, and more preferred specific examples thereof include a region within the second extracellular loop (primary structure or three-dimensional structure).

As a preferred specific example of the recognition region of the antibody of the present invention, a region within the first extracellular loop is, for example, a region of amino acids (proline) to 80 (alanine) at position 28 from the N-terminus, and preferably a region containing amino acid (aspartic acid) at position 68 from the N-terminus, in the human CLDN-5 protein having the amino acid sequence represented by sequence number 27. As another specific example of the CLDN-5 protein, a region corresponding thereto can be mentioned.

The region within the second extracellular loop, which is a preferred example of the recognition region of the antibody of the present invention, includes, for example, a region of amino acids 147 th to 163 th amino acids (phenylalanine) from the N-terminus, and preferably a region containing amino acid 151 th (serine) from the N-terminus of the human CLDN-5 protein having the amino acid sequence represented by seq id No. 27. As another specific example of the CLDN-5 protein, a region corresponding thereto can be mentioned.

When the recognition region of the antibody of the present invention is a region within the first extracellular loop, as a preferred embodiment, in the case of an antibody against human CLDN-5 protein, the following antibodies are exemplified: the binding property of the antibody to a protein (1-1-5: examples 3-4) comprising the amino acid sequence represented by SEQ ID NO. 45 is 1/5 or less, 1/20 or less, 1/100 or less, 1/500 or less, 1/2000 or less or 1/10000 or less of the binding property to a human CLDN-5 protein comprising the amino acid sequence represented by SEQ ID NO. 27, wherein in the protein (1-1-5: examples 3-4), the first extracellular loop of the human CLDN-5 protein is replaced with the sequence of the human CLDN-1 protein.

When the recognition region of the antibody of the present invention is a region within the first extracellular loop, as a preferred embodiment, in the case of an antibody against human CLDN-5 protein, the following antibodies are exemplified: the binding property of the antibody to human CLDN-5 protein point mutant D68E (examples 3 to 5) having the amino acid sequence represented by sequence No. 41 is 1/5 or less, 1/20 or less, 1/100 or less, 1/500 or less, 1/2000 or 1/10000 or less, of the binding property to human CLDN-5 protein having the amino acid sequence represented by sequence No. 27.

When the antibody of the present invention is an antibody against a human CLDN-5 protein having a region within the first extracellular loop as a recognition region, an antibody having binding to a protein (5-5-1: example 3-4) having an amino acid sequence represented by SEQ ID NO. 48, wherein the second extracellular loop of the human CLDN-5 protein in the protein (5-5-1: example 3-4) is replaced with a sequence of the human CLDN-1 protein, is a preferred embodiment.

When the recognition region of the antibody of the present invention is a region within the second extracellular loop, as a preferred embodiment, in the case of an antibody against human CLDN-5 protein, the following antibodies are exemplified: the binding property of the antibody to a protein (5-5-1: examples 3-4) comprising the amino acid sequence represented by SEQ ID NO. 48 is 1/5 or less, 1/20 or less, 1/100 or less, 1/500 or less, 1/2000 or less or 1/10000 or less of the binding property to a human CLDN-5 protein comprising the amino acid sequence represented by SEQ ID NO. 27, wherein in the protein (5-5-1: examples 3-4), the second extracellular loop of the human CLDN-5 protein is replaced with the sequence of the human CLDN-1 protein.

When the recognition region of the antibody of the present invention is a region within the second extracellular loop, as a preferred embodiment, in the case of an antibody against human CLDN-5 protein, the following antibodies are exemplified: the binding property of the antibody to the human CLDN-5 protein point mutant S151T (examples 3 to 5) having the amino acid sequence represented by sequence No. 43 is 1/5 or less, 1/20 or less, 1/100 or less, 1/500 or less, 1/2000 or 1/10000 or less, of the binding property to the human CLDN-5 protein having the amino acid sequence represented by sequence No. 27.

When the antibody of the present invention is an antibody against a human CLDN-5 protein having a region within the second extracellular loop as a recognition region, an antibody having binding ability to a protein (1-1-5: examples 3-4) having an amino acid sequence represented by SEQ ID NO. 45, wherein the first extracellular loop of the human CLDN-5 protein in the protein (1-1-5: examples 3-4) is replaced by a sequence of the human CLDN-1 protein, is a preferred embodiment.

In addition, the binding of the antibody to be detected to the target CLDN protein can be detected by fluorescence staining cells expressing the target CLDN protein using the antibody to be detected (or a solution for diluting the antibody), and performing FACS analysis on the fluorescence stained cells, as in examples 1 to 4. The sum of the fluorescent signals of the cells can be regarded as the binding of the detected antibody to the target CLDN protein. When the total of the fluorescence signals obtained by using the antibody to be detected is 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times, 1000 times, 5000 times, or 10000 times or more the total of the fluorescence signals obtained by using the antibody diluent (Vehicle), it can be determined that the antibody to be detected "has binding properties to the target protein". The binding property is the same as below.

The three-dimensional structure formed by the first extracellular loop and/or the second extracellular loop, which is a preferred example of the recognition region of the antibody of the present invention, is, for example, a three-dimensional structure formed by a region of N-terminal amino acids (proline) at positions 28 to 80 and a region of N-terminal amino acids (phenylalanine) at positions 147 to 163 (alanine) in human CLDN-5 protein having an amino acid sequence represented by seq id No. 27, a three-dimensional structure formed by a region of N-terminal amino acids (proline) at positions 28 to 67 (tyrosine) and a region of N-terminal amino acids (phenylalanine) at positions 147 to 163 (alanine), and a three-dimensional structure formed by a region of N-terminal amino acids (proline) at positions 28 to 55 (valine) and a region of N-terminal amino acids (phenylalanine) at positions 147 to 163 (alanine) The structure is more preferably a three-dimensional structure formed by a region from the 28 th amino acid (proline) to the 48 th amino acid (lysine) at the N-terminus and a region from the 147 th amino acid (phenylalanine) to the 163 th amino acid (alanine) at the N-terminus. As another specific example of the CLDN-5 protein, a region corresponding thereto can be mentioned.

When the recognition region of the antibody of the present invention is a three-dimensional structure formed by a first extracellular loop and/or a second extracellular loop, as a preferred embodiment, in the case of an antibody against a human CLDN-5 protein, the following antibodies can be exemplified: the binding properties of the antibody to a mutant of the amino acid sequence represented by SEQ ID NO. 44 (TM: examples 3 to 5) wherein an amino acid of mouse CLDN-5 is introduced into the transmembrane region of the human CLDN-5 protein are 1/5 or less, 1/20 or less, 1/100 or less, 1/500 or less, 1/2000 or less, and 1/10000 or less, the binding properties of the antibody to the human CLDN-5 protein of the amino acid sequence represented by SEQ ID NO. 27.

When the antibody of the present invention is an antibody against human CLDN-5 protein having a three-dimensional structure formed by the first extracellular loop and/or the second extracellular loop as a recognition region, the following antibodies may be exemplified as a preferred embodiment: the binding properties of the antibody to human CLDN-5 protein mutants (1-5-5 and 5-1-5: examples 3-4) having the amino acid sequences represented by SEQ ID Nos. 46 and 47 are 1/5 or less, 1/20 or less, 1/100 or less, 1/500 or less, 1/2000 or less, and 1/10000 or less, which are the binding properties to the human CLDN-5 protein having the amino acid sequence represented by SEQ ID No. 27.

Preferably, the antibodies of the invention have a higher specificity for CLDN-5 proteins. This can further reduce side effects caused by the opening of tight junctions other than the blood-brain barrier. From this viewpoint, preferred examples of the antibody of the present invention include an antibody having a binding property to a CLDN family protein other than the CLDN-5 protein of not more than 1/5, which is a binding property to a CLDN-5 protein.

Examples of "proteins of the CLDN family other than the CLDN-5 protein" to be compared for determining the binding properties include, in the case of human, CLDN-1, CLDN-2, CLDN-3, CLDN-4, CLDN-6, and CLDN-7, and in the case of mouse, CLDN-1, CLDN-2, CLDN-3, and CLDN-4. In the determination of the binding property, the CLDN family protein to be compared may be only one type, two or more types may be arbitrarily combined, or all types may be used.

From the viewpoint of specificity for a CLDN-5 protein, it is preferable that the antibody of the present invention does not bind to extracellular regions of human and mouse CLDN-1 proteins, human and mouse CLDN-2 proteins, human and mouse CLDN-3 proteins, human and mouse CLDN-4 proteins, human CLDN-6 proteins, and human CLDN-7 proteins.

The dissociation constant (Kd) of the antibody of the present invention is not particularly limited. Kd is, for example, 1X 10^-7(M) or less, preferably 3X 10^-8(M) or less, more preferably 1X 10^-8(M) or less, more preferably 5X 10^-9(M) is as follows.

Preferred examples of the antibody of the present invention include the following antibodies A to M, from the viewpoints of binding to CLDN-5 protein, blood-brain barrier controlling activity, and the like. Further, as the antibodies a to M, mouse anti-CLDN-5 antibody 5 clone and rat anti-CLDN-5 antibody 8 clone can be more preferably exemplified. The antibodies were named M3, M11, M23, M29, M48, R2, R3, R8, R9, R11, R14, R20, R28 according to the clone names of the respective hybridoma cells. Among them, M48 and R9 are preferable. The antibodies A to M will be described below.

(antibody A)

Antibody A comprises a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 50 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 52 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 54 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 141 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 143 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 145 or an amino acid sequence having 95% or more identity to said amino acid sequence.

The antibody A may further contain FRs of the heavy chain variable region and the light chain variable region, as long as it contains at least the CDRs1-3 of the heavy chain and the light chain. The sequences of FR1 to FR4 in the heavy chain variable region, the sequences of FR1 to FR4 in the light chain variable region, the entire sequences of the heavy chain variable region, and the entire sequences of the light chain variable region include the sequences of antibody M3 shown in tables 2 and 4, and amino acid sequences having an identity of 95% or more with respect to the sequences. More specifically, antibody a includes antibody M3.

(M3)

The sequence of the entire heavy chain variable region (a sequence obtained by arranging FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 in this order from the N-terminal side) of mouse anti-CLDN-5 antibody M3 was the amino acid sequence represented by sequence number 1, and the sequence of the entire light chain variable region (a sequence obtained by arranging FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 in this order from the N-terminal side) was the amino acid sequence represented by sequence number 2. The present antibody is presumed to recognize the three-dimensional structure of the second extracellular loop. Furthermore, it is presumed that the present antibody recognizes a three-dimensional structure produced by the transmembrane region of hLDN-5.

(antibody B)

Antibody B comprises a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 57 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 59 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 61 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 148 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 150 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 152 or an amino acid sequence having 95% or more identity to said amino acid sequence.

The antibody B may further contain FRs of the heavy chain variable region and the light chain variable region, as long as it contains at least the CDRs1-3 of the heavy chain and the light chain. The sequences of FR1 to FR4 in the heavy chain variable region, the sequences of FR1 to FR4 in the light chain variable region, the entire sequences of the heavy chain variable region, and the entire sequences of the light chain variable region include the sequences of antibody M11 shown in tables 2 and 4, and amino acid sequences having an identity of 95% or more with respect to the sequences. More specifically, antibody B includes antibody M11.

(M11)

The sequence of the entire heavy chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) of mouse anti-CLDN-5 antibody M11 is the amino acid sequence represented by sequence number 3, and the sequence of the entire light chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) is the amino acid sequence represented by sequence number 4. The present antibody is presumed to recognize the three-dimensional structure of the first extracellular loop.

(antibody C)

Antibody C comprises a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 64 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 66 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 68 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 155 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 157 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 159 or an amino acid sequence having 95% or more identity to said amino acid sequence.

The antibody C may further contain FRs of the heavy chain variable region and the light chain variable region, as long as it contains at least the CDRs1-3 of the heavy chain and the light chain. The sequences of FR1 to FR4 in the heavy chain variable region, the sequences of FR1 to FR4 in the light chain variable region, the entire sequences of the heavy chain variable region, and the entire sequences of the light chain variable region include the sequences of antibody M23 shown in tables 2 and 4, and amino acid sequences having an identity of 95% or more with respect to the sequences. More specifically, antibody C includes antibody M23.

(M23)

The sequence of the entire heavy chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) of mouse anti-CLDN-5 antibody M23 is the amino acid sequence represented by sequence number 5, and the sequence of the entire light chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) is the amino acid sequence represented by sequence number 6. The present antibody is presumed to recognize the three-dimensional structure of the second extracellular loop.

(antibody D)

Antibody D comprises a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 71 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 73 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 75 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 162 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 164 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 166 or an amino acid sequence having 95% or more identity to said amino acid sequence.

The antibody D may further contain FRs of the heavy chain variable region and the light chain variable region, as long as it contains at least the CDRs1-3 of the heavy chain and the light chain. The sequences of FR1 to FR4 in the heavy chain variable region, the sequences of FR1 to FR4 in the light chain variable region, the entire sequences of the heavy chain variable region, and the entire sequences of the light chain variable region include the sequences of antibody M29 shown in tables 2 and 4, and amino acid sequences having an identity of 95% or more with respect to the sequences. More specifically, antibody D includes antibody M29.

(M29)

The sequence of the entire heavy chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) of mouse anti-CLDN-5 antibody M29 is the amino acid sequence represented by sequence number 7, and the sequence of the entire light chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) is the amino acid sequence represented by sequence number 8. The antibody is presumed to recognize the second extracellular loop.

(antibody E)

Antibody E comprises a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 78 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 80 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 82 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID No. 169 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 171 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID No. 173 or an amino acid sequence having 95% or more identity to said amino acid sequence.

The antibody E may further contain FRs of the heavy chain variable region and the light chain variable region, as long as it contains at least the CDRs1-3 of the heavy chain and the light chain. The sequences of FR1 to FR4 in the heavy chain variable region, the sequences of FR1 to FR4 in the light chain variable region, the entire sequences of the heavy chain variable region, and the entire sequences of the light chain variable region include the sequences of antibody M48 shown in tables 2 and 4, and amino acid sequences having an identity of 95% or more with respect to the sequences. More specifically, antibody E includes antibody M48.

(M48)

The sequence of the entire heavy chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) of mouse anti-CLDN-5 antibody M48 is the amino acid sequence represented by sequence number 9, and the sequence of the entire light chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) is the amino acid sequence represented by sequence number 10. The present antibody is presumed to recognize the three-dimensional structure of the second extracellular loop.

(antibody F)

Antibody F comprises a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 85 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 87 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 89 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 176 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 178 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 180 or an amino acid sequence having 95% or more identity to said amino acid sequence.

The antibody F may further contain FRs of the heavy chain variable region and the light chain variable region, as long as it contains at least the CDRs1-3 of the heavy chain and the light chain. The sequences of FR1 to FR4 in the heavy chain variable region, the sequences of FR1 to FR4 in the light chain variable region, the entire sequences of the heavy chain variable region, and the entire sequences of the light chain variable region include the sequences of antibody R2 shown in tables 2 and 4, and amino acid sequences having an identity of 95% or more with respect to the sequences. More specifically, the antibody F includes an antibody R2.

(R2)

The sequence of the entire heavy chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) of rat anti-CLDN-5 antibody R2 is the amino acid sequence represented by sequence number 11, and the sequence of the entire light chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) is the amino acid sequence represented by sequence number 12. The present antibody is presumed to recognize the three-dimensional structure of the first extracellular loop.

(antibody G)

Antibody G comprises a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 92 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 94 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 96 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 183 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID No. 185 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 187 or an amino acid sequence having 95% or more identity to the amino acid sequence.

The antibody G may further contain FRs of the heavy chain variable region and the light chain variable region, as long as it contains at least the CDRs1-3 of the heavy chain and the light chain. The sequences of FR1 to FR4 in the heavy chain variable region, the sequences of FR1 to FR4 in the light chain variable region, the entire sequences of the heavy chain variable region, and the entire sequences of the light chain variable region include the sequences of antibody R3 shown in tables 2 and 4, and amino acid sequences having an identity of 95% or more with respect to the sequences. More specifically, antibody G includes antibody R3.

(R3)

The sequence of the entire heavy chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) of rat anti-CLDN-5 antibody R3 is the amino acid sequence represented by sequence number 13, and the sequence of the entire light chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) is the amino acid sequence represented by sequence number 14. The present antibody is presumed to recognize the three-dimensional structure of the second extracellular loop.

(antibody H)

Antibody H comprises a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 99 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 101 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 103 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 190 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 192 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 194 or an amino acid sequence having 95% or more identity to said amino acid sequence.

The antibody H may further contain the FRs of the heavy chain variable region and the light chain variable region, as long as it contains at least the CDRs1-3 of the heavy chain and the light chain. The sequences of FR1 to FR4 in the heavy chain variable region, the sequences of FR1 to FR4 in the light chain variable region, the entire sequences of the heavy chain variable region, and the entire sequences of the light chain variable region include the sequences of antibody R8 shown in tables 3 and 5, and amino acid sequences having an identity of 95% or more with respect to the sequences. More specifically, antibody H includes antibody R8.

(R8)

The sequence of the entire heavy chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) of rat anti-CLDN-5 antibody R8 is the amino acid sequence represented by sequence number 15, and the sequence of the entire light chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) is the amino acid sequence represented by sequence number 16.

(antibody I)

Antibody I comprises a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 106 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 108 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 110 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID No. 197 or an amino acid sequence having 95% or more identity to said amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID No. 199 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 201 or an amino acid sequence having 95% or more identity to said amino acid sequence.

The antibody I may contain at least the CDRs1-3 of the heavy and light chains, and may further contain FRs of the heavy chain variable region and the light chain variable region. The sequences of FR1 to FR4 in the heavy chain variable region, the sequences of FR1 to FR4 in the light chain variable region, the entire sequences of the heavy chain variable region, and the entire sequences of the light chain variable region include the sequences of antibody R9 shown in tables 3 and 5, and amino acid sequences having an identity of 95% or more with respect to the sequences. More specifically, antibody I includes antibody R9.

(R9)

The sequence of the entire heavy chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) of rat anti-CLDN-5 antibody R9 is the amino acid sequence represented by sequence number 17, and the sequence of the entire light chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) is the amino acid sequence represented by sequence number 18. The antibody is presumed to recognize the primary structure of the second extracellular loop.

(antibody J)

Antibody J comprises a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 113 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 115 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 117 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 204 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 206 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 208 or an amino acid sequence having 95% or more identity to said amino acid sequence.

The antibody J may further contain FRs of the heavy chain variable region and the light chain variable region, as long as it contains at least the CDRs1-3 of the heavy chain and the light chain. The sequences of FR1 to FR4 in the heavy chain variable region, the sequences of FR1 to FR4 in the light chain variable region, the entire sequences of the heavy chain variable region, and the entire sequences of the light chain variable region include the sequences of antibody R11 shown in tables 3 and 5, and amino acid sequences having an identity of 95% or more with respect to the sequences. More specifically, antibody J includes antibody R11.

(R11)

The sequence of the entire heavy chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) of rat anti-CLDN-5 antibody R11 is the amino acid sequence represented by sequence number 19, and the sequence of the entire light chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) is the amino acid sequence represented by sequence number 20.

(antibody K)

Antibody K comprises a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 120 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 122 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 124 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 211 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 213 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 215 or an amino acid sequence having 95% or more identity to said amino acid sequence.

The antibody K may further contain FRs of the heavy chain variable region and the light chain variable region, as long as it contains at least the CDRs1-3 of the heavy chain and the light chain. The sequences of FR1 to FR4 in the heavy chain variable region, the sequences of FR1 to FR4 in the light chain variable region, the entire sequences of the heavy chain variable region, and the entire sequences of the light chain variable region include the sequences of antibody R14 shown in tables 3 and 5, and amino acid sequences having an identity of 95% or more with respect to the sequences. More specifically, antibody K includes antibody R14.

(R14)

The sequence of the entire heavy chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) of rat anti-CLDN-5 antibody R14 is the amino acid sequence represented by sequence number 21, and the sequence of the entire light chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) is the amino acid sequence represented by sequence number 22. The antibody is presumed to recognize the primary structure and three-dimensional structure of the second extracellular loop. Furthermore, it is presumed that the present antibody recognizes a three-dimensional structure produced by the transmembrane region of hLDN-5.

(antibody L)

Antibody L comprises a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 127 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 129 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 131 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 218 or an amino acid sequence having an identity of 95% or more with respect to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 220 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 222 or an amino acid sequence having 95% or more identity to said amino acid sequence.

The antibody L may further contain the FRs of the heavy chain variable region and the light chain variable region, as long as it contains at least the CDRs1-3 of the heavy chain and the light chain. The sequences of FR1 to FR4 in the heavy chain variable region, the sequences of FR1 to FR4 in the light chain variable region, the entire sequences of the heavy chain variable region, and the entire sequences of the light chain variable region include the sequences of antibody R20 shown in tables 3 and 5, and amino acid sequences having an identity of 95% or more with respect to the sequences. More specifically, antibody L includes antibody R20.

(R20)

The sequence of the entire heavy chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) of rat anti-CLDN-5 antibody R20 is the amino acid sequence represented by sequence number 23, and the sequence of the entire light chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) is the amino acid sequence represented by sequence number 24.

(antibody M)

Antibody M comprises a heavy chain variable region and/or a light chain variable region,

the heavy chain variable region comprises:

a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 134 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 136 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 138 or an amino acid sequence having 95% or more identity to the amino acid sequence,

the light chain variable region comprises:

a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO. 225 or an amino acid sequence having 95% or more identity to the amino acid sequence,

A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO. 227 or an amino acid sequence having 95% or more identity to the amino acid sequence, and

a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO. 229 or an amino acid sequence having 95% or more identity to said amino acid sequence.

The antibody M may further contain the FRs of the heavy chain variable region and the light chain variable region, as long as it contains at least the CDRs1-3 of the heavy chain and the light chain. The sequences of FR1 to FR4 in the heavy chain variable region, the sequences of FR1 to FR4 in the light chain variable region, the entire sequences of the heavy chain variable region, and the entire sequences of the light chain variable region include the sequences of antibody R28 shown in tables 3 and 5, and amino acid sequences having an identity of 95% or more with respect to the sequences. More specifically, antibody M includes antibody R28.

(R28)

The sequence of the entire heavy chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) of rat anti-CLDN-5 antibody R28 is the amino acid sequence represented by sequence number 25, and the sequence of the entire light chain variable region (the sequence in which FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 are arranged in this order from the N-terminal side) is the amino acid sequence represented by sequence number 26.

In the above-mentioned preferred examples (SEQ ID NOS: 1 to 26) of the sequences of the entire heavy chain variable regions and the sequences of the entire light chain variable regions of the antibodies M3, M11, M23, M29, M48, R2, R3, R8, R9, R11, R14, R20 and R28, the amino acid sequences thereof may be mutated. For example, a sequence having preferably 90% or more identity, more preferably 95% or more identity, still more preferably 98% or more identity, and still more preferably 99% or more identity to the above preferred examples (SEQ ID NOS: 1 to 26) can be used as the sequence of the whole heavy chain variable region and the sequence of the whole light chain variable region of the antibodies M3, M11, M23, M29, M48, R2, R3, R8, R9, R11, R14, R20, and R28. The mutation site may be arbitrary, and is preferably a site other than the CDR in order not to reduce the affinity of the antibody.

The antibody of the present invention may be either a monoclonal antibody or a polyclonal antibody, and is preferably a monoclonal antibody in view of Kd value, specificity, and the like.

The molecular weight of the antibody of the present invention is not particularly limited, and the lower limit is, for example, 20,000, preferably 50,000, preferably 100,000, more preferably 120,000, and the upper limit is, for example, 1,000,000, preferably 500,000, more preferably 200,000.

The structure of the antibody of the present invention is not particularly limited. The antibodies of the invention may or may not contain constant regions. When the constant region is contained, all of the constant regions (CH1, CH2, and CH3) of the heavy chain and the constant region (CL) of the light chain may be contained, or any one or a combination of two or more of them may be contained.

Specific examples of the structure of the antibody of the present invention include immunoglobulin, Fab, F (ab')₂A minibody (minibody), scFv-Fc, Fv, scFv, diabody (diabody), triabody (triabody), tetrabody (tetrabody), and the like. Among them, immunoglobulins are preferably used from the viewpoint of the effects of the present invention.

The immunoglobulin has a structure in which two structures including 1 heavy chain having a heavy chain variable region and a heavy chain constant region and 1 light chain having a light chain variable region and a light chain constant region are combined.

The Fab comprises a heavy chain fragment containing CH1 in the heavy chain variable region and the heavy chain constant region, and a light chain containing the light chain variable region and the light chain constant region (CL), and has a structure in which the heavy chain variable region and the light chain variable region are associated with each other by the above-mentioned intermolecular interaction of non-covalent binding, or are bonded by a disulfide bond. In Fab, the sulfhydryl groups present in the cysteine residues of CH1 and CL, respectively, can be disulfide bonded to each other.

F(ab’)₂This means that the Fab has two pairs of the above-mentioned Fab, and CH1 has a disulfide bond formed between them via the thiol group of the cysteine residue contained in them.

The minibody is a structure in which two fragments having CH3 bound to the heavy chain variable region constituting the scFV described below are associated with each other between CH3 by non-covalently bound intermolecular interaction.

The scFv-Fc is a structure in which two antibody fragments containing scFv, CH2, and CH3 described below are associated with each other between CH3 by non-covalently bound intermolecular interaction in the same manner as the above minibody, and a disulfide bond is formed between thiol groups included in cysteine residues of CH 3.

Fv, also called the minimal structural unit of an antibody, is the structure of the variable region of the heavy chain in association with the variable region of the light chain by non-covalently bound intermolecular interactions. In Fv, the thiol groups of cysteine residues present in the heavy chain variable region and the light chain variable region may form disulfide bonds with each other.

The scFv is a structure in which the C-terminus of the heavy chain variable region and the N-terminus of the light chain variable region are connected to each other by a linker, or a structure in which the N-terminus of the heavy chain variable region and the C-terminus of the light chain variable region are connected to each other by a linker, and is also referred to as a single-chain antibody.

Diabodies, triabodies, and tetrabodies are structures in which the aforementioned scfvs form dimers, trimers, and tetramers, respectively, and associate in a structurally stable state, such as by intermolecular interactions through non-covalent binding between variable regions, in the same manner as in Fv and the like.

When the antibody of the present invention is an immunoglobulin, the class thereof is not particularly limited. Examples of the class include IgA, IgD, IgE, IgG, IgM, and subclasses of these classes. Preferred classes include, for example, IgG and IgM, IgG is preferably used, IgG2 is more preferably used, and IgG2a is even more preferably used.

The source of the antibody of the present invention is not particularly limited. The antibody of the present invention may be, for example, a human-derived antibody, a mouse-derived antibody, a rat-derived antibody, a rabbit-derived antibody, a monkey-derived antibody, a chimpanzee-derived antibody, or the like. The antibody of the present invention may be a chimeric antibody (e.g., an antibody obtained by replacing the amino acid sequence of the constant region of an antibody derived from a living body other than a human (e.g., a mouse) with the amino acid sequence of the constant region of an antibody derived from a human), a humanized antibody, a fully humanized antibody, or the like.

The antibody of the present invention can be prepared, for example, by a method according to or based on a conventional method, except that a plasmid expressing the human CLDN-5 protein represented by sequence number 27 is used as an immunogen. (preparation method 1). Specifically, when the antibody of the present invention is a polyclonal antibody, a non-human animal such as a rabbit can be immunized with the plasmid and obtained from the serum of the immunized animal by a conventional method. On the other hand, when the antibody of the present invention is a monoclonal antibody, a non-human animal such as a mouse is immunized with a plasmid, and a hybridoma is prepared by cell fusion of a lymphocyte recovered from the obtained lymph node or spleen with a myeloma cell, and obtained therefrom (Current protocols in Molecular Biology apparatus. Ausubel et al (1987) publish. John Wiley and sons. section 11.4 to 11.11).

The animal to be immunized with the plasmid is not particularly limited as long as it is an animal capable of producing an antibody. The target animal is preferably an autoimmune disease animal (for example, a BXSB mouse in the case of a mouse).

When at least the amino acid sequence of the antibody CDR of the present invention is known, it can also be prepared by a method (preparation method 2) comprising the following steps: culturing a host transformed with a polynucleotide encoding the antibody of the present invention, and recovering a fraction containing the antibody of the present invention.

The polynucleotide encoding the antibody of the present invention is not particularly limited as long as it contains the antibody of the present invention in an expressible state, and may contain other sequences in addition to the coding sequence of the antibody of the present invention. Examples of the other sequences include a secretory signal peptide coding sequence, a promoter sequence, an enhancer sequence, a repressor sequence (repressor sequence), an insulator sequence, a replication origin, and a drug resistance gene coding sequence, which are arranged adjacent to the antibody coding sequence of the present invention. The polynucleotide encoding the antibody of the present invention may be a linear polynucleotide or a cyclic polynucleotide (such as a vector).

Specific examples of the polynucleotide of the present invention include (I) a polynucleotide containing a nucleotide sequence encoding at least one selected from the group consisting of the heavy chain, the heavy chain variable region and the heavy chain CDRs1-3 of the antibody of the present invention, (II) a polynucleotide containing a nucleotide sequence encoding at least one selected from the group consisting of the light chain, the light chain variable region and the light chain CDRs1-3 of the antibody of the present invention, (III) a nucleic acid containing a nucleotide sequence encoding at least one selected from the group consisting of the heavy chain, the heavy chain variable region and the heavy chain CDRs1-3 of the antibody of the present invention, and (III) a polynucleotide containing a nucleotide sequence encoding at least one selected from the group consisting of the light chain, the light chain variable region and the light chain CDRs1-3 of the antibody of the present invention.

The host is not particularly limited, and examples thereof include insect cells, eukaryotic cells, and mammalian cells. Among them, HEK cells, CHO cells, NSO cells, SP2/0 cells, P3U1 cells, and the like are preferable as mammalian cells from the viewpoint of more efficient expression of antibodies.

The method of transformation, culture and recovery is not particularly limited, and a known method for producing an antibody can be used.

After recovery, the antibody of the present invention may be purified as necessary. Purification can be carried out by a known method for antibody production, for example, chromatography, dialysis, or the like.

3. Composite body

In one embodiment, the present invention relates to a complex of an antibody of the present invention and a drug (also referred to as "complex of the present invention" in the present specification). The complex will be described below.

The drug is not particularly limited and may be appropriately selected as needed. Examples of the drug include physiologically active substances such as nucleic acids, polynucleotides, genes and their analogs, glycosaminoglycans and their derivatives, oligosaccharides, polysaccharides and their derivatives, proteins, and peptides; an anti-neurologic agent, an anti-viral agent, an anti-cancer agent, an anti-biomass agent, an enzyme preparation, an antioxidant, an anti-inflammatory agent, a steroid preparation, an angiotensin converting enzyme inhibitor, a vasodilator, an inhibitor of proliferation and/or migration of smooth muscle cells, a platelet aggregation inhibitor, an anti-coagulant, an inhibitor of allergic mediator release, examples of the pharmaceutically active substance include immunosuppressants, lipid uptake inhibitors, hormones, angiotensin receptor antagonists, vascular endothelial cell proliferation inhibitors, aldose reductase inhibitors, lipoxygenase inhibitors, immunostimulants, maillard reaction inhibitors, amyloidosis inhibitors, Nitric Oxide Synthase (NOS) inhibitors, Advanced glycation end products (AGEs) inhibitors, antibodies against neurological diseases such as a β antibody and a Tau antibody, and pharmacologically active substances such as radical scavengers. The complex of the present invention contains the antibody of the present invention as a partial structure and is thus capable of more effectively penetrating the blood-brain barrier, and therefore an anti-neurological agent (for example, a drug capable of being used for treatment and/or diagnosis of the central nervous system) is preferable among drugs.

Specific examples of the anti-nerve agent include anxiolytics such as Constan, clorazolam (Sepazon), diazepam, oxazalan (sernal), Solanax, etizolam (Depas), isoxaflutole (Balance), lexan (meilac), chlordiazepam (Rize), clonazepam (Rivotril), bromazepam (Lexotan), lorazepam (Wypax), tandospirone (Sediel), tofacibenzone (Grandaxin), and fludizepam (Erispan); antidepressants such as anafennil (Anafranil), imipramine hydrochloride (Tofranil), amitriptyline (Trypanol), amoxapine, lofepramine (Amplit), Doryopin (Prothiaden), spetriptyline (Tecipul), mianserin hydrochloride (Tetramide), Ludomam, trazodone (Desyrel), trazodone hydrochloride (Reslin), sulpirine (Abilit), demonomethyl (Dogmatyl), sulpiride (Miradol), ritalin, Depromel, paroxetine, fluvoxamine maleate, and milnacipran hydrochloride (Toledomine); anti-insomnia drugs such as zopiclone (Amoban), euphoria (halcinon), Evamyl, zolpidem tartrate (Myslee), limazane (Rhythmy), lenitron, lenalidomide, flunitrazepam (Silec), quazepam (Doral), nitrodiazepam (Benzalin), estazolam, flunitrazepam (Rohypnol), fluvalin hydrochloride (Insulin), halooxazolam (Somelin), fluazepam hydrochloride (Dalmate), phenobarbital (Phenobal), and isoparbital (Isoytal); sedatives such as chlorpromazine, chlorpromazine (Contomin), piperazines (Neuleptil), levopromazine (Hirnamin), PZC, thioridazine (Mellaril), brepiridon (Impromen), haloperidol (Serenace), pimozide (Orap), moxapamide (Cremine), chlorocarpamine hydrochloride (Clofekton), carpipamine hydrochloride (Defekton), Oxipatin (Forit), zotepin (Lodopin), and hydroxyzine (Atarax); antimanic agents such as lithium carbonate (Limas) and carbamazepine; antiepileptic drugs such as ethaphenytoin, phenytoin, acetylphenylbutyluride, epileptone, sutamine, ethosuximide, clonazepam, carbamazepine, sodium valproate, and zonisamide; antiparkinsonian drugs such as levodopa, pergolide mesylate, amantadine hydrochloride, diphenhydramine hydrochloride, piroheptadine hydrochloride, mazatine hydrochloride, methathixene hydrochloride, biperiden, prophenamine, droxidopa, and the like.

The complex of the present invention may be formed by directly binding the antibody of the present invention to a drug, or by indirectly binding the antibody of the present invention to a drug via a linker or the like. The binding mode is not particularly limited, and examples thereof include a covalent bond, a coordinate bond, and an ionic bond. Furthermore, the antibody of the present invention may be modified on the surface of microparticles such as liposomes to contain a drug in the microparticles. The binding of the antibody of the present invention to a drug can be carried out according to a known method or by a known method, depending on the binding mode.

The covalent bond can be carried out, for example, by reacting the antibody of the present invention with a functional group of each of the drugs or a functional group introduced as needed. Examples of the combination of such functional groups include amino groups and carboxyl groups, carboxyl groups and hydroxyl groups, maleimide groups and mercapto groups, mercapto groups and mercapto groups, hydrazide groups and ketone groups, hydrazide groups and aldehyde groups, amino groups and aldehyde groups, mercapto groups and carboxyl groups, amino groups and squaric acid derivatives, enal groups and amino groups, halogenated esters and mercapto groups, azide groups and alkyne groups.

4. Medicine

In one embodiment, the present invention relates to a drug containing at least one selected from the group consisting of the antibody of the present invention and the complex of the present invention (in the present specification, also referred to as "the drug of the present invention").

The antibody of the invention can open the connection between cerebrovascular endothelial cells and promote the substance transmission of blood brain barrier. Therefore, the antibody of the present invention and the complex of the present invention can be suitably used as an active ingredient of a medicament, and in particular, can be suitably used as an active ingredient of a medicament for the following uses: blood brain barrier regulation (inhibition), brain vascular endothelial cell barrier function regulation (inhibition), blood brain barrier drug permeation promotion, CLDN-5 protein localization change (i.e., the use of changing the location of CLDN-5 usually present in the intercellular space, reducing the adhesion of the intercellular space), and the like.

When the drug of the present invention contains the antibody of the present invention but does not contain the complex of the present invention, the drug can be used in combination with a drug to promote the permeation of the drug through the blood-brain barrier. When the drug of the present invention contains the complex of the present invention, the drug in the complex or the drug to be used in combination can be promoted to permeate the blood-brain barrier by using the drug as it is or in combination with the drug.

The content of the active ingredient in the drug of the present invention may be appropriately set in consideration of the kind of the disease to be treated, the target therapeutic effect, the administration method, the treatment time, the age of the patient, the weight of the patient, and the like. For example, the content of the active ingredient in the drug of the present invention may be about 0.0001 to 100 parts by weight, based on 100 parts by weight of the entire drug of the present invention.

The administration form of the drug of the present invention is not particularly limited as long as the desired effect can be obtained, and the drug can be administered to mammals including humans by any administration route of oral administration and non-oral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, topical administration). Preferred forms of administration are non-oral, more preferably intravenous. Dosage forms for oral administration and non-oral administration and methods for producing the same are well known to those skilled in the art, and can be produced by a conventional method by mixing an active ingredient with a pharmaceutically acceptable carrier or the like.

Examples of dosage forms for parenteral administration include preparations for injection (e.g., intravenous drip, intravenous injection, intramuscular injection, subcutaneous injection, and intradermal injection), external preparations (e.g., ointment, cataplasm, and lotion), suppositories, inhalants, eye drops, eye ointments, nasal drops, ear drops, and liposome preparations. For example, the antibody or cell can be dissolved in distilled water for injection to prepare a preparation for injection, and if necessary, a cosolvent, a buffer, a pH adjuster, an isotonic agent, an analgesic, a preservative, a stabilizer, and the like can be added. The medicine can also be prepared into a freeze-dried preparation prepared in use.

The drug of the present invention preferably further contains other components capable of improving the blood-brain barrier regulating (inhibiting) activity, the brain vascular endothelial cell layer barrier function regulating (inhibiting) activity, the blood-brain barrier drug permeation promoting activity, and the like. Examples of such a component include an occludin (occludin) antibody.

The medicament of the present invention may further contain other pharmaceutical agents effective for the treatment or prevention of diseases. The drug of the present invention may further contain, as necessary, components such as a bactericide, an anti-inflammatory agent, a cell activator, vitamins, and amino acids.

As a vehicle for formulating the drug to be used in the present invention, there can be used an excipient, a binder, a disintegrating agent, a lubricant, a coloring agent, a taste-modifying agent, which are generally used in the art, or a stabilizer, an emulsifier, an absorption promoter, a surfactant (surfactant), a pH adjuster, a preservative, an antioxidant, an extender, a wetting agent, a surfactant (surfactant), a dispersant, a buffer, a preservative, a solubilizer, an analgesic, and the like, as required.

The dose of the drug of the present invention can be determined by a clinician based on various factors such as the route of administration, the type of disease, the degree of symptoms, the age, sex, body weight, severity of disease, pharmacological findings such as pharmacokinetic and toxicological profiles, the presence or absence of a drug delivery system, and whether or not the drug is administered as part of another drug combination. The dose of the drug of the present invention can be, for example, about 1. mu.g/kg (body weight) to about 10g/kg (body weight) per day. The dosage regimen of the drug of the present invention can be determined in consideration of the same important factors as the amount thereof to be administered. For example, the administration may be performed 1 time every 1 day to 1 month at the above-mentioned daily dose.

5. Reagent

In one embodiment, the present invention relates to a reagent containing at least one selected from the group consisting of the antibody of the present invention and the complex of the present invention (also referred to as "the reagent of the present invention" in the present specification), and more particularly to a reagent for detecting CLDN-5 or the like in a cell expressing CLDN-5 or in a solubilized state. Herein, the "reagent" also includes a "diagnostic reagent" for use in examination, detection, diagnosis or the like by detecting CLDN-5 or the like.

The reagent of the present invention may be in the form of a composition containing at least one selected from the group consisting of the antibody of the present invention and the complex of the present invention. The composition may contain other ingredients as necessary. Examples of the other components include a base (base), a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a thickener, a humectant, a coloring material, a perfume, and a chelating agent.

The reagent of the present invention may be in the form of a kit containing at least one selected from the group consisting of the antibody of the present invention and the complex of the present invention. The kit may contain instruments, reagents and the like that can be used for detecting and separating CLDN-5 in cells expressing CLDN-5 or in a solubilized state. Examples of such instruments and reagents include test tubes, microtiter plates, agarose beads, latex beads, chromatography columns for purification, labeled antibodies, standard samples (positive control and negative control), and reagents for extracting exosomes (exosomes) (WO 2016/088689).