JP7370569B2 - Humanized antibody that binds amyloid β protein oligomers - Google Patents

Description

The present invention relates to humanized antibodies that bind amyloid beta protein oligomers.

Alzheimer's disease (AD) is the most common form of dementia, and today it is estimated that there are 47 million dementia patients worldwide, of whom AD accounts for approximately 70%. There is. The main pathological feature of AD patient brains is the formation of senile plaques or amyloid plaques consisting of fibrillated insoluble amyloid β (Aβ) peptides and neurofibrillary tangle deposits consisting of hyperphosphorylated tau protein. In AD, Aβ peptide, which is physiologically produced in monomeric form by protease cleavage from “amyloid precursor protein” (APP), a precursor membrane protein that penetrates and localizes through nerve cell membranes, polymerizes to form soluble Aβ oligomers. It is thought that synaptic dysfunction triggers the fatal tau cascade, leading to the development of cognitive dysfunction. Recently, direct PET imaging analysis of Aβ oligomers and tau proteins in the brain has become possible, and it has been revealed that the accumulation of each in the brain begins approximately 15 years before the onset of dementia, with Aβ oligomers preceding the onset of dementia. .

Soluble Aβ oligomers start accumulating in the entorhinal cortex from the stage of preclinical AD (Non-Patent Document 1), and are localized within neurons in the brain, but diffusely appear outside neurons in the brains of AD patients. A plaque-like localization is observed (Non-patent Document 1; Non-Patent Document 2). Controlling extracellular soluble Aβ oligomers prevents the accumulation of intraneuronal Aβ oligomers, which in turn prevents neuronal degeneration and synaptic degeneration, and prevents the onset of memory disorders (Non-patent Document 1) and improves memory disorders (Non-Patent Document 2). has been proven to be achieved.

Humanized antibodies that do not bind to Aβ monomers but specifically bind only to Aβ oligomers have been reported (Patent Documents 1 and 2). This humanized antibody can be used as an active ingredient of an anti-cognitive dysfunction agent, a therapeutic agent for Alzheimer's disease, a senile plaque formation inhibitor, and an amyloid β amyloid fibril formation inhibitor. Furthermore, by detecting Aβ oligomers in a sample collected from a subject using this humanized antibody, it becomes possible to obtain data useful for diagnosing whether or not the subject is an AD candidate.

International Publication No. 2011/016567 International Publication No. 2011/016568

Takamura A. et al. , Mol. Neurodegener. ,2011,6:20 Takamura A. et al. , Life Sci. , 2012, 91: 1177-1186

An object of the present invention is to provide a new humanized antibody that does not bind to Aβ monomers but specifically binds only to Aβ oligomers. Another object of the present invention is to provide an anti-cognitive dysfunction agent, a therapeutic agent for Alzheimer's disease, an inhibitor of senile plaque formation, and an inhibitor of amyloid β-amyloid fibril formation using such an antibody as an active ingredient. Furthermore, another object of the present invention is to provide a method for diagnosing AD in a subject by detecting Aβ oligomers in a sample collected from the subject using this humanized antibody.

In order to solve the above problems, the present inventor conducted intensive studies and found that by humanizing the mouse antibody 6H4, which does not bind to Aβ monomers but specifically binds only to Aβ oligomers, it is possible to bind to Aβ oligomers. It has been found that humanized monoclonal antibodies with significantly high binding specificity can be obtained. The present invention was completed by further studies based on such knowledge, and includes the following aspects.

Item 1.
A humanized monoclonal antibody that binds to amyloid beta protein (Aβ) oligomers, the antibody comprising:
A heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 as CDR1, the amino acid sequence set forth in SEQ ID NO: 2 as CDR2, and the amino acid sequence set forth in SEQ ID NO: 3 as CDR3;
A humanized monoclonal antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 4 as CDR1, the amino acid sequence set forth in SEQ ID NO: 5 as CDR2, and the amino acid sequence set forth in SEQ ID NO: 6 as CDR3.
Item 2.
A heavy chain having the amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having 70% or more identity with the amino acid sequence;
Item 2. The humanized monoclonal antibody according to Item 1, comprising the amino acid sequence set forth in SEQ ID NO: 8, or a light chain having an amino acid sequence having 70% or more identity with the amino acid sequence.
Item 3.
A heavy chain having an amino acid sequence set forth in SEQ ID NO: 7;
The humanized monoclonal antibody according to claim 1, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO: 8.
Item 4.
4. The humanized monoclonal antibody according to any one of Items 1 to 3, which exhibits selective binding to Aβ oligomers.
Item 5.
An anti-cognitive dysfunction agent comprising the humanized monoclonal antibody according to any one of Items 1 to 4.
Item 6.
Item 5. A therapeutic agent for Alzheimer's disease, comprising the humanized monoclonal antibody according to any one of Items 1 to 4.
Section 7.
Item 5. A senile plaque formation inhibitor comprising the humanized monoclonal antibody according to any one of Items 1 to 4.
Section 8.
Item 5. An Aβ amyloid fibril formation inhibitor comprising the humanized monoclonal antibody according to any one of Items 1 to 4.
Item 9.
Detecting Aβ oligomers contained in a sample collected from a subject using the humanized monoclonal antibody according to any one of items 1 to 4,
By comparing the detected amount of Aβ oligomer with the standard that if it is higher than the standard value, it is Alzheimer's type dementia (AD), and if it is lower than the standard value, it is not Alzheimer's type dementia (AD). A method of testing whether a subject has developed AD.
Item 10.
A kit comprising the humanized monoclonal antibody according to any one of claims 1 to 4, which is used for the method according to claim 9.

According to the present invention, humanized monoclonal antibodies with significantly high binding specificity to Aβ oligomers can be obtained.

The amino acid sequence of the heavy chain variable region VH0 and amino acid residues modified from the amino acid sequence shown in SEQ ID NO: 7 in VH1, VH2, VH3, and VH4 are shown. The first and second lines in the figure represent the amino acid numbers of the H chain variable region, and the alphabet in each line represents the modified amino acid (single letter notation). The amino acid sequence of the light chain variable region VL0 and the amino acid residues modified from the amino acid sequence shown in SEQ ID NO: 8 in VL1, VL2, VL3, and VL4 are shown. The first and second lines in the figure represent the amino acid numbers of the L chain variable region, and the alphabet in each line represents the modified amino acid (single letter notation). It is a graph showing the Aβ oligomer selective binding ability of five types of antibodies (6H4, commercially available 6E10, VH0VL0, VH3VL4, VH4VL3). The vertical axis shows the absorbance at a wavelength of 450 nm, and the horizontal axis shows the concentration of Aβ oligomer or Aβ monomer used as an inhibitor. The solid line in each graph represents the Aβ oligomer, and the broken line represents the antibody binding activity using the Aβ monomer as an inhibitor. The IC50 value was determined using Prizm 6.0 (GraphPad) and the selective binding ability of the Aβ oligomer is presented. The left half is a graph showing the neutralizing activity of the 6H4 antibody and its humanized antibody against Aβ oligomer-induced cell death. The vertical axis shows the amount of dead cell-derived LDH released into the medium (% comparison with when Triton-X was added), and the horizontal axis shows the added antibody. The right half shows the results of examining cell damage due to cytotoxicity induced by Aβ oligomers using a Live/Dead two-color fluorescence assay (left column: live cells, middle column: photoreceptor cells, right column: overlapping images). The top horizontal row is without addition of Aβ oligomer, the second horizontal row is with Aβ oligomer added, the third horizontal row is Aβ oligomer + 6H4, the fourth horizontal row is Aβ oligomer + VH0VL0, and the fourth horizontal row is Aβ oligomer + VH4V.

1. Humanized monoclonal antibodies of the invention
The humanized monoclonal antibody of the present invention is a humanized antibody that binds to amyloid β protein (Aβ) oligomer, and has the amino acid sequence set forth in SEQ ID NO: 1 as CDR1, the amino acid sequence set forth in SEQ ID NO: 2 as CDR2, and CDR3. A heavy chain having the amino acid sequence set forth in SEQ ID NO: 3 as CDR1, the amino acid sequence set forth in SEQ ID NO: 5 as CDR2, and the amino acid sequence set forth in SEQ ID NO: 6 as CDR3. It is a humanized monoclonal antibody containing a light chain.

In the present invention, Aβ oligomer refers to human-derived Aβ oligomer.

Aβ protein, the main component of amyloid, is a peptide consisting of 40 to 42 amino acids, and is known to be produced from a precursor protein called amyloid precursor protein (APP) by the action of protease. There is. In addition to the amyloid fibrils recovered in the ultracentrifugation fraction, the amyloid molecules produced from APP include soluble monomers and oligomeric non-fibrillar polymers. "Aβ oligomer" in the present invention refers to a non-fibrous polymer.

In the present invention, "Aβ oligomer" includes, for example, Aβ40 (Aβ1-40) oligomer, Aβ42 (Aβ1-42) oligomer, and Aβ40 (Aβ1-40) Aβ40 labeled with the fluorescent dye 6-carboxytetramethylrhodamine (6-TAMRA) at the N-terminus. (Aβ1-40) was polymerized with Aβ40 (Aβ1-40) or Aβ42 (Aβ1-42) to prepare a specimen containing a large amount of oligomer (Aβ1-40 or Aβ1-42 oligomer). When observed with an electron microscope, a mixture of granular and amorphous molecules can be seen. In Blue Native-PAGE, the molecules show molecular weights of 13.5 to 720 kDa (Aβ1-42 oligomer) and 13.5 to 1,000 kDa, respectively, and in immunoblot analysis after Native-PAGE, 6H4 selectively targets 3-mer and 5-mer. recognize.

The amino acid sequences of SEQ ID NOs: 1 to 6 are shown in Table 1, respectively.

SEQ ID NOs: 1-3 are CDRs 1-3 of the heavy chain of mouse monoclonal antibody 6H4 (Patent No. 5113853) against Aβ oligomer. Furthermore, SEQ ID NOS: 4 to 6 are CDRs 1 to 3 of the light chain of mouse monoclonal antibody 6H4 against Aβ oligomer.

In the humanized monoclonal antibody of the present invention, the heavy chain preferably has an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO: 7. Further, in the humanized antibody of the present invention, the heavy chain more preferably has an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 7. It is further preferred that the humanized antibody of the present invention has an amino acid sequence in which the heavy chain has 90% or more identity with the amino acid sequence set forth in SEQ ID NO:7. Most preferably, the heavy chain of the humanized antibody of the present invention has the amino acid sequence set forth in SEQ ID NO:7.

In the humanized monoclonal antibody of the present invention, the light chain preferably has an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:8. Further, in the humanized antibody of the present invention, the light chain more preferably has an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO:8. It is further preferred that the light chain of the humanized antibody of the invention has an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO:8. Most preferably, the light chain of the humanized antibody of the present invention has the amino acid sequence set forth in SEQ ID NO:8.

As used herein, the percent identity of two amino acid sequences is determined using a computer program. As such a computer program, BLAST is used in the present invention. Various conditions (parameters) for identity search using the BLAST program can be set as appropriate.

A method well known to those skilled in the art for preparing a polypeptide having an activity equivalent to that of a certain polypeptide is a method of introducing a mutation into a polypeptide. For example, a person skilled in the art would be able to introduce appropriate mutations into the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8 using site-directed mutagenesis, etc. to obtain the same activity as the amino acid sequences. Heavy and light chains can be prepared separately.

The amino acid residue to be mutated is preferably mutated to another amino acid that preserves the properties of the amino acid side chain. For example, the properties of amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains (C, M), amino acids with carboxylic acid- and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), and aromatic-containing side chains. Mention may be made of amino acids having chains (H, F, Y, W). If the amino acid mutation is a substitution between amino acids belonging to the same group as described above, the properties of the amino acid side chain will be preserved before and after the introduction of the mutation at the relevant site.

Preferred amino acid sites at which mutations are introduced are 2, 5, 10-13, 15, 19, 23, 43, 45, 46, 72, 77, 81, in the amino acid sequence set forth in SEQ ID NO: 7 for the heavy chain. Examples include amino acid positions 83, 84, 86, 87, 89, 90, and 117.

Preferred amino acid sites at which mutations are introduced are 2, 3, 7, 4, 5, 7, 8, 41, 44, 50, 51, 79, 86, in the amino acid sequence set forth in SEQ ID NO: 8 for the light chain. Examples include amino acid positions 88, 90, 105, 109, and 111.

In addition to the above-mentioned modifications, the humanized monoclonal antibodies of the invention may be further linked to other substances as long as they retain activity. Other substances include, for example, peptides, lipids, sugars and sugar chains, acetyl groups, natural and synthetic polymers, and the like. These modifications can be made to confer additional functionality or to stabilize the antibody.

2. A medicament containing the humanized monoclonal antibody of the present invention
The humanized monoclonal antibody of the present invention has the property of specifically binding to Aβ oligomers. Utilizing this property, it is possible to provide a medicament containing the humanized monoclonal antibody of the present invention as an active ingredient.

Such drugs include anti-cognitive dysfunction agents, anti-cognitive dysfunction agents, senile plaque formation inhibitors, Aβ amyloid fibril formation inhibitors, and the like.

When the humanized monoclonal antibody of the present invention is used as an active ingredient of a medicine, it can be formulated by a method known to those skilled in the art. It can be prepared into an injectable form that can be administered parenterally, for example, by making it into a sterile solution or suspension in water or any other pharmaceutically acceptable liquid, if desired. . For example, the humanized monoclonal antibodies of the invention may be transported in an acceptable carrier or solvent, such as sterile water, physiological saline, vegetable oils, emulsifying agents, suspending agents, surfactants, stabilizers, flavoring agents, excipients, etc. , solvents, preservatives, binders, etc., to form generally acceptable unit doses necessary for pharmaceutical use. The term "pharmaceutically acceptable" indicates that the material is inert and includes conventional materials used as diluents or vehicles for drugs.

Physiological saline, glucose, and other isotonic solutions containing adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as aqueous solutions for injection. These are used with suitable solubilizers such as alcohols, specifically ethanol and polyalcohols (e.g. propylene glycol and polyethylene glycol), and nonionic surfactants (e.g. Polysorbate 80™ or HCO-50). can do.

Sesame oil or soybean oil can be used as the oily liquid, together with benzyl benzoate or benzyl alcohol as a solubilizing agent. Buffers (phosphate buffers, sodium acetate buffers, etc.), analgesics (procaine hydrochloride, etc.), stabilizers (benzyl alcohol, phenol, etc.), and antioxidants can be used in the formulation. The prepared injection solution can be filled into suitable ampoules.

Administration is preferably parenteral, and specific examples include injection, nasal, pulmonary, and transdermal administration. Examples of injection formulations include systemic or local administration, such as intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection.

3. Test method of the present invention
The test method of the present invention includes:
A step of detecting Aβ oligomers contained in a sample collected from a subject using the humanized monoclonal antibody of the present invention,
By comparing the detected amount of Aβ oligomer with the standard that if it is higher than the standard value, it is Alzheimer's type dementia (AD), and if it is lower than the standard value, it is not Alzheimer's type dementia (AD). This is a method of testing whether a subject has developed AD.

Measurement of Aβ oligomers in a sample can be performed using the humanized monoclonal antibody of the present invention, and specific methods include, for example, sandwich solid-phase enzyme immunoassay (chemiluminescence-ELISA) that uses chemiluminescence. Methods such as , immunoprecipitation, immunoblot, flow cytometry, mass spectrometry, and immunohistochemistry can be used.

In this method, a sample collected from a subject is brought into contact with the humanized monoclonal antibody of the present invention. Here, "contact" can be carried out, for example, by adding each of the above antibodies to a sample collected from a subject in a test tube. In this case, the form of the antibody to be added may be a solution or a solid obtained by freeze-drying or the like, as appropriate. When added as an aqueous solution, it may be an aqueous solution containing only the humanized monoclonal antibody of the present invention; for example, a surfactant, excipient, coloring agent, flavoring agent, preservative, stabilizer, The solution may further contain at least one additive selected from the group consisting of buffering agents, suspending agents, tonicity agents, binders, disintegrants, lubricants, fluidity promoters, and flavoring agents. . The concentration of the antibody added is not particularly limited. For example, similar to human immunoglobulin preparations, 500 mg, 1000 mg, 2500 mg preparations, etc. can be suitably used in a freeze-dried state.

The "reference value" used in the test method of the present invention is determined by preparing enough samples from a human individual who has developed AD and a human individual who has not developed AD, and determining the amount contained in each of these samples. It can be set by detecting the amount of Aβ oligomer. At this time, the amount of Aβ oligomer can be detected using the humanized monoclonal antibody of the present invention.

Although the test method of the present invention can be carried out either in vitro or in vivo, it is preferable to carry out it in vitro since it can further reduce the burden on the patient.

The "sample" in the test method of the present invention is not particularly limited as long as it is a tissue derived from a subject. Examples include the subject's brain (brain parenchyma, etc.), organs, and body fluids (blood, cerebrospinal fluid, etc.). In the present invention, blood (more preferably plasma) or cerebrospinal fluid is preferred.

4. Kit of the invention
The kit of the present invention is a kit containing the humanized monoclonal antibody of the present invention used for the test method of the present invention.

In addition to the humanized monoclonal antibody of the present invention, the kit of the present invention includes a procedure manual for the test of the present invention, and may further contain reagents and/or instruments necessary for the test of the present invention.

The above procedure manual may include a description of the "reference value" required in the test method of the present invention. Such a description may be a direct description of the "reference value" or may be an explanation of how to set the "reference value".

In addition to the above, the kit of the present invention may optionally further contain reagents necessary for the test method of the present invention. Such reagents include, for example, sterile water, physiological saline, vegetable oil, surfactants, lipids, solubilizing agents, buffers, protein stabilizers, preservatives, blocking solutions, reaction solutions, and reaction stop solutions. Can be mentioned. The kit of the present invention may contain one or more of these reagents.

<Example 1>
Preparation of humanized anti-Aβ oligomer antibody (1) Design of VH and VL amino acid sequences of anti-Aβ oligomer 6H4 humanized antibody First, IMGT and Kabat [Sequences of Proteins of Immunological Internet, US Dept. Health and Human Services ( [1991)], the CDR sequence of the VH amino acid sequence (SEQ ID NO: 7) of the previously produced anti-Aβ oligomer mouse monoclonal antibody 6H4 was determined. As a result, CDRs 1 to 3 of VH were assigned SEQ ID NOS: 1 to 3.

First, referring to the amino acid sequence of the VH of the anti-Aβ oligomer 6H4 mouse antibody 6H4HV0 and the human germline gene (IGVH2-5) that is most homologous to this, the amino acid sequence of the VH of the anti-Aβ oligomer 6H4 humanized antibody is determined as follows. It was designed as follows. Key FR amino acid sequences were selected from the top 200 candidates obtained by BLAST search algorithms for the amino acid sequences of the human antibody VH framework region (FR) for transplanting the CDR1-3 amino acid sequences (SEQ ID NOs: 1-3) of 6H4VH. We selected 4 acceptor frameworks by considering combinations of homology that can maintain the canonical loop structure. The C-terminal sequence of the variable region is repaired or removed to create charge pairs suitable for the light chain, and the amino acid sequence of murine 6H4 VH CDR (sequence 1 to 3), and the amino acid sequences 6H4VH1 to VH4 (SEQ ID NOs. 9 to 12) of the VH of the anti-Aβ oligomer 6H4 humanized antibody were designed.

As a result, among the amino acid residues in the FR of VH0VL0, the amino acid residue shown in SEQ ID NO: 7 in 6H4VH0 was selected as an amino acid residue that is thought to change the three-dimensional structure of the antigen-binding site and affect antibody binding activity. 2nd Val, 5th Lys, 10th Gly, 11th Ile, 12th Leu, 13th Gln, 15th Ser, 19th Ser, 23rd Ser, 43rd Ser , 45th Glu, 46th Gly, 72nd Ser, 77th Arg, 81st Phe, 83rd Lys, 84th Ile, 86th Ser, 87th Val, 89th Thr , 90th Ala, and 117th Ala were selected, respectively.

Among these selected amino acid residues, at least one amino acid sequence was modified to an amino acid residue present at the same site in 6H4, and humanized antibody VHs containing amino acid sequences with various modifications were designed.

Specifically, for VH, the second Val of the amino acid sequence shown in SEQ ID NO: 7 is Ile, the fifth Lys is Arg, the 10th Gly is Thr or Ala, and the 11th Ile is Leu. , 12th Leu to Val, 13th Gln to Lys, 15th Ser to Thr, 19th Ser to Thr, 23rd Ser to Thr, 43rd Ser to Pro, 45th Glu to Gln, 46th Gly to Ala, 72nd Ser to Thr, 77th Arg to Lys, 81st Phe to Val, 83rd Lys to Thr, 84 th Ile to Met or Leu, 86th Ser->Asn, Asp, 87th Val->Pro, 89th Thr to Pro, 90th Ala->Val, 117th Ala to Ser At least one substituting amino acid modification was introduced.

Next, in the same manner as for the H chain, the sequences of CDRs 1 to 3 of the VL amino acid sequence (SEQ ID NO: 8) were determined as SEQ ID NOs: 4 to 6, and the VL amino acid sequence of the anti-Aβ oligomer 6H4 humanized antibody was determined as follows. It was designed like this.

Using the amino acid sequence 6H4HL0 of the VL of the anti-Aβ oligomer 6H4 mouse antibody and the human germline gene (IGKV2-29) with the highest homology thereto, the amino acid sequence of CDR1 to 3 of the 6H4 VL (SEQ ID NOs: 4 to 6) was transplanted. From the top 200 candidate amino acid sequences of human antibody VL FRs obtained using BLAST search algorithms, we selected 4 acceptor frameworks by considering combinations of key FR amino acid sequences and homology that can maintain the canonical loop structure. Selected. The amino acid sequence of murine 6H4 VL CDR (SEQ ID NOs: 4 to 6) is grafted onto the appropriate position of the FR amino acid sequence of the human antibody VL common sequence, and the anti-Aβ oligomer 6H4 humanized represented by SEQ ID NO: Antibody VL amino acid sequences 6H4VL1 to VL4 (SEQ ID NOs: 13 to 16) were designed.

As a result, among the amino acid residues in the FR of VH0VL0, the second Val and third amino acid residues in 6H4VL0 are thought to change the three-dimensional structure of the antigen-binding site and affect antibody binding activity. Leu, 7th Thr, 14th Ser, 15th Leu, 17th Asp, 18th Glu, 41st Thr, 44th Lys, 50th Lys, 51st Leu, 79th Lys, Glu at position 86, Leu at position 88, Val at position 90, Ala at position 105, Leu at position 109, and Leu at position 111 were selected, respectively.

Among these selected amino acid residues, at least one amino acid sequence was modified to an amino acid residue present at the same site in 6H4, and VL of a humanized antibody containing amino acid sequences with various modifications was designed.

Specifically, in 6H4VL0, 2nd Val to Ile, 3rd Leu to Val, 7th Thr to Ser, 14th Ser to Thr, 15th Leu to Pro, 17th Asp to Gln or Glu or Ala, 18th Glu to Pro, 41st Thr to Phe, 44th Lys to Arg, 50th Lys to Arg, Gln or Lys, 51st Leu 79th Lys to Arg, 86th Glu to Asp, 88th Leu to Val, 90th Val to Ile, 105th Ala to Gln or Pro, 109th Leu At least one amino acid modification, substituting Leu at position 111 with Ile, was introduced into Val.

VH0VL0, VH1VL1, VH1VL2, VH1VL3, VH1VL4, VH2VL1, VH2VL2, VH2VL3, VH2VL4, VH3VL1, VH3VL2 as the antibody V region of the anti-Aβ oligomer 6H4 humanized antibody with modified at least one amino acid residue present in the FR of HV0LV0. , ]VH3VL3, VH3VL4, VH4VL1, VH4VL2, VH4VL3, and VH4VL4 were designed respectively.

The amino acid sequences of the heavy chain variable regions VH0, VH1, VH2, VH3, VH4 and the light chain variable regions VL0, VL1, VL2, VL3, VL4 are shown in FIGS. 1 and 2, respectively.

(2) Preparation of anti-Aβ oligomer humanized antibody The DNA encoding the amino acid sequence of the variable region of the anti-Aβ oligomer humanized antibody is used in the DNA encoding the amino acid sequences of 6H4VH and 6H4VL (SEQ ID NO: 17, 18). When making amino acid modifications using codons that exist, codons that are frequently used in mammalian cells were used.

The DNA sequences encoding the amino acid sequences of 6H4VH0 and 6H4VL0 of the anti-Aβ oligomer 6H4 humanized antibody are shown in SEQ ID NOs: 17 and 18, respectively. Furthermore, variable regions with amino acid modifications were created using codons used in DNA encoding the amino acid sequences of 6H4VH and 6H4VL.

Using these DNA sequences, an expression vector for a humanized antibody was constructed and the humanized antibody was expressed.

(3) Construction of cDNA encoding the VH of the anti-Aβ oligomer humanized antibody The amino acid sequence 6H4VH0 of the VH of the anti-Aβ oligomer humanized antibody shown in SEQ ID NO: 7 designed in (1) above, and the amino acid sequence 6H4VH0 of the VH in the above (2). cDNAs encoding VH1, VH2, VH3, and VH4 shown in FIG. 1 designed by the method were created by total synthesis.

(4) Construction of cDNA encoding the VL of the anti-Aβ oligomer humanized antibody The amino acid sequence 6H4VL0 of the VL of the anti-Aβ oligomer humanized antibody shown in SEQ ID NO: 8 designed in (1) above, and the cDNAs encoding VL1, VL2, VL3, and VL4 shown in FIG. 2 designed by the method were created by total synthesis.

(5) Construction of anti-Aβ oligomer humanized antibody expression vector 6H4VH0 or VH1, VH2, VH3, VH4 obtained in (3) and (4) above is encoded at an appropriate position of the humanized antibody expression vector pVITRO-DHFR3. Various anti-Aβ oligomer humanized antibody expression vectors were constructed by inserting the cDNA encoding 6H4VL0, VL1, VL2, VL3, and VL4.

(6) Expression of anti-Aβ oligomer humanized antibody using animal cells Expression of anti-Aβ oligomer humanized antibody in animal cells is routinely performed using the anti-Aβ oligomer humanized antibody expression vector obtained in (5) above. anti-Aβ oligomer chimeric antibody (VH0VL0) and anti-Aβ oligomer humanized antibody (VH1VL1, VH1VL2, VH1VL3, VH1VL4, VH2VL1, VH2VL2, VH2VL3, A transformed strain producing VH2VL4, VH3VL1, VH3VL2, ]VH3VL3, VH3VL4, VH4VL1, VH4VL2, VH4VL3, VH4VL4) was obtained.

(7) Obtaining purified anti-Aβ oligomer humanized antibody After culturing the transformed strain obtained in Example (6) using CHO-S cells by a normal culture method, the culture supernatant was collected and protein An IgG1 anti-Aβ oligomer humanized antibody fraction was obtained by A purification, and the protein concentration was measured after dialysis.

As a result, the anti-Aβ oligomer humanized antibody HV0LV0 whose VH is 6H4VH0 and whose VL is 6H4VL0, HV0LV2a whose VH is 6H4HV0 and whose VL is LV2a, and the antibody VH0VL4 whose VH is 6H4VH0 and whose VL is VL4; Seventeen types of VH12VL4, consisting of VH of VH12 and VL of VL4, were prepared.

<Example 2> Inhibition ELISA
The Aβ oligomer used in this assay was prepared by diluting synthetic Aβ1-40 (HCl form) with PBS to a concentration of 0.1 mg/ml and incubating at 37° C. for 1 hour. Aβ monomer was prepared by diluting synthetic Aβ1-40 (TFA form) to a concentration of 0.1 mg/ml with PBS and incubating at 37° C. for 1 hour. First, 400 ng/well of Aβ oligomer was immobilized on a 96-well immunoplate, and then blocking was performed using BSA. Aβ monomer or Aβ oligomer prepared by serially diluting humanized antibody, 6H4 antibody, and anti-Aβ antibody (6E10) in the range of 100 pg/ml to 100 μg/ml as a control were mixed, kept warm for 2 hours, and then added to the above 96-well immunoplate. The mixture was then kept warm at room temperature for 10 minutes. The binding strength between the immobilized Aβ oligomer and each antibody was detected by measuring the absorbance at 450 nm in a color reaction using HRP-labeled anti-mouse or anti-human IgG antibody and TMB solution.

Using this method, 6H4 antibody and 17 anti-Aβ oligomer humanized antibodies (VH0VL0, VH1VL1, VH1VL2, VH1VL3, VH1VL4, VH2VL1, VH2VL2, VH2VL3, VH2VL4, VH3VL1, VH3VL2, VH3VL3, VH3VL4, VH4VL1 , VH4VL2, VH4VL3 , VH4VL4), a premixed solution of each antibody and serially diluted Aβ oligomer or Aβ monomer (inhibitor) was added to a 96-well immunoplate with Aβ oligomer solid phase and reacted. I let it happen. In addition, a commercially available 6E10 antibody was used as a control antibody that binds to Aβ oligomers and Aβ monomers without discrimination. When antibodies selectively bind to Aβ oligomers, antibodies that have been mixed with Aβ monomers in advance are not absorbed by Aβ monomers in solution and can therefore bind to immobilized Aβ oligomers. Conversely, antibodies that have been mixed with Aβ oligomers in advance are absorbed by the Aβ oligomers in the solution, so that as the concentration increases, the binding force to the immobilized Aβ oligomers decreases. As a result, for all humanized antibodies, a concentration-dependent decrease in antibody binding strength was detected with Aβ oligomers, whereas a decrease in binding strength as large as that with Aβ monomers was not detected (Fig. 3, left graph). reference). On the other hand, in general, when producing humanized antibodies, simply transplanting the CDR amino acid sequence of a mouse antibody to the FRs of a human antibody often results in a decrease in binding activity, and this is the same in this case. A decrease in IC50 was observed compared to 6H4. The binding activity of VH3VL4 and VH4VL3 was significantly improved (see the table on the right of Figure 3). These results suggest that, like VH0VL, the two antibodies (VH3VL4, VH4VL3) are antibodies that selectively bind to Aβ oligomers, and unlike the commercial control 6E10, the anti-Aβ oligomer mouse antibody 6H4 It was revealed that the protein had the same specific and selective binding ability to Aβ oligomers (see Figure 3).

<Example 3> Aβ oligomer-induced neurotoxicity neutralizing activity In addition, the 6H4 antibody and its humanized antibody (VH0VL0 and the humanized antibody VH1VL4, which has a Kd value inferior to that of the chimeric antibody, have approximately the same Kd value) The Aβ oligomer-induced neurotoxicity neutralizing activity of five types of VH2VL3, VH2VL4, VH3VL1, VH3VL2 and VH4VL1, and two types of VH3VL4 and VH4VL3 with superior Kd values, was evaluated using the following method. First, human Neuroblastoma cells (SH-SY5Y) were cultured in a 24-well plate at a density of 15,000 cells/well for 24 hours in DMEM containing 10% FBS.Next, the medium was cultured with Aβ1-42 (37.5 μg) with or without antibody. The medium was replaced with serum-free medium containing 12.5 μM) + Tamura-labeled Aβ1-40 (12.5 μg 12.5 μM) and cultured for another 24 hours.Cell death induced by Aβ oligomers is caused by LDH derived from dead cells released into the medium. The amount of Aβ oligomer-induced cytotoxicity was determined using the CytoTox96 kit (Promega) (see the left half of Figure 4). (See the right half of Figure 4: 6H4, VH0VL, and VH4VL3 are presented as representative examples).

SEQ ID NO: 1; HCDR1 amino acid sequence SEQ ID NO: 2; HCDR2 amino acid sequence SEQ ID NO: 3; HCDR3 amino acid sequence SEQ ID NO: 4; LCDR1 amino acid sequence SEQ ID NO: 5; LCDR2 amino acid sequence SEQ ID NO: 6; LCDR3 amino acid sequence SEQ ID NO: 7; 6H4VH0 amino acid sequence SEQ ID NO: 8; 6H4VL0 amino acid sequence SEQ ID NO: 9; 6H4VH1 amino acid sequence SEQ ID NO: 10; 6H4VH2 amino acid sequence SEQ ID NO: 11; 6H4VH3 amino acid sequence SEQ ID NO: 12; 6H4VH4 amino acid sequence SEQ ID NO: 13; 6H4VL1 amino acid sequence SEQ ID NO: 14; 6H4VL2 amino acid sequence SEQ ID NO: 15; 6H4VL3 amino acid sequence SEQ ID NO: 16; 6H4VL4 amino acid sequence SEQ ID NO: 17; DNA sequence encoding 6H4VH0 SEQ ID NO: 18; DNA sequence encoding 6H4VL0

Claims (8)

A humanized monoclonal antibody that specifically binds to amyloid β protein (Aβ) oligomer,
A heavy chain having the amino acid sequence set forth in SEQ ID NO: 1 as CDR1, the amino acid sequence set forth in SEQ ID NO: 2 as CDR2, and the amino acid sequence set forth in SEQ ID NO: 3 as CDR3, and the amino acid sequence set forth in SEQ ID NO: 4 as CDR1, A light chain having the amino acid sequence set forth in SEQ ID NO: 5 as CDR2 and the amino acid sequence set forth in SEQ ID NO: 6 as CDR3,
(A) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 11;
(B) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 12;
and a light chain having the amino acid sequence set forth in SEQ ID NO: 15.
Humanized monoclonal antibody. 2. The humanized monoclonal antibody of claim 1, which exhibits selective binding to Aβ oligomers. An anti-cognitive dysfunction agent comprising the humanized monoclonal antibody according to claim 1 or 2 as an active ingredient . A therapeutic agent for Alzheimer's disease, comprising the humanized monoclonal antibody according to claim 1 or 2 as an active ingredient . A senile plaque formation inhibitor comprising the humanized monoclonal antibody according to claim 1 or 2 as an active ingredient . An Aβ amyloid fibril formation inhibitor comprising the humanized monoclonal antibody according to claim 1 or 2 as an active ingredient . Detecting Aβ oligomers contained in a sample collected from a subject using the humanized monoclonal antibody according to claim 1 or 2,
By comparing the detected amount of Aβ oligomer with the standard that if it is higher than the standard value, it is Alzheimer's type dementia (AD), and if it is lower than the standard value, it is not Alzheimer's type dementia (AD). A method of testing whether a subject has developed AD. A kit comprising the humanized monoclonal antibody according to claim 1 or 2, used for the method according to claim 7.