JP7127272B2 - Antagonists against interleukin-6 family cytokines - Google Patents

Description

The present invention relates to antagonists against interleukin-6 (IL-6) family cytokines. More specifically, it relates to an antagonist capable of suppressing signal transduction through the human gp130 receptor by binding to the extracellular region of the human gp130 receptor involved in the signal transduction of IL-6 and other IL-6 family satokines.

Interleukin-6 (IL-6) is associated with rheumatoid arthritis, autoimmune diseases such as Castleman's disease, diseases of the skeletal system, cancer, heart disease, obesity, diabetes, asthma, Alzheimer's disease, and multiple sclerosis. It is believed to be involved in many diseases. These suggest that suppression of IL-6 signaling may be beneficial in the treatment of these diseases.

IL-6 is transduced by binding to the IL-6 receptor (IL-6R) and then dimerizing the gp130 receptor, a signal transducing receptor (Non-Patent Document 1). In addition, IL-6R and gp130 receptors are present not only on cell membranes but also in soluble form, and are believed to transduce and suppress IL-6 signals in vivo (Non-Patent Document 2). In addition to IL-6, the gp130 receptor is interleukin 11 (IL-11) belonging to the IL-6 family, interleukin 27 (IL-27), leukocyte migration inhibitory factor (LIF), and oncostatin M (OSM). , ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), etc. (Non-Patent Document 3).

In recent years, the development of pharmaceuticals (antibody pharmaceuticals) using monoclonal antibodies is progressing. For example, antibody drugs against tumor necrosis factor (TNF-α) and IL-6R are marketed as antibody drugs against inflammation caused by rheumatoid arthritis, and have shown high therapeutic effects.

Taga, T. et al. , Cell, 58, 573-581 (1989) Narzaki, M.; et al. , Blood, 82, 1120-1126 (1993) Heinrich, P.; C. et al. , Biochem. J. , 334, 297-314 (1998)

An object of the present invention is to provide an antagonist that binds to the human gp130 receptor, which is important for signal transduction of human interleukin-6 and its family cytokines, and that is capable of suppressing the signaling of said cytokines.

As a result of intensive studies aimed at solving the above problems, the present inventors found an antagonist that suppresses IL-6 family cytokine signaling by binding to a specific region of the extracellular region of the human gp130 receptor, and have completed the present invention. Completed.

That is, the first aspect of the present invention is an interleukin that can bind to at least the amino acid residues shown in (1) and/or (2) below of the human gp130 receptor containing the amino acid sequence set forth in SEQ ID NO: 1. -6 family cytokines.
(1) amino acid residues from the 252nd valine to the 262nd arginine of SEQ ID NO: 1 (2) amino acid residues from the 288th aspartic acid to the 300th arginine of SEQ ID NO: 1 An embodiment is the antagonist of the first embodiment, wherein the antagonist is an antibody.

In a third aspect of the present invention, the antibody heavy chain complementarity determining region (CDR) 1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 each have the following amino acid sequences: Or an antagonist according to the second aspect, comprising an amino acid sequence having 80% or more homology with said amino acid sequence.
Heavy chain CDR1: amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 28 or SEQ ID NO: 38 Heavy chain CDR2: amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 29 or SEQ ID NO: 39 Heavy chain CDR3: SEQ ID NO: 20, SEQ ID NO Amino acid sequence light chain CDR1 set forth in SEQ ID NO: 30 or SEQ ID NO: 40: amino acid sequence set forth in SEQ ID NO: 23, SEQ ID NO: 33 or SEQ ID NO: 43 Light chain CDR2: amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 34 or SEQ ID NO: 44 Light chain CDR3: amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 35 or SEQ ID NO: 45 In addition, in the fourth aspect of the present invention, the amino acid sequences of the heavy and light chain CDRs of the antibody are from the following (a) The antagonist according to the second or third aspect, which is any of (c).
(a) heavy chain CDR1 in SEQ ID NO:18, heavy chain CDR2 in SEQ ID NO:19, heavy chain CDR3 in SEQ ID NO:20, light chain CDR1 in SEQ ID NO:23, light chain CDR2 in SEQ ID NO:24, and light chain CDR2 in SEQ ID NO:24 (b) heavy chain CDR1 in SEQ ID NO:28, heavy chain CDR2 in SEQ ID NO:29, heavy chain CDR3 in SEQ ID NO:30, and light chain CDR1 in SEQ ID NO:30. (c) heavy chain CDR1 in SEQ ID NO: 38 and heavy chain CDR2 in SEQ ID NO: 39, comprising the amino acid sequences set forth in SEQ ID NO: 33, light chain CDR2 in SEQ ID NO: 34, and light chain CDR3 in SEQ ID NO: 35, respectively; , heavy chain CDR3 in SEQ ID NO: 40, light chain CDR1 in SEQ ID NO: 43, light chain CDR2 in SEQ ID NO: 44, and light chain CDR3 in SEQ ID NO: 45, respectively. A fifth aspect is the antagonist according to any of the second to fourth aspects, wherein the antibody heavy and light chain amino acid sequences are any of (A) to (C) below.
(A) heavy chain in SEQ ID NO: 16 and light chain in SEQ ID NO: 21, respectively (B) heavy chain in SEQ ID NO: 26 and light chain in SEQ ID NO: 31, respectively (C) the heavy chain comprising the amino acid sequence of SEQ ID NO: 36, and the light chain comprising the amino acid sequence of SEQ ID NO: 41, respectively. The antagonist of any of the second to fourth aspects, wherein the sequence comprises at least the amino acid sequence from tryptophan at position 34 to tyrosine at position 48 of SEQ ID NO:170.

A seventh aspect of the present invention is the antagonist according to any one of the second to fourth and sixth aspects, wherein the amino acid sequence of the light chain variable (VL) region of the antibody comprises at least the amino acid sequence set forth in SEQ ID NO: 170. is.

The eighth aspect of the present invention is the second to fourth, sixth and seventh aspects, wherein the antibody heavy chain variable (VH) region amino acid sequence comprises at least the amino acid sequence set forth in SEQ ID NO: 142 or SEQ ID NO: 152. An antagonist according to any of the preceding claims.

A ninth aspect of the present invention is the antagonist according to any one of the second to fourth aspects, wherein the amino acid sequences of the VH region and the VL region of the antibody are any of the following (i) to (ix): be.
(i) the VH region is SEQ ID NO: 140, and the VL region is SEQ ID NO: 170, each containing the amino acid sequences described (ii) the VH region is SEQ ID NO: 142, and the VL region is SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, or SEQ ID NO: 204 at positions 1 to 107, respectively (iii) a VH region having the amino acid sequence described in SEQ ID NO: 144 , wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168 or SEQ ID NO: 170, respectively; (iv) the VH region comprises SEQ ID NO: 146 , the VL region is SEQ ID NO: 170, and (v) the VH region is SEQ ID NO: 148, and the VL region is SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, or SEQ ID NO: 170 , (vi) the VH region in SEQ ID NO: 150, the VL region in SEQ ID NO: 170, and (vii) the VH region in SEQ ID NO: 152, and the VL region in SEQ ID NO: 170, each containing the amino acid sequence described. 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170 or SEQ ID NO: 172, respectively; The region contains the amino acid sequence set forth in SEQ ID NO: 158, SEQ ID NO: 164 or SEQ ID NO: 170, respectively (ix) The VH region contains the amino acid sequence set forth in SEQ ID NO: 156, and the VL region contains the amino acid sequence set forth in SEQ ID NO: 170 (x ) containing the amino acid sequences described in positions 1 to 120 of SEQ ID NO: 212 for the VH region and positions 1 to 107 of SEQ ID NO: 200, 202 or 206 for the VL region, respectively

A further tenth aspect of the present invention is a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36 or SEQ ID NO: 41.

A further eleventh aspect of the present invention is a vector comprising at least the polynucleotide according to the tenth aspect.

A twelfth aspect of the present invention is a transformant obtained by transforming a host with the vector according to the eleventh aspect.

Furthermore, in a thirteenth aspect of the present invention, the transformant according to the twelfth aspect is cultured to express the antibody, and then the expressed antibody is recovered from the culture of the transformant. manufacturing method.

The present invention will be described in detail below.

The antagonist of the present invention comprises at least the following (1 ) and/or (2):
(1) amino acid residues from the 252nd valine to the 262nd arginine (specifically, amino acid residues consisting of VIILKYNIQYR), or
(2) Amino acid residues from the 288th aspartic acid to the 300th arginine (specifically DLKPFTEYVFRIR)
An antagonist capable of suppressing signaling through the human gp130 receptor by binding to the amino acid residue shown in , preferably capable of binding to both the residue (1) and the residue (2) is an antagonist. The antagonist of the present invention is not particularly limited as long as it is a substance that binds to the extracellular region of the human gp130 receptor and has an inhibitory effect on signal transduction through the human gp130 receptor. , RNA aptamers, antibodies, and minibodies. Among them, antibodies or minibodies are preferred, and antibodies are more preferred.

When a low-molecular-weight compound, DNA, or RNA aptamer is used as the antagonist of the present invention, it can be produced using an organic synthesis reaction, and can be purified by using various types of chromatography after a chemical reaction.

As used herein, an antibody refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable (VH) region and a heavy chain constant region. The heavy chain constant region contains three domains (CH1, CH2 and CH3). Each light chain comprises a light chain variable (VL) region and a light chain constant region. The light chain constant region is composed of one domain (CL1). VH and VL regions are further divided into regions of hypervariability, called complementarity determining regions (CDR), and highly conserved regions, called framework regions (FR). As for the definition of CDR, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest 5th edition), Chothia, AbM, contact definition are most commonly used, and CDR in this specification is defined by Kabat is based on The VH and VL regions are each composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. ing. The antibodies of the present invention may be antibodies derived from mice, rats, humans, rabbits, chickens, goats, or the like. Bispecific antibodies, having two specificities within, and the like can also be used. Furthermore, VHH antibodies consisting only of camelid-derived heavy chains can also be used.

As used herein, a minibody means a fragment of an antibody that retains the ability to specifically bind to an antigen (eg, human gp130 receptor). For example,
(i) Fab fragment: a fragment consisting of the VL, VH, CL1 and CH1 domains (ii) F(ab′) ² fragment: a fragment containing two Fab fragments linked by disulfide bridges in the hinge region (iii) Fd fragment: Fragment consisting of VH region and CH1 domain (iv) Fv fragment: Fragment consisting of VL region and VH region of a single arm of antibody. Of these, (iv) the two regions of the Fv fragment, the VL region and the VH region, are usually encoded by separate genes. A conjugated version (single-chain Fv (scFv)) may be produced using genetic recombination techniques.

The antibodies of the present invention are not particularly limited by subclass (for example, there are four types in humans: IgG1, IgG2, IgG3, and IgG4). It may be appropriately selected depending on the necessity of activity (CDC). When producing an antibody intended to kill target cells by ADCC or CDC, a subclass with high ADCC or CDC may be selected, and when producing an antibody that does not require ADCC or CDC, ADCC or CDC is required. Choose a lower subclass. The ADCC and CDC of an antibody may be enhanced or decreased by substituting amino acid residues in the constant region of the antibody and changing sugar chains added to the antibody.

Since the antibodies and low-molecular-weight antibodies described above are proteins, polynucleotides encoding these proteins may be directly introduced into host cells, but cloned into expression vectors containing promoters and signal peptides, It is preferable to produce by culturing a transformant obtained by transforming a host with. When designing a polynucleotide encoding an antibody or low-molecular-weight antibody, it is preferable to consider the frequency of codon usage in the host to be transformed. As an example, when the host is Escherichia coli, AGA/AGG/CGG/CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), and GGA for glycine (Gly) However, in proline (Pro), CCC is used less frequently (because it is a so-called rare codon), so these codons should be converted to avoid them. The codon usage frequency can also be analyzed by using a public database (eg, Codon Usage Database on the homepage of Kazusa DNA Research Institute). The host cells are not particularly limited, and examples include mammalian cells (CHO (Chinese Hamster Ovary) cells, HEK cells, Hela cells, COS cells, etc.), yeast (Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces japonic, Schizosaccharomyces octosporus, Schizosaccharomyces pombe, etc.), insect cells (Sf9, Sf21, etc.), Escherichia coli (JM109 strain, BL21 (DE3) strain, W3110 strain, etc.) and Bacillus subtilis, Bacillus brevis. Among them, mammalian cells are preferred, and CHO cells are particularly preferred, when antibody molecules are expressed.

The expression vector may contain a promoter or signal peptide suitable for the host. For example, when mammals are used as hosts, CMV promoter, SV40 promoter, CAG promoter and EF1α promoter can be exemplified, and signal peptides added to various secretory proteins may be combined. When Escherichia coli is used as a host cell, T7 promoter, trc promoter, lac promoter and the like can be exemplified, and signal peptides can be exemplified by PelB, DsbA, MalE and TorT. Drug markers such as antibiotics may also be introduced into the expression vector, if necessary, to select host cells into which the polynucleotide encoding the expressed protein has been introduced. Furthermore, the expression vector may contain a marker for gene amplification (for example, the dihydrofolate reductase (dhfr) gene) that is performed when producing a high-expressing cell line in CHO cells.

The antibody expressed by culturing the transformant can be purified and recovered from the culture of the transformant by a combination of affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, and the like. can.

Preferred embodiments of the antibody of the present invention include antibodies in which the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 of the antibody each comprise the amino acid sequences shown below.
Heavy chain CDR1: amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 28 or SEQ ID NO: 38 Heavy chain CDR2: amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 29 or SEQ ID NO: 39 Heavy chain CDR3: SEQ ID NO: 20, SEQ ID NO Amino acid sequence light chain CDR1 set forth in SEQ ID NO: 30 or SEQ ID NO: 40: amino acid sequence set forth in SEQ ID NO: 23, SEQ ID NO: 33 or SEQ ID NO: 43 Light chain CDR2: amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 34 or SEQ ID NO: 44 Light chain CDR3: amino acid sequence set forth in SEQ ID NO: 25, SEQ ID NO: 35 or SEQ ID NO: 45 amino acid residues from valine to arginine at position 262 and/or amino acid residues from aspartic acid at position 288 to arginine at position 300 in SEQ ID NO: 1), as long as the amino acid sequences exemplified above have the ability to bind to may have amino acid substitutions, deletions, insertions or additions, specifically, antibody heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 The amino acid sequence may be an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more homology with the amino acid sequences exemplified above.

A more preferred embodiment of the antibody of the present invention includes an antibody in which the heavy chain CDR and light chain CDR amino acid sequences of the antibody are any one of (a) to (c) below.
(a) heavy chain CDR1 in SEQ ID NO:18, heavy chain CDR2 in SEQ ID NO:19, heavy chain CDR3 in SEQ ID NO:20, light chain CDR1 in SEQ ID NO:23, light chain CDR2 in SEQ ID NO:24, and light chain CDR2 in SEQ ID NO:24 (b) heavy chain CDR1 in SEQ ID NO:28, heavy chain CDR2 in SEQ ID NO:29, heavy chain CDR3 in SEQ ID NO:30, and light chain CDR1 in SEQ ID NO:30. (c) heavy chain CDR1 in SEQ ID NO: 38 and heavy chain CDR2 in SEQ ID NO: 39, comprising the amino acid sequences set forth in SEQ ID NO: 33, light chain CDR2 in SEQ ID NO: 34, and light chain CDR3 in SEQ ID NO: 35, respectively; , heavy chain CDR3 in SEQ ID NO: 40, light chain CDR1 in SEQ ID NO: 43, light chain CDR2 in SEQ ID NO: 44, and light chain CDR3 in SEQ ID NO: 45, respectively. An even more preferred embodiment of is an antibody in which the heavy chain and light chain amino acid sequences of the antibody are any one of (A) to (C) below.
(A) heavy chain in SEQ ID NO: 16 and light chain in SEQ ID NO: 21, respectively (B) heavy chain in SEQ ID NO: 26 and light chain in SEQ ID NO: 31, respectively (C) A heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a light chain of SEQ ID NO: 41 comprising the amino acid sequences of SEQ ID NO: 41. Antibodies in which the amino acid sequences of chains other than the CDRs are changed from mouse antibody amino acid sequences to human antibody amino acid sequences (humanized antibodies) (FIG. 4). By increasing the proportion of the amino acid sequence of a human antibody in the entire antibody, it is possible to obtain the advantage of being less likely to be eliminated as a foreign substance when applied to the human body.

Specifically, the amino acid sequence of the framework region in the light chain variable (VL) region of the antibody has at least the amino acid sequence from tryptophan at position 34 to tyrosine at position 48 in SEQ ID NO: 170 (specifically WFQQRPGQAPKVLIY). Antibodies comprising

Another preferred embodiment of the antibody of the present invention includes an antibody in which the amino acid sequence of the VL region of the antibody comprises at least the amino acid sequence set forth in SEQ ID NO:170.

Still another embodiment of the antibody of the present invention includes an antibody in which the amino acid sequence of the heavy chain variable (VH) region of the antibody comprises at least the amino acid sequence set forth in SEQ ID NO:142 or SEQ ID NO:152.

Still another preferred embodiment of the antibody of the present invention includes an antibody in which the amino acid sequences of the VH region and VL region of the antibody are any one of the following (i) to (ix).
(i) the VH region is SEQ ID NO: 140, and the VL region is SEQ ID NO: 170, each containing the amino acid sequences described (ii) the VH region is SEQ ID NO: 142, and the VL region is SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, or SEQ ID NO: 204 at positions 1 to 107, respectively (iii) a VH region having the amino acid sequence described in SEQ ID NO: 144 , wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168 or SEQ ID NO: 170, respectively; (iv) the VH region comprises SEQ ID NO: 146 , the VL region is SEQ ID NO: 170, and (v) the VH region is SEQ ID NO: 148, and the VL region is SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, or SEQ ID NO: 170 , (vi) the VH region in SEQ ID NO: 150, the VL region in SEQ ID NO: 170, and (vii) the VH region in SEQ ID NO: 152, and the VL region in SEQ ID NO: 170, each containing the amino acid sequence described. 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170 or SEQ ID NO: 172, respectively; (ix) the VH region contains the amino acid sequence set forth in SEQ ID NO: 156, and the VL region contains the amino acid sequence set forth in SEQ ID NO: 170; The constant regions other than the variable regions of the heavy and light chains of the humanized antibody described above may be selected from the amino acid sequences of human antibody subclasses IgG1, IgG2, IgG3, and IgG4.
(x) The VH region comprises amino acid sequences 1 to 120 of SEQ ID NO: 212, and the VL region comprises amino acid sequences 1 to 107 of SEQ ID NO: 200, 202, or 206.

Furthermore, the antibodies of the present invention are not limited to the amino acid sequences described above, and substitutions that do not substantially change the functional properties (conservative amino acid substitutions) are also included in the antibodies of the present invention. Examples of conservative amino acid substitutions include valine-leucine-isoleucine substitutions, phenylalanine-tyrosine substitutions, lysine-arginine substitutions, alanine-valine substitutions, glutamic acid-aspartic acid substitutions, and asparagine-glutamine substitutions. substitution.

The antagonist of the present invention can suppress the signaling of IL-6 and other cytokines belonging to the IL-6 family by binding to a specific region of the extracellular domain of the human gp130 receptor. It is expected that it will be useful for the treatment of diseases (for example, autoimmune diseases such as rheumatoid arthritis).

FIG. 2 shows the results of evaluating the binding properties of the antibodies of the present invention to human gp130 receptors. FIG. 10 shows the results of confirming IL-6 signal suppression by the antibody of the present invention (antibody A). FIG. 10 shows the result of confirming IL-6 signal suppression by the antibody of the present invention (antibody B). FIG. 1 shows a schematic diagram of a humanized antibody, according to one embodiment of the present invention.

EXAMPLES The present invention will be described in more detail below using examples, but the present invention is not limited to the examples.

Example 1 Preparation of Soluble Human gp130 Receptor (1) Extracellular Region of Human gp130 Receptor Consisting of the Amino Acid Sequence of SEQ ID NO: 1 (Region from 1st Methionine to 619th Glutamic Acid of SEQ ID NO: 1) A vector for expressing was constructed by the following method.
(1-1) Genes encoding dihydrofolate reductase (dhfr) shown in SEQ ID NO: 2 and PolyA of SV40 were totally synthesized (entrusted to Integrated DNA Technologies) and cloned into a plasmid.
(1-2) Escherichia coli JM109 strain was transformed with the plasmid prepared in (1-1). The resulting transformant was cultured, and the plasmid was extracted and digested with restriction enzyme SacII to prepare a gene encoding dhfr-SV40PolyA and named dhfr-SV40PolyA-P1.
(1-3) Using pIRES vector (manufactured by Clontech) as a template, oligonucleotide primers consisting of the sequences set forth in SEQ ID NO: 3 (5'-TCCCCGCGGGGCGGGACTCTGGGGTTCGAAATGACCG-3') and SEQ ID NO: 4 (5'-TCCCCGCGGGGTGGCTCTAGCCTTAAGTTCGAGACTG-3') PCR was performed using Specifically, a reaction solution having the composition shown in Table 1 was prepared, and after heat treatment of the reaction solution at 98° C. for 30 seconds, a first step at 98° C. for 10 seconds, a second step at 55° C. for 5 seconds, 72 The reaction was carried out by repeating 25 cycles of the third step of 5 minutes at °C. This PCR amplified the region of the pIRES vector excluding the neomycin resistance gene.

(1-4) The PCR product prepared in (1-3) was purified, digested with restriction enzyme SacII, and ligated with dhfr-SV40PolyA-P1 prepared in (1-2). Escherichia coli strain JM109 was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain an expression vector pIRES-dhfr containing the dhfr gene.

(2) Using the pIRES-dhfr prepared in (1) as a template, an oligonucleotide primer consisting of the sequences described in SEQ ID NO: 5 (5'-ACGCGTCGACACTAGAAGCTTTATTGCGGTAGTTTATCAC-3') and SEQ ID NO: 6 (5'-ACGCGTCGACAGATCTGTCGAGCCAGTGAGCAAAAGGCC-3') PCR was performed using Specifically, a reaction solution having the composition shown in Table 2 was prepared, and after heat treatment of the reaction solution at 98° C. for 5 minutes, a first step at 98° C. for 10 seconds, a second step at 55° C. for 5 seconds, 72 The reaction was carried out by repeating 30 cycles of the third step of 7 minutes at °C. By this PCR, the region of the pIRES-dhfr vector excluding the CMV promoter gene was amplified and named pIRES-dhfr-P1.

(3) Using pEBMulti-Neo (manufactured by Wako Pure Chemical Industries, Ltd.) as a template, oligos consisting of the sequences described in SEQ ID NO: 7 (5'-ACGCGTCGACGGATCTCGACATTGATTATTGACTAG-3') and SEQ ID NO: 8 (5'-ACGCGTCGACCAAAATGATGAGACAGCACAATAACC-3') PCR was performed using nucleotide primers. Specifically, a reaction solution having the composition shown in Table 3 was prepared, and after heat treatment of the reaction solution at 98° C. for 5 minutes, a first step at 98° C. for 10 seconds, a second step at 55° C. for 5 seconds, 72 The reaction was carried out by repeating 30 cycles of the third step of 2 minutes at °C. By this PCR, the gene encoding the CAG promoter (Japanese Patent No. 2824433) contained in pEBMulti-Neo was amplified and named CAG-P1.

(4) pIRES-dhfr-P1 prepared in (2) and CAG-P1 prepared in (3) were each digested with restriction enzyme SalI, purified and ligated. Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain an expression vector pCAG-dhfr containing the CAG promoter gene.

(5) A polynucleotide encoding the extracellular region (the region from the first methionine to the 619th glutamic acid of SEQ ID NO: 1) of the human gp130 receptor consisting of the amino acid sequence set forth in SEQ ID NO: 1, six An oligonucleotide encoding histidine and a polynucleotide (SEQ ID NO: 9) added with restriction enzymes NheI and NotI recognition sequences were totally synthesized (entrusted to FASMAC), cloned into a plasmid, and then E. coli JM109 strain was transformed using the plasmid. did. The resulting transformant was cultured, and the plasmid was extracted, digested with restriction enzymes NheI and NotI, and purified to obtain the gene encoding the extracellular region of the human gp130 receptor.

(6) The pCAG-dhfr prepared in (4) was digested with restriction enzymes NheI and NotI, and the gene encoding the extracellular region of the human gp130 receptor prepared in (5) was ligated. Strains were transformed. An expression vector pCAG-hgp130 for expressing the extracellular region of the human gp130 receptor was obtained by extracting the plasmid from the transformant culture.

(7) The pCAG-hgp130 prepared in (6) was transfected into CHO cells (DG44 strain) using NeonTransfection System (manufactured by Thermo Fisher Scientific). Thereafter, the transformed cells were cultured in CD OptiCHO Medium (manufactured by Thermo Fisher Scientific), gene amplification was performed using methotrexate (MTX), and then stable soluble human gp130 receptor protein-producing cells were obtained by the limiting dilution method. got stock.

(8) The cell line obtained in (7) was subjected to shaking culture (37° C., 8% CO ₂ ) in a CO ₂ incubator using CD OptiCHO Medium and an Erlenmeyer flask to secretly express soluble human gp130 receptor protein. let me The supernatant obtained by removing cells and impurities by centrifugation was treated with AKTAprime plus (manufactured by GE Healthcare) to 20 mM Tris-HCl containing 20 mM imidazole and 150 mM sodium chloride in advance (pH 7.0). It was applied to a 1 mL HisTrap HP column (manufactured by GE Healthcare) equilibrated in 4) at a flow rate of 1 mL/min. After washing with the buffer used for the equilibration, elution was performed with 20 mM Tris-HCl (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride. The eluate was subjected to buffer exchange with 20 mM Tris-HCl (pH 7.4) containing 150 mM sodium chloride through an ultrafiltration membrane to obtain a highly pure soluble human gp130 receptor protein.

Example 2 Production of Anti-gp130 Receptor Antibody-Producing Hybridoma (1) 50 μg of the soluble gp130 receptor protein produced in Example 1 is injected into the peritoneal cavity of BALB/c mice for a total of 4 times once every 10 days. I was immunized by

(2) Spleen cells were excised from mice and fused with mouse-derived myeloma cells (SP2/0 strain) using polyethylene glycol. After cell fusion, hybridomas were produced by culturing in DMEM medium (manufactured by Thermo Fisher Scientific) containing HAT (manufactured by Gibco) and 10% (w/v) bovine serum.

(3) The hybridomas prepared in (2) are cultured in a 96-well plate, and the culture supernatant is subjected to the following Enzyme-Linked Immunosorbent Assay (ELISA) to secrete an antibody that binds to the human gp130 receptor protein. Wells in which hybridomas with cytoplasmic activity were present were selected.
(3-1) The soluble human gp130 receptor prepared in Example 1 was immobilized to wells of a 96-well microplate at 1 µg/well (overnight at 4°C). After completion of immobilization, blocking was performed with 20 mM Tris-HCl buffer (pH 7.4) containing 2% (w/v) SKIM MILK (manufactured by Becton Dickinson) and 150 mM sodium chloride.
(3-2) After washing with a washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% [w/v] Tween 20 and 150 mM NaCl), on hybridomas containing antibodies The supernatant was added and the antibody was allowed to react with the immobilized recombinant human gp130 receptor protein (1 hour at 30°C).
(3-3) After completion of the reaction, the cells were washed with the washing buffer, and 100 μL/well of a peroxidase-labeled anti-mouse antibody (manufactured by Bethyl) diluted to 100 ng/mL was added.
(3-4) After reacting at 30° C. for 1 hour and washing with the washing buffer, TMB Peroxidase Substrate (manufactured by KPL) was added at 50 μL/well. Thereafter, 50 μL/well of 1 M phosphoric acid was added to stop color development, absorbance at 450 nm was measured using a microplate reader (manufactured by Tecan), and hybridomas with high measured values were selected.

(4) The hybridomas selected in (3) were subjected to the following operations.
(4-1) A mouse-derived anti-human gp130 receptor monoclonal AM64 antibody (JP-A-3-219894) was immobilized on each well of a 96-well plate.
(4-2) The soluble human gp130 receptor prepared in Example 1 was added to each well and allowed to bind to the human gp130 receptor antibody.
(4-3) A mixture of recombinant human interleukin-6 (IL-6) (manufactured by Wako Pure Chemical Industries, Ltd.) and recombinant human IL-6 receptor (IL-6R) (manufactured by PeproTech) in each well , and the culture supernatant of each hybridoma were added simultaneously.
(4-4) IL-6R immobilized via AM64 antibody and soluble human gp130 receptor under the above conditions, with anti-IL-6R polyclonal antibody and alkaline phosphatase produced by immunizing guinea pigs with IL-6R A labeled anti-guinea pig immunoglobulin antibody was added, and after washing, an alkaline phosphatase substrate was added to measure the IL-6 binding inhibitory effect of the antibody on the added soluble human gp130 receptor protein.

Cells in 3 wells that exhibited an IL-6 binding inhibitory effect were monocloned by the limiting dilution method to obtain 3 types of hybridomas.

Example 3 Sequence Analysis of Each Antibody Chain (1) The monoclonal antibodies obtained from the three types of hybridomas obtained in Example 2 were named Antibody A, Antibody B, and Antibody C, respectively. Each of the three types of hybridomas was cultured in a 90 mmφ culture dish supplemented with 10 mL of DMEM medium (manufactured by Thermo Fisher Scientific) containing 100 μg/mL kanamycin and 10% (w/v) bovine serum. The subclass of each antibody was confirmed using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (manufactured by Roche) for the culture supernatant. was κ, antibody C confirmed that the H chain was IgG2a and the L chain was κ.

(2) The cultured cells were collected, total RNA was extracted using RNeasy Mini Kit (manufactured by QIAGEN), and a cDNA library was constructed using cDNA Library Construction Kit (manufactured by Takara Bio).

(3) Using the cDNA library prepared in (2) as a template, the antibody gene was amplified by PCR.
Specifically, for amplification of the antibody A heavy chain (H chain) gene, a cDNA library of a hybridoma expressing antibody A was used as a template, and SEQ ID NO: 10 (5'-GCATAGAATTCCCCGGG-3') was used as a PCR primer. and SEQ ID NO: 11 (5'-ATGAATGCGGCCGCTCATTTACCAGGAGAGTGGGGAGAGGCTCTTC-3').
In addition, for amplification of the heavy chain genes of antibody B and antibody C, cDNA libraries of hybridomas expressing antibody B or antibody C were used as templates, and SEQ ID NO: 10 and SEQ ID NO: 12 (5'-ATGAATGCGGCCGCTCATTTACCGGAGWCCGGGAGAWGSTCTTMKTC-3) were used as PCR primers. ') was used.
For amplification of the light chain (L chain) genes of antibody A, antibody B and antibody C, cDNA libraries of hybridomas expressing each antibody were used as templates, and PCR primers of SEQ ID NO: 10 and SEQ ID NO: 13 (5 '-ATGAATGCGGCCGCCTAACACTCATTCCTGTTTGAAGCTCTTGAC-3') was used.
For PCR, a reaction solution having the composition shown in Table 4 was prepared, and the reaction solution was heat-treated at 98 ° C. for 5 minutes, followed by a first step at 98 ° C. for 10 seconds, a second step at 55 ° C. for 5 seconds, and a second step at 72 ° C. The reaction was carried out by repeating 30 cycles of 1 cycle of 3 steps for 1.5 minutes.

(4) The PCR product obtained in (3) is purified, digested with restriction enzymes EcoRI and NotI, and then ligated into an expression vector pAP3neo (manufactured by Takara Bio Inc.) previously digested with restriction enzymes EcoRI and NotI, and the ligation product is was used to transform E. coli strain JM109.

(5) After culturing the obtained transformant in LB medium containing 100 μg / mL ampicillin, using QIAprep Spin Miniprep kit (manufactured by QIAGEN), a vector capable of expressing the heavy chain or light chain of each antibody pAP-AH (antibody A heavy chain), pAP-AL (antibody A light chain), pAP-BH (antibody B heavy chain), pAP-BL (antibody B light chain), pAP-CH (antibody C heavy chain) and pAP-CL (antibody C light chain) was extracted.

(6) Of the expression vector prepared in (5), the region around the gene encoding each antibody chain was treated with BigDye Terminator Ver. 3.1 Cycle Sequencing Kit (manufactured by Applied Biosystems) was used for cycle sequencing reaction, and the nucleotide sequence was analyzed using a fully automatic DNA sequencer ABI Prism 3700 DNA analyzer (manufactured by Applied Biosystems). During the analysis, an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 14 (5'-TAATACGACTCACTATAGGG-3') or SEQ ID NO: 15 (5'-ATTAACCCTCACTAAAGGGGCG-3') was used as a sequencing primer.

The amino acid sequence of the antibody A heavy chain polypeptide expressed in the expression vector pAP-AH is shown in SEQ ID NO: 16, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 17, respectively. The complementarity-determining region (CDR) of the antibody A heavy chain polypeptide consisting of the sequence set forth in SEQ ID NO: 16 is from the 50th aspartic acid (Asp) to the 54th aspartic acid (Asp) (SEQ ID NO: 18, CDR1), the region from the 69th aspartic acid (Asp) to the 85th aspartic acid (Asp) (SEQ ID NO: 19, CDR2) and the 118th threonine (Thr) to the 128th tyrosine (Tyr) (SEQ ID NO: 20, CDR3).

The amino acid sequence of the antibody A light chain polypeptide expressed in the expression vector pAP-AL is shown in SEQ ID NO:21, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO:22. In the antibody A light chain polypeptide consisting of the sequence set forth in SEQ ID NO: 21, the CDRs are the region from the 46th asparagine (Asn) to the 55th tyrosine (Tyr) (SEQ ID NO: 23, CDR1), The region from leucine (Leu) to 77th serine (Ser) (SEQ ID NO: 24, CDR2) and the region from 110th glutamine (Gln) to 118th threonine (Thr) (SEQ ID NO: 25, CDR3) .

The amino acid sequence of the antibody B heavy chain polypeptide expressed in the expression vector pAP-BH is shown in SEQ ID NO:26, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO:27. The CDRs of the antibody B heavy chain polypeptide consisting of the sequence set forth in SEQ ID NO: 26 are the region from the 50th aspartic acid (Asp) to the 54th histidine (His) (SEQ ID NO: 28, CDR1), the 69th The region from arginine (Arg) to the 85th aspartic acid (Asp) (SEQ ID NO: 29, CDR2) and the region from the 116th alanine (Ala) to the 127th tyrosine (Tyr) (SEQ ID NO: 30, CDR3) is.

The amino acid sequence of the antibody B light chain polypeptide expressed in the expression vector pAP-BL is shown in SEQ ID NO:31, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO:32. The CDRs of the antibody B light chain polypeptide consisting of the sequence set forth in SEQ ID NO: 31 are the region from arginine (Arg) at position 44 to glutamine (Gln) at position 58 (SEQ ID NO: 33, CDR1), The region from valine (Val) to the 80th serine (Ser) (SEQ ID NO: 34, CDR2) and the region from the 113th glutamine (Gln) to the 121st threonine (Thr) (SEQ ID NO: 35, CDR3) .

The amino acid sequence of the antibody C heavy chain polypeptide expressed in the expression vector pAP-CH is shown in SEQ ID NO:36, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO:37. The CDRs of the antibody C heavy chain polypeptide consisting of the sequence set forth in SEQ ID NO: 36 are the region from the 50th alanine (Ala) to the 54th serine (Ser) (SEQ ID NO: 38, CDR1), The region from threonine (Thr) to glycine (Gly) at position 85 (SEQ ID NO: 39, CDR2) and the region from phenylalanine (Phe) at position 118 to tyrosine (Tyr) at position 124 (SEQ ID NO: 40, CDR3) .

The amino acid sequence of the antibody C light chain polypeptide expressed in the expression vector pAP-CL is shown in SEQ ID NO:41, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO:42. In the antibody C light chain polypeptide consisting of the sequence set forth in SEQ ID NO: 41, the CDRs are the region from lysine (Lys) at position 44 to alanine (Ala) at position 52 (SEQ ID NO: 43, CDR1), A region from tryptophan (Trp) to 76th threonine (Thr) (SEQ ID NO: 44, CDR2) and a region from 109th glutamine (Gln) to 117th threonine (Thr) (SEQ ID NO: 45, CDR3) .

Example 4 Evaluation of Recombinant Antibody Binding to gp130 Receptor (1) DMEM medium containing 50 μg/mL kanamycin and 10% (w/v) bovine serum (manufactured by Thermo Fisher Scientific) was added 6 Using a well plate, COS1 cell line was added to each well and cultured under conditions of 37° C. and 5% CO ₂ .

(2) After the cells became confluent, the medium in each well was replaced with 0.5 mL of Opti-MEM (manufactured by Thermo Fisher Scientific).

(3) After exchanging the medium, the expression vector containing the heavy chain gene of antibody A and the expression vector containing the light chain gene (pAP-AH, pAP-AL) obtained in Example 3, the heavy chain gene of antibody B and an expression vector containing the light chain gene (pAP-BH, pAP-BL) or an expression vector containing the antibody C heavy chain gene and an expression vector containing the light chain gene (pAP-CH, pAP- CL) 250 μL of Opti-MEM medium containing a complex of 1 μg each and 8 μL of Lipofectamine 2000 (manufactured by Thermo Fisher Scientific) was added to each well and cultured for 2 days at 37° C. and 5% CO ₂ to obtain antibody A, Antibody B and antibody C proteins were obtained.

(4) The binding of the antibody obtained in (2) to human gp130 receptor was evaluated by the ELISA method described in Example 2 (3).

The results are shown in FIG. The black bars are the results when the human soluble gp130 receptor was immobilized (gp130(+)), and the white bars are the results when the gp130 receptor was not immobilized (gp130(-), negative control). Antibody A, antibody B, and antibody C were all confirmed to bind to the soluble human gp130 receptor. Antibody A (antibody having a heavy chain consisting of the amino acid sequence of SEQ ID NO: 16 and a light chain consisting of the amino acid sequence of SEQ ID NO: 21), antibody B (amino acid sequence of SEQ ID NO: 26) and an antibody having a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 31) and antibody C (a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 36 and the amino acid sequence set forth in SEQ ID NO: 41 ), all of which bind to the human gp130 receptor.

Example 5 Antibody Purification (1) The three types of hybridomas obtained in Example 2 were placed in five 150 mmφ dishes (manufactured by Sumitomo Bakelite) containing 30 mL of Hybridoma-SFM medium (manufactured by Thermo Fisher Scientific). cultured. After culturing, the culture supernatant was obtained by centrifugation.

(2) 1 mL of the supernatant obtained in (1) was pre-equilibrated with 20 mM Tris-HCl (pH 7.4) containing 150 mM sodium chloride using AKTA prime plus (manufactured by GE Healthcare). HiTrap Protein G HP Columns (manufactured by GE Healthcare) at a flow rate of 1 mL/min.

(3) After washing with the buffer used for the equilibration, elution was performed with 0.1 M glycine hydrochloride buffer (pH 2.8). The obtained eluate was returned to near neutral pH by adding 1M Tris-HCl buffer (pH 8.0) in an amount of 1/4 of the volume of the eluate.

About 5 mg of antibody A, about 20 mg of antibody B, and about 2 mg of antibody C of high purity were obtained by this purification.

Example 6 Epitope Mapping (1) Region from 124th leucine (Leu) to 324th aspartic acid (Asp) in the amino acid sequence of the human gp130 receptor described in SEQ ID NO: 1 (201 amino acid residues in total) , a total of 94 oligopeptides consisting of 15 amino acid residues were synthesized so as to cover the region with a gap of 2 amino acid residues (Table 5, consisting of the amino acid sequences set forth in SEQ ID NO: 46 to SEQ ID NO: 139 oligopeptide).

(2) The 94 peptides synthesized in (1) were immobilized on a microarray slide by adding linkers.

(3) Antibody A, antibody B and antibody C purified in Example 5 were diluted to 10 μg/mL, 1 μg/mL, 0.5 μg/mL or 0.1 μg/mL with a blocking buffer (manufactured by Pierce). , was added to the microarray slide.

(4) After standing at 30° C. for 1 hour, wash with washing buffer (150 mM sodium chloride, Tris-HCl (pH 7.2) containing 0.1% Tween 20), and use DyLight as a secondary antibody. 650-bound anti-mouse IgG (H+L) (manufactured by Thermo Fisher Scientific) was added after being diluted to 1 μg/mL with a blocking buffer.

(5) Leave at 30° C. for 1 hour, wash with washing buffer, dry, and scan fluorescence with Genepix Scanner 4200AL (manufactured by Molecular Devices).

Table 6 shows the results for each antibody at 10 μg/mL. All of the antibodies examined this time (antibody A, antibody B, antibody C) have high binding to oligopeptides consisting of the amino acid sequences of SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 127 and SEQ ID NO: 128. had a high fluorescence intensity (high fluorescence intensity). The common region of the oligopeptides consisting of the amino acid sequences set forth in SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110 is the region from 252nd to 262nd of the human gp130 receptor amino acid sequence set forth in SEQ ID NO: 1 (specifically is VIILKYNIQYR), and the common region of the oligopeptides consisting of the amino acid sequences set forth in SEQ ID NO: 127 and SEQ ID NO: 128 is the region from 288th to 300th of the human gp130 receptor amino acid sequence set forth in SEQ ID NO: 1 (specifically DLKPFTEYVFRIR). From the above results, the antibodies examined this time (antibody A, antibody B, antibody C) are all VIILKYNIQYR regions (regions from 252nd to 262nd of SEQ ID NO: 1) of human gp130 receptor (SEQ ID NO: 1) and DLKPFTEYVFRIR It can be seen that at least the region (region from 288th to 300th of SEQ ID NO: 1) is recognized.

Example 7 Suppression of IL-6 Signal by Antibody (1) 7-TD-1 (Monbukagakusho National RIKEN BRC Cell Bank No. RCB1190 provided by RIKEN BRC through the BioResource Project) was cultured in DMEM medium containing 10% (w/v) bovine serum (manufactured by Thermo Fisher Scientific).

(2) After culturing, cells were counted and added to a 24-well plate at 1×10 ⁵ cells/well (1 mL of medium).

(3) Human IL-6 (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the wells at a final concentration of 50 ng/mL, and antibody A or antibody B purified in Example 5 was added at a final concentration of 500 ng/well or 1000 ng/ml. After adding to the wells, the 24-well plate was statically cultured for 2 days under conditions of 37° C. and 5% CO ₂ .

(4) After the culture in (3), the number of cells in the wells to which human IL-6 was not added, human IL-6 alone was added, and human IL-6 and antibody (antibody A or antibody B) were added was counted using an automatic cell measuring device, Countness. (manufactured by Thermo Fisher Scientific).

FIG. 2 shows the results of measuring cell counts when using antibody A, and FIG. 3 shows the results of measuring cell counts when using antibody B, respectively. The addition of human IL-6 increased the number of 7-TD-1 cells in the well compared to when human IL-6 was not added, suggesting that 7-TD-1 is induced by human IL-6. It was confirmed that the number increased. On the other hand, when antibody A or antibody B was added to the wells to which human IL-6 was added, the growth of 7-TD-1 in the wells was suppressed compared to when no antibody was added, and the degree of suppression was The result was dependent on the amount of addition of (Figs. 2 and 3). From the above results, of the human gp130 receptor consisting of the amino acid sequence set forth in SEQ ID NO: 1, amino acid residues from valine (Val) at position 252 to arginine (Arg) at position 262 (specifically, VIILKYNIQYR) and /or Antibody A and Antibody B, which are antibodies that bind at least amino acid residues from aspartic acid (Asp) at position 288 to arginine (Arg) at position 300 (specifically DLKPFTEYVFRIR), bind to the human gp130 receptor It can be seen that IL-6 signaling can be suppressed by doing so.

Example 8 Affinity Measurement of Antibody A (1) The antibody A solution prepared in Example 5 was concentrated by ultrafiltration and replaced with HBS-EP+ buffer (manufactured by GE Healthcare), followed by HBS-EP+. Diluted to 16 μg/mL, 8 μg/mL, 4 μg/mL, 2 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.25 μg/mL and 0.125 μg/mL with buffer solution (manufactured by GE Healthcare) did.

(2) The human gp130 receptor prepared in Example 1 was diluted with 10 mM acetate buffer (pH 5.0) to 10 μg/mL, and the sensor chip CM5 (manufactured by GE Healthcare) was subjected to an amine coupling method. was immobilized using (corresponding to immobilized amount of 10 ⁸ RU).

(3) The antibody A diluted solution prepared in (1) was flowed to the chip on which human gp130 was immobilized under the conditions of a contact time of 200 seconds, a dissociation time of 800 seconds, and a flow rate of 30 μL / min, and the immobilized human gp130 receptor Interaction was measured using Biacore T200 (manufactured by GE Healthcare). As a result of affinity analysis using analysis software, the affinity of antibody A was ka = 1.74 × 10 ⁵ [1/Ms], kd = 1.16 × 10 ^-4 [1/s], KD = It was 6.67×10 ⁻¹⁰ [M], confirming that it has a high affinity.

Example 9 Production of Humanized Antibody Gene (1) Heavy chain variable (VH) region of antibody A, consisting of amino acid residues from glutamic acid at position 20 to serine at position 139 in the amino acid sequence shown in SEQ ID NO: 16 and a framework in the light chain variable (VL) region of Antibody A, consisting of amino acid residues from glutamine at position 23 to arginine at position 129 in the amino acid sequence set forth in SEQ ID NO: 21. The region (FR) is changed from mouse antibody amino acid residues to human antibody amino acid residues (hereinafter also referred to as humanized antibody A), integrated computational chemistry system "MOE" (manufactured by Chemical Computing Group). It was designed using

Among the 9 designed VH regions of humanized antibody A (AH1 to AH9) and the 8 VL regions of humanized antibody A (AL1 to AL8), the amino acid sequence of AH1 is shown in SEQ ID NO: 140, and AH1 is encoded The polynucleotide sequence is SEQ ID NO: 141, the amino acid sequence of AH2 is SEQ ID NO: 142, the polynucleotide sequence encoding AH2 is SEQ ID NO: 143, the amino acid sequence of AH3 is SEQ ID NO: 144, and the polynucleotide encoding AH3. The sequence of AH4 is SEQ ID NO: 145, the amino acid sequence of AH4 is SEQ ID NO: 146, the sequence of the polynucleotide encoding AH4 is SEQ ID NO: 147, the amino acid sequence of AH5 is SEQ ID NO: 148, the sequence of the polynucleotide encoding AH5 is SEQ ID NO: 149, the amino acid sequence of AH6 is SEQ ID NO: 150, the sequence of the polynucleotide encoding AH6 is SEQ ID NO: 151, the amino acid sequence of AH7 is SEQ ID NO: 152, the sequence of the polynucleotide encoding AH7 is sequence The amino acid sequence of AH8 is SEQ ID NO: 154, the polynucleotide sequence encoding AH8 is SEQ ID NO: 155, the amino acid sequence of AH9 is SEQ ID NO: 156, and the polynucleotide sequence encoding AH9 is SEQ ID NO: 157. Furthermore, the amino acid sequence of AL1 is SEQ ID NO: 158, the polynucleotide sequence encoding AL1 is SEQ ID NO: 159, the amino acid sequence of AL2 is SEQ ID NO: 160, the polynucleotide sequence encoding AL2 is SEQ ID NO: 161, The amino acid sequence of AL3 is SEQ ID NO: 162, the polynucleotide sequence encoding AL3 is SEQ ID NO: 163, the amino acid sequence of AL4 is SEQ ID NO: 164, the polynucleotide sequence encoding AL4 is SEQ ID NO: 165, AL5 The amino acid sequence is SEQ ID NO: 166, the sequence of the polynucleotide encoding AL5 is SEQ ID NO: 167, the amino acid sequence of AL6 is SEQ ID NO: 168, the sequence of the polynucleotide encoding AL6 is SEQ ID NO: 169, and the amino acid sequence of AL7. is shown in SEQ ID NO: 170, the polynucleotide sequence encoding AL7 is shown in SEQ ID NO: 171, the amino acid sequence of AL8 is shown in SEQ ID NO: 172, and the polynucleotide sequence encoding AL8 is shown in SEQ ID NO: 173.

(2) A polynucleotide encoding the VH region of humanized antibody A designed in (1) (SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153 , SEQ ID NO: 155, SEQ ID NO: 157) added with a restriction enzyme EcoRI recognition sequence (GAATTC) and NheI recognition sequence (GCTAGC), or a polynucleotide encoding a VL region (SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171 and SEQ ID NO: 173 (polynucleotides consisting of sequences set forth in SEQ ID NO: 171 and SEQ ID NO: 173) with restriction enzyme EcoRI recognition sequence (GAATTC) and BsiWI recognition Polynucleotides added with a sequence (CGTACG) and adjusted so as not to undergo frameshift after cloning were totally synthesized (consigned to FASMAC) and cloned into plasmids.

(3) Using the pCAG-dhfr prepared in Example 1 as a template, SEQ ID NO: 174 (5'-TTTAAATCAGCGGCCGCGCAGCACCATGGCCTGAAATAACCTCTG-3') and SEQ ID NO: 175 (5'-GCAAGTAAAACCTCTACAAATGTGGTAAACGATCGCTCCGGTGCCCGT-3') oligonucleotide primers consisting of the sequences described PCR was performed using Specifically, a reaction solution having the composition shown in Table 7 was prepared, and after heat treatment of the reaction solution at 98° C. for 1 minute, a first step at 98° C. for 10 seconds, a second step at 55° C. for 5 seconds, 72 The reaction was carried out by repeating 30 cycles of the third step at 1 minute at °C. The PCR product (SV40 promoter, dhfr, region up to PolyA of SV40) amplified by this PCR was named dhfr-P1.

(4) pFUSEss-CHIg-hG1 containing a human antibody heavy chain constant region (manufactured by InvivoGen), pFUSE2ss-CLIg-hk containing a human antibody light chain constant region (manufactured by InvivoGen) and (3) The resulting dhfr-P1 was digested with restriction enzymes NotI and PvuI, purified, and ligated. Escherichia coli strain JM109 was transformed with the ligation products, and plasmids were extracted from the cultured transformants to obtain pFUSEss-CHIg-hG1 and pFUSE2ss-CLIg-hk containing the SV40 promoter, dhfr, and SV40 PolyA. A plasmid obtained by integrating the SV40 promoter, dhfr and SV40 PolyA into pFUSEss-CHIg-hG1 was named pFU-CHIg-dhfr, and a plasmid obtained by integrating the SV40 promoter, dhfr and SV40 PolyA into pFUSE2ss-CLIg-hk was named pFU. -CLIg-dhfr.

(5) Escherichia coli strain JM109 was transformed with each of the plasmids prepared in (2) and containing the polynucleotide encoding the VH region of humanized antibody A. The resulting transformant was cultured, plasmid was extracted, and digested with restriction enzymes EcoRI and NheI to obtain polynucleotides encoding VH regions of humanized antibody A (SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155 and SEQ ID NO: 157) were prepared.

(6) pFU-CHIg-dhfr prepared in (4) was digested with restriction enzymes EcoRI and NheI, purified, and ligated with the polynucleotide prepared in (5). Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid expressing the heavy chain (H chain) of humanized antibody A was prepared by extracting the plasmid from the cultured transformant.

Among the prepared plasmids, a plasmid containing a polynucleotide (SEQ ID NO: 141) encoding AH1 (SEQ ID NO: 140) was named pFU-AH1, and a polynucleotide (SEQ ID NO: 143) encoding AH2 (SEQ ID NO: 142) was named pFU-AH1. The plasmid containing was named pFU-AH2, the plasmid containing the polynucleotide (SEQ ID NO: 145) encoding AH3 (SEQ ID NO: 144) was named pFU-AH3, and the polynucleotide encoding AH4 (SEQ ID NO: 146) (SEQ ID NO: 146) was named pFU-AH3. No. 147) was named pFU-AH4, and the plasmid containing the polynucleotide (SEQ ID NO: 149) encoding AH5 (SEQ ID NO: 148) was named pFU-AH5, encoding AH6 (SEQ ID NO: 150). The plasmid containing the polynucleotide (SEQ ID NO: 151) was named pFU-AH6, the plasmid containing the polynucleotide (SEQ ID NO: 153) encoding AH7 (SEQ ID NO: 152) was named pFU-AH7, AH8 (SEQ ID NO: 154) ) was designated pFU-AH8, and the plasmid containing the polynucleotide (SEQ ID NO: 157) encoding AH9 (SEQ ID NO: 156) was designated pFU-AH9.

(7) Escherichia coli strain JM109 was transformed with each plasmid containing the polynucleotide encoding the VL region of humanized antibody A produced in (2). The resulting transformant was cultured, plasmid was extracted, and digested with restriction enzymes EcoRI and BsiWI to obtain polynucleotides encoding the VL region of humanized antibody A (SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, Polynucleotides consisting of the sequences set forth in SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171 and SEQ ID NO: 173) were prepared.

(8) pFU-CLIg-dhfr prepared in (4) was digested with restriction enzymes EcoRI and BsiWI, purified, and then ligated with the polynucleotide prepared in (7). Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid expressing the light chain (L chain) of humanized antibody A was prepared by extracting the plasmid from the cultured transformant.

Among the prepared plasmids, a plasmid containing a polynucleotide (SEQ ID NO: 159) encoding AL1 (SEQ ID NO: 158) was named pFU-AL1, and a polynucleotide (SEQ ID NO: 161) encoding AL2 (SEQ ID NO: 160) was designated as pFU-AL1. The plasmid containing was named pFU-AL2, the plasmid containing the polynucleotide (SEQ ID NO: 163) encoding AL3 (SEQ ID NO: 162) was named pFU-AL3, the polynucleotide encoding AL4 (SEQ ID NO: 164) (sequence No. 165) was named pFU-AL4, and the plasmid containing the polynucleotide (SEQ ID NO: 167) encoding AL5 (SEQ ID NO: 166) was named pFU-AL5, encoding AL6 (SEQ ID NO: 168). The plasmid containing the polynucleotide (SEQ ID NO: 169) was named pFU-AL6, the plasmid containing the polynucleotide (SEQ ID NO: 171) encoding AL7 (SEQ ID NO: 170) was named pFU-AL7, AL8 (SEQ ID NO: 172) ) (SEQ ID NO: 173) was designated pFU-AL8.

Example 10 Evaluation of binding property of humanized antibody A to human gp130 receptor (1) Prepared with 100 ng of plasmid expressing H chain of humanized antibody A prepared in Example 9 (6) and Example 9 (8) 25 μL of Opti-MEM medium (manufactured by Thermo Fisher Scientific) added with 100 ng of a plasmid expressing the L chain of humanized antibody A, and 25 μL of 1 μL of Lipofectamine 2000 (manufactured by Thermo Fisher Scientific) containing The mixture was mixed with Opti-MEM medium and allowed to stand at room temperature for 20 minutes.

(2) The medium of COS1 cells (renal fibroblasts of African green monkeys) cultured in DMEM medium containing 10% (w/v) bovine serum using a 96-well culture plate was added to 90 μL of Opti-MEM medium. Exchange, transfection is performed by adding Opti-MEM medium containing the antibody expression plasmid and Lipofectamine 2000 described in (1), humanized antibody by standing for 2 days at 37 ° C., 5% CO ₂ environment A was expressed.

(3) The binding ability of the expressed humanized antibody A to the human gp130 receptor was confirmed using the same ELISA method as described in Example 2(3). The ELISA method was the same as described in Example 2(3), except that a peroxidase-labeled anti-human antibody (manufactured by Bethyl) was used to detect the humanized antibody.

Table 8 shows the results. In Table 8, the numerical value is the absorbance of ELISA, and the larger the numerical value, the stronger the binding to gp130. From Table 8, the combination of the VH region and VL region of humanized antibody A is AH1 (SEQ ID NO: 140) -AL7 (SEQ ID NO: 170), AH2 (SEQ ID NO: 142) -AL1 (SEQ ID NO: 158), AH2 (sequence 142)-AL2 (SEQ ID NO: 160), AH2 (SEQ ID NO: 142)-AL3 (SEQ ID NO: 162), AH2 (SEQ ID NO: 142)-AL4 (SEQ ID NO: 164), AH2 (SEQ ID NO: 142)-AL5 (SEQ ID NO: 164) 166), AH2 (SEQ ID NO: 142)-AL6 (SEQ ID NO: 168), AH2 (SEQ ID NO: 142)-AL7 (SEQ ID NO: 170), AH2 (SEQ ID NO: 142)-AL8 (SEQ ID NO: 172), AH3 (SEQ ID NO: 144 )-AL1 (SEQ ID NO: 158), AH3 (SEQ ID NO: 144)-AL2 (SEQ ID NO: 160), AH3 (SEQ ID NO: 144)-AL3 (SEQ ID NO: 162), AH3 (SEQ ID NO: 144)-AL4 (SEQ ID NO: 164) , AH3 (SEQ ID NO: 144)-AL5 (SEQ ID NO: 166), AH3 (SEQ ID NO: 144)-AL6 (SEQ ID NO: 168), AH3 (SEQ ID NO: 144)-AL7 (SEQ ID NO: 170), AH4 (SEQ ID NO: 146)- AL7 (SEQ ID NO: 170), AH5 (SEQ ID NO: 148)-AL1 (SEQ ID NO: 158), AH5 (SEQ ID NO: 148)-AL3 (SEQ ID NO: 162), AH5 (SEQ ID NO: 148)-AL4 (SEQ ID NO: 164), AH5 (SEQ ID NO: 148)-AL5 (SEQ ID NO: 166), AH5 (SEQ ID NO: 148)-AL7 (SEQ ID NO: 170), AH6 (SEQ ID NO: 150)-AL7 (SEQ ID NO: 170), AH7 (SEQ ID NO: 152)-AL1 ( SEQ ID NO: 158), AH7 (SEQ ID NO: 152)-AL2 (SEQ ID NO: 160), AH7 (SEQ ID NO: 152)-AL3 (SEQ ID NO: 162), AH7 (SEQ ID NO: 152)-AL4 (SEQ ID NO: 164), AH7 (SEQ ID NO: 164) 152)-AL5 (SEQ ID NO: 166), AH7 (SEQ ID NO: 152)-AL6 (SEQ ID NO: 168), AH7 (SEQ ID NO: 152)-AL7 (SEQ ID NO: 170), AH7 (SEQ ID NO: 152)-AL8 (SEQ ID NO: 152)-AL8 (SEQ ID NO: 168) 172), AH8 (SEQ ID NO: 154)-AL1 (SEQ ID NO: 158), AH8 (SEQ ID NO: 154)-AL4 (SEQ ID NO: 164), AH8 (SEQ ID NO: 154)-AL7 (SEQ ID NO: 170) and AH9 (SEQ ID NO: 156 )-AL7 (SEQ ID NO: 170) was confirmed to bind to the human gp130 receptor. From this fact, it is possible to obtain a humanized anti-gp130 receptor antibody that exhibits antigen-binding ability even if the framework region (FR) of the antibody is changed from the amino acid residues of the mouse antibody to the amino acid residues of the human antibody. Recognize.

From the results in Table 8, among the VL regions of humanized antibody A examined this time, AL7 (SEQ ID NO: 170) has a high human gp130 receptor in combination with all the VH regions of humanized antibody A examined this time. showed connectivity. Therefore, when the amino acid sequences of FR were compared between AL7 (SEQ ID NO: 170) and the VL region (AL1 to AL6 and AL8) of the other humanized antibody A designed this time, the 34th to 48th amino acids of SEQ ID NO: 170 A characteristic sequence was found in the region of the sequence (Table 9). This suggests that an antibody having a light chain FR containing the 34th to 48th amino acid sequences of SEQ ID NO: 170 is a preferred embodiment as an antibody against the human gp130 receptor.

Example 11 Evaluation of affinity of humanized antibody A (SEQ ID NO: 162) and AH2 (SEQ ID NO: 142)-AL4 (SEQ ID NO: 164) combinations were measured for affinity.

(1) Escherichia coli JM109 strains transformed with pFU-AH2, pFU-AL3 and pFU-AL4 prepared in Example 9 were cultured, respectively, and plasmids were extracted to obtain pFU-AH2, pFU-AL3 and pFU-AL4. got

(2) 12 μg of pFU-AH2 and 12 μg of pFU-AL3, or 12 μg of pFU-AH2 and 12 μg of pFU-AL4 were added to 1.5 mL of Opti-MEM medium (manufactured by Thermo Fisher Scientific) and 60 μL of 1.5 mL of Opti-MEM medium containing Lipofectamine 2000 (manufactured by Thermo Fisher Scientific) was mixed and allowed to stand at room temperature for 20 minutes.

(3) Using a 90 mm diameter culture dish (manufactured by Sumitomo Bakelite Co., Ltd.), 9 mL of COS1 cell (African green monkey kidney fibroblast) cultured in DMEM medium containing 10% (w/v) bovine serum was added. and the combination of pFU-AH2 and pFU-AL3 or the combination of pFU-AH2 and pFU-AL4 and Opti-MEM medium containing Lipofectamine 2000 was added to perform transfection. AH2-AL3 and AH2-AL4 were expressed by standing at 37° C. and 5% CO ₂ environment for 3 days.

(4) The culture supernatant obtained by removing the cells by centrifugation from the culture solution containing AH2-AL3 and AH2-AL4 expressed in (3) was added to 20 mM Tris-HCl buffer (pH 7) containing 150 mM sodium chloride. .4) was applied to 1 mL of TOYOPEARL AF-rProtein A-650F (manufactured by Tosoh Corporation) equilibrated. After washing with 10 mL of 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride, purification was performed by elution with 0.1 M glycine-HCl (pH 3.0). Immediately after the purification, 1M Tris-HCl buffer (pH 8.0) was added in an amount of 1/4 of the eluted amount to restore the pH to neutral.

(5) Purified AH2-AL3 and AH2-AL4 are replaced with HBS-EP+buffer (manufactured by GE Healthcare) while being concentrated by ultrafiltration, and HBS-EP+buffer (manufactured by GE Healthcare). to 16 μg/mL, 8 μg/mL, 4 μg/mL, 2 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.25 μg/mL and 0.125 μg/mL.

(6) The human gp130 receptor prepared in Example 1 was diluted to 10 μg/mL in 10 mM acetate buffer (pH 5.0), and the sensor chip CM5 (manufactured by GE Healthcare) was subjected to amine coupling method. was immobilized using (corresponding to immobilized amount of 266 RU).

(7) The AH2-AL3 and AH2-AL4 diluted solutions of each concentration prepared in (5) were flowed to the human gp130 receptor-immobilized sensor chip under conditions of contact time of 230 seconds, dissociation time of 800 seconds, and flow rate of 30 μL/min, Interaction with immobilized human gp130 receptor was measured using Biacore T200 (manufactured by GE Healthcare).

As a result of affinity analysis using analysis software, the affinity of AH2-AL3 is ka = 7.0
6×10 ⁴ [1/Ms], kd=2.68×10 ⁻⁴ [1/s], KD=3.80×10
⁻⁹ [M], and the affinity of AH2-AL4 is ka=6.27×10 ⁴ [1/Ms], k
d=2.89×10 ⁻⁴ [1/s] and KD=4.61×10 ⁻⁹ [M]. From this, the affinity of humanized antibody AH2-AL3 and humanized antibody AH2-AL4 is the affinity of antibody A (mouse antibody) before humanization described in Example 8 (KD = 6.67 × 10 ^{- 10} [M
]), it was confirmed to have almost the same affinity as

Example 12 Preparation of L Chain Frame Library (1) Antibody A light chain consisting of amino acid residues from glutamine (Gln) at position 23 to arginine (Arg) at position 129 in the amino acid sequence set forth in SEQ ID NO:21. Five amino acid residues of an antibody in which the framework region (FR) in the variable chain (VL) region was humanized were designed using the integrated computational chemistry system "MOE" (manufactured by Chemical Computing Group).
Among the designed humanized antibody A VL regions (AL9 to AL13), the amino acid sequence of AL9 is shown in SEQ ID NO: 176, the polynucleotide sequence encoding AL9 is shown in SEQ ID NO: 177, and the amino acid sequence of AL10 is shown in SEQ ID NO: 178. , the sequence of the polynucleotide encoding AL10 in SEQ ID NO: 179, the amino acid sequence of AL11 in SEQ ID NO: 180, the sequence of the polynucleotide encoding AL11 in SEQ ID NO: 181, the amino acid sequence of AL12 in SEQ ID NO: 182, AL12 The sequence of the polynucleotide encoding is shown in SEQ ID NO: 183, the amino acid sequence of AL13 is shown in SEQ ID NO: 184, and the sequence of the polynucleotide encoding AL13 is shown in SEQ ID NO: 185.

(2) Polynucleotides of SEQ ID NOS: 177, 179, 181, 183 and 185 were added with a restriction enzyme EcoRI recognition sequence (GAATTC) and a BsiWI recognition sequence (CGTACG) to prevent frameshift after cloning. Each nucleotide was totally synthesized (commissioned by FASMAC) and cloned into a plasmid.

(3) Escherichia coli strain JM109 was transformed with each of the plasmids prepared in (2) and containing each of the five types of polynucleotides encoding the VL region of humanized antibody A. The resulting transformants were cultured, plasmids were extracted, and digested with restriction enzymes EcoRI and BsiWI to obtain five polynucleotides encoding the VL regions of humanized antibody A (SEQ ID NOS: 177, 179, 181, 183 and 185) were prepared.

(4) pFU-CLIg-dhfr prepared in Example 9 (4) was digested and purified with restriction enzymes EcoRI and BsiWI, and then ligated with the prepared polynucleotides encoding the VL regions of humanized antibody A, respectively. . Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid expressing the L chain of humanized antibody A was prepared by extracting the plasmid from the cultured transformant.
Among the prepared plasmids, a plasmid containing a polynucleotide (SEQ ID NO: 177) encoding AL9 (SEQ ID NO: 176) was named pFU-AL9, and a polynucleotide (SEQ ID NO: 179) encoding AL10 (SEQ ID NO: 178) was designated as pFU-AL9. The plasmid containing was named pFU-AL10, the plasmid containing the polynucleotide (SEQ ID NO: 181) encoding AL11 (SEQ ID NO: 180) was named pFU-AL11, and the polynucleotide encoding AL12 (SEQ ID NO: 182) was named pFU-AL11. No. 183) was designated pFU-AL12, and the plasmid containing the polynucleotide (SEQ ID NO: 185) encoding AL13 (SEQ ID NO: 184) was designated pFU-AL13.

(5) Using the plasmid pFUSE2ss-CLIg-hk (manufactured by InvivoGen) containing the light chain constant region of a human antibody as a template, SEQ ID NO: 186 (5'-GACAGATCTGCTCCGGTGCCCGTCAGTGGGCAGAG-3') and SEQ ID NO: 187 (5'-TAGGCGGCCGCTCCCTCTAACACTCTCCCCTGTTGAAG-3 '), PCR was performed using the oligonucleotides described in 1.1. Specifically, a reaction solution having the composition shown in Table 7 was prepared, and after heat treatment of the reaction solution at 98°C for 5 minutes, the first step was at 98°C for 10 seconds, the second step was at 55°C for 5 seconds, and the heat treatment was performed at 72°C. The reaction was carried out by repeating 30 cycles of the third step of 1.5 minutes at .

(6) After purifying the PCR product containing the gene encoding the promoter, IL-2 secretion signal, and human Igκ chain obtained in (5), it was digested with the restriction enzymes BglII and NotI, and then digested with the same restriction enzymes. It was ligated with pCAG-dhfr prepared in Example 1 (4). Escherichia coli JM109 strain was transformed with the ligation product, and pEF-hL capable of expressing the light chain constant region of a human antibody was obtained by extracting the plasmid from the cultured transformant.

(7) 8 humanized L chains prepared in Example 9 (pFU-AL1, pFU-AL2, pFU-AL3, pFU-AL4, pFU-AL5, pFU-AL6, pFU-AL7, and pFU-AL8) and the five types of humanized L chains (pFU-AL9, pFU-AL10, pFU-AL11, pFU-AL12 and pFU-AL13) prepared in (2) were mixed so that each concentration was 50 ng/μL as a template. , SEQ ID NO: 188 (5′-CACTTGTCACGAATTC-3′), 189 (5′-AACGTGAGCTCCTCCGTAACCTC-3′), 191 (5′-CTGACCTCCAATCTGGCATCT-3′) and 193 (5′-CAGCAGTGGTCCACCAATCCCTTGAC-3′) Using an oligonucleotide consisting of the described sequence as a forward primer, SEQ ID NOS: 190 (5'-GAGGTTACGGAGGAGCTCACGTT-3'), 192 (5'-AGATGCCAGATTGGAGGTCAG-3'), 194 (5'-GTCAAGGGATTGGTGGACCACTGCTG-3') and 195 (5 '-GTGCAGCCACCGTACG-3') was used as a reverse primer for PCR. Specifically, a reaction solution having the composition shown in Table 7 was prepared, and after heat treatment of the reaction solution at 98°C for 5 minutes, the first step was at 98°C for 10 seconds, the second step was at 55°C for 5 seconds, and the heat treatment was performed at 72°C. The reaction was carried out by repeating 30 cycles of the reaction in which one cycle was the third step for 30 seconds at .
A PCR product obtained by using an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 188 as a forward primer and an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 190 as a reverse primer contains four FRs in the variable region of the humanized L chain. Among them, it encodes FR1 and was named AL-FR1. Further, a PCR product obtained by using an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 189 as a forward primer and an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 192 as a reverse primer was obtained by four FRs in the variable region of the humanized L chain. Among them, it encodes FR2 and was designated as AL-FR2. Further, a PCR product obtained by using an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 191 as a forward primer and an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 194 as a reverse primer is the four FRs in the variable region of the humanized L chain. Among them, it encodes FR3 and was named AL-FR3. Further, a PCR product obtained by using an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 193 as a forward primer and an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 195 as a reverse primer was obtained by four FRs in the variable region of the humanized L chain. Among them, it encodes FR4 and was named AL-FR4.

(8) After purifying the PCR products obtained in (7), 1 μL of each of these mixtures was used as a template, the oligonucleotide set forth in SEQ ID NO: 188 was used as a forward primer, and the oligonucleotide set forth in SEQ ID NO: 195 was used as a reverse primer. PCR was performed as Specifically, a reaction solution having the composition shown in Table 7 was prepared, and after heat treatment of the reaction solution at 98°C for 5 minutes, the first step was at 98°C for 10 seconds, the second step was at 55°C for 5 seconds, and the heat treatment was performed at 72°C. The reaction was carried out by repeating 30 cycles of the third step of 1 minute at .

(9) The PCR product obtained in (8) was purified, digested with restriction enzymes EcoRI and BsiWI, and ligated with pEF-hL prepared in (6), which was similarly digested with restriction enzymes EcoRI and BsiWI and purified.

(10) E. coli JM109 strain was transformed with the ligation product obtained in (9) and spread on an LB plate medium containing 50 µg/mL carbenicillin to humanize the L chain frame region of antibody A and live. A rally of humanized L chain frame library colonies was obtained.

Example 13 Humanized L Chain Frame Library Screening (1) SEQ ID NO: 196 (5′-AGATGCTAGCACCATGGGATGGAGCTGGATC-3′ using pAP-AH (antibody A heavy chain, mouse antibody before humanization) prepared in Example 3 as a template ) and SEQ ID NO: 197 (5'-AAGGGCGGCCGCTCATTTACCAGGAGAGTG-3'). Specifically, a reaction solution having the composition shown in Table 7 was prepared, and after heat treatment of the reaction solution at 98° C. for 1 minute, a first step at 98° C. for 10 seconds, a second step at 55° C. for 5 seconds, 72 The reaction was carried out by repeating 30 cycles of the third step of 1.5 minutes at °C. The PCR product amplified by this PCR (polynucleotide encoding the amino acid sequence of antibody A heavy chain shown in SEQ ID NO: 16) was named AH-P.

(2) SEQ ID NO: 198 (5'-AGATGCTAGCACCATGGATTTTCAAGTGC-3') and SEQ ID NO: 199 (5'-AAGGGCGGCCGCCTAACACTCATTCCTG-) using pAP-AL (antibody A light chain, mouse antibody before humanization) prepared in Example 3 as a template 3') was performed using oligonucleotide primers having the sequences described in 3'). Specifically, a reaction solution having the composition shown in Table 7 was prepared, and after heat treatment of the reaction solution at 98° C. for 1 minute, a first step at 98° C. for 10 seconds, a second step at 55° C. for 5 seconds, 72 The reaction was carried out by repeating 30 cycles of the third step of 1.5 minutes at °C. A PCR product amplified by this PCR (polynucleotide encoding the amino acid sequence of the antibody A light chain shown in SEQ ID NO: 21) was named AL-P.

(3) After purifying AH-P prepared in (1) and AL-P prepared in (2), digested with restriction enzymes NheI and NotI, similarly digested with restriction enzymes NheI and NotI and purified Example 1 It was ligated with pCAG-dhfr prepared in . Escherichia coli strain JM109 was transformed with the ligation product, and the plasmid was extracted from the transformant cultured in LB medium containing 50 μg/mL carbenicillin. A plasmid pCAG-AL was constructed to express the L chain.

(4) A colony of the L chain frame library obtained in Example 12 was picked up in a 96-well deep well containing 1.5 mL/well of 2xYT containing 100 µg/mL carbenicillin and cultured overnight at 37°C with shaking at 1000 rpm. did. After culturing, the cells were collected by centrifugation at 2000 rpm for 20 minutes, and plasmids were prepared using Direct Prep96 Kit (manufactured by Qiagen).

(5) 150 ng of H chain expression plasmid pFU-AH2 of humanized antibody A in which the heavy chain variable (VH) region prepared in Example 9 is AH2 (SEQ ID NO: 142), the humanized L chain prepared in (4) 20 μL of Opti-MEM containing 2 μL of frame library expression plasmid and 0.2 μL of P3000 reagent (manufactured by Thermo Fisher Scientific) and 20 μL of Opti-MEM containing 0.2 μL of Lipofectamine 3000 (manufactured by Thermo Fisher Scientific) were mixed and allowed to stand at room temperature for 15 minutes. The mixture was cultured in a 96-well plate at 37°C, 5% CO ₂ using DMEM medium (manufactured by Thermo Fisher Scientific) containing 50 μg/mL kanamycin and 10% (w/v) bovine serum. Humanized antibody A was expressed by exchanging the culture supernatant of confluent COS1 cells with 90 μL of Opti-MEM, followed by static culture at 37° C. and 5% CO ₂ for 2 days. .

(6) The binding of the expressed humanized antibody A to the human gp130 receptor was evaluated using the ELISA described in Example 2 (3), except that the labeled antibody used for detection was an anti-human antibody (manufactured by Bethyl). It was evaluated by the same method. As a result of evaluating about 1400 clones from the humanized L chain frame library, 49 clones that showed binding to the human gp130 receptor were obtained.

(7) 250 ng of pFU-AH2 prepared in Example 9, 250 ng of the humanized A antibody L chain expression clone selected in (6) as a plasmid, and 50 μL of Opti-MEM containing 1 μL of P3000 reagent and 1 μL 50 μL of Opti-MEM containing Lipofectamine 3000 was mixed and allowed to stand at room temperature for 15 minutes. COS1 cells were cultured in 24-well plates at 37° C. and 5% CO ₂ .

(8) After replacing the culture supernatant of the confluent cells with 400 μL of Opti-MEM, the humanized antibody A was expressed by static culture for 2 days under conditions of 37° C. and 5% CO ₂ . rice field.

(9) In the same manner as described in (5) and (6), pCAG-AH and pCAG-AL prepared in (3) were used to express mouse antibody A as a control.

(10) Using an amine coupling kit, the human gp130 receptor prepared by the method described in Example 1 was immobilized on a sensor chip CM5 (manufactured by GE Healthcare) (immobilization amount: equivalent to 4023 RU), ( The human antibody A expressed in 8) and the mouse antibody A expressed in (9) are flowed to the immobilized sensor chip CM5 under the conditions of a contact time of 200 seconds, a dissociation time of 800 seconds, and a flow rate of 30 μL / min, and immobilized. The interaction with human gp130 receptor was measured using Biacore T200 (manufactured by GE Healthcare). Based on the measurement results, the L chain of humanized antibody A, which has a slower dissociation characteristic with humanized antibody A bound to the immobilized human gp130 receptor than mouse antibody A, was screened, and four types of clones were screened. got

Clones expressing the L chain of humanized antibody A obtained by the above method were named AL-4F1, AL-6E6, AL-13H10 and AL-14A10. In addition, the humanized antibody L chain expression plasmid possessed by AL-4F1 is named pFU-AL-4F1, the humanized antibody L chain expression plasmid possessed by AL-6E6 is named pFU-AL-6E6, and AL-13H10 possesses The humanized antibody L chain expression plasmid was named pFU-AL-13H10, and the humanized antibody L chain expression plasmid possessed by AL-14A10 was named pFU-AL-14A10. The amino acid sequence of the humanized antibody L chain AL-L4F1 expressed in pFU-AL-4F1 is SEQ ID NO: 200, the sequence of the polynucleotide encoding AL-L4F1 is SEQ ID NO: 201, and the human expressed in pFU-AL-6E6. The amino acid sequence of the humanized antibody L chain AL-L6E6 in SEQ ID NO: 202, the sequence of the polynucleotide encoding AL-L6E6 in SEQ ID NO: 203, and the amino acids of the humanized antibody L chain AL-L13H10 expressed in pFU-AL-13H10 The sequence is SEQ ID NO: 204, the sequence of the polynucleotide encoding AL-L13H10 is SEQ ID NO: 205, the amino acid sequence of the humanized antibody L chain AL-L14A10 expressed in pFU-AL-14A10 is SEQ ID NO: 206, AL- The sequences of polynucleotides encoding L14A10 are shown in SEQ ID NO: 207, respectively.

Example 14 Affinity measurement of humanized antibody AH2-L13H10 (1) Select pFU-AL-13H10 from among the humanized antibody L chain expression plasmids obtained in Example 13, and prepare 2 μg of the plasmid in Example 9 The heavy chain variable (VH) region is mixed with 1 μg of H chain expression plasmid pFU-AH2 of humanized antibody A whose heavy chain variable (VH) region is AH2 (SEQ ID NO: 142), and electroporation using NeonTransfection System (manufactured by Thermo Fisher Scientific) (1700 V, 20 ms, 1 time) to introduce the gene into CHO cells (DG44 strain).

(2) The transfected cells were shake-cultured (37°C, 8% CO ₂ , 130 rpm) in CD OptiCHO Medium in an Erlenmeyer flask to secretly express the humanized antibody AH2-L13H10.

(3) Cells and impurities contained in the resulting culture medium were removed by centrifugation, and the resulting supernatant was equilibrated with 20 mM Tris-HCl (pH 7.4) containing 150 mM sodium chloride. It was applied to an open column packed with 1 mL of protein A gel (manufactured by Tosoh Corporation).

(4) After washing with the buffer used for the equilibration, elution was performed with 0.1 M glycine hydrochloride buffer (pH 3.0). The eluate was neutralized in pH by adding 1/4 of 1M Tris-HCl (pH 8.0) to the eluate.

(5) The neutralized eluate was concentrated by ultrafiltration and buffer exchanged with 20 mM Tris-HCl (pH 7.4) containing 150 mM sodium chloride to obtain humanized antibody AH2-L13H10 protein. rice field.

(6) After measuring the concentration at 280 nm absorbance and calculating the humanized antibody protein concentration as an extinction coefficient = 1.4, using an amine coupling kit, human gp130 receptor prepared by the method described in Example 1 The body was immobilized on a sensor chip CM5 (manufactured by GE Healthcare) (immobilization amount: equivalent to 389 RU), and the humanized antibody (AH2-L13H10) prepared in (2) was applied for a contact time of 200 seconds and a dissociation time of 800 seconds. Flow was performed at a flow rate of 30 μL/min, and interaction with immobilized human gp130 receptor was measured using Biacore T200 (manufactured by GE Healthcare).

As a result of affinity analysis using analysis software, the affinity of humanized antibody AH2-13H10 was ka = 8.93 × 10 ⁴ [1/Ms], kd = 2.03 × 10 ^-4 [1/s ], KD=2.27×10 ⁻⁹ [M], confirming high affinity. The affinity of the humanized antibody AH2-13H10 is determined by the KD values of the humanized antibodies AH2-AL3 and AH2-AL4 prepared in Example 11 (AH2-AL3: 3.80 × 10 ^-9 [M], AH2-AL4: It had a higher affinity than 4.61×10 ⁻⁹ [M].

Example 15 Improvement of VH Region and Affinity Measurement (1) H chain expression plasmid pFU-AH2 of humanized antibody A having AH2 (SEQ ID NO: 142) as the heavy chain variable (VH) region prepared in Example 9 was used as a template. and SEQ ID NO: 208 (5'-CACTTGTCACGAATTCGCAGGTTCAACTCCAGCAACCAGG-3') or SEQ ID NO: 209 (5'-GCGGTTTACTACTGCGCTCGTACCTACTAC-3') as a forward primer, SEQ ID NO: 210 (5'-GTAGTAGGTACGAGCGCAGTAGTAAACCGC-3 ') or an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 211 (5'-GGCCCTTGGTGCTAGCCGCAGAAACAGTAACGAGGGTACC-3') was used as a reverse primer to perform PCR. Specifically, a reaction solution having the composition shown in Table 10 was prepared, and after heat treatment of the reaction solution at 98°C for 5 minutes, the first step was at 98°C for 10 seconds, the second step was at 55°C for 5 seconds, and the heat treatment was performed at 72°C. The reaction was carried out by repeating 30 cycles of the reaction in which one cycle was the third step for 30 seconds at .
A PCR product obtained by using an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 208 as a forward primer and an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 210 as a reverse primer was named AH2-2P1, and is designated as SEQ ID NO: 209. A PCR product obtained by using an oligonucleotide consisting of the sequence as a forward primer and an oligonucleotide consisting of the sequence shown in SEQ ID NO: 211 as a reverse primer was named AH2-2P2.

(2) Using the purified products of AH2-2P1 and AH2-2P2 prepared in (1) as templates, using an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 208 as a forward primer, and an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 211. was used as a reverse primer for PCR. Specifically, a reaction solution having the composition shown in Table 7 was prepared, and after heat treatment of the reaction solution at 98°C for 5 minutes, the first step was at 98°C for 10 seconds, the second step was at 55°C for 5 seconds, and the heat treatment was performed at 72°C. The reaction was carried out by repeating 30 cycles of the third step of 1 minute at . The resulting PCR product was named AH2-2P3.

(3) AH2-2P3 and pFU-CLIg-dhfr prepared in Example 9(4) were digested with restriction enzymes EcoRI and NheI, purified, and AH2-2P3 and pFU-CLIg-dhfr were ligated.

(4) Escherichia coli strain JM109 was transformed with the ligation product, and pFU-AH2-2 containing humanized antibody AH2-2 was obtained by extracting the plasmid from the transformant cultured in LB medium containing Zeocin. rice field. As a result of sequence analysis, the amino acid sequence of AH2-2, which is the H chain of humanized antibody A cloned into pFU-AH2-2, is shown in SEQ ID NO: 212, and the sequence of the polynucleotide encoding AH2-2 is shown in SEQ ID NO: 213. rice field. The amino acid sequence of the VH region (from 1st glutamine (Gln) to 120th alanine (Ala)) in the amino acid sequence of AH2-2 set forth in SEQ ID NO: 212 is the VH region amino acid sequence set forth in SEQ ID NO: 142 ( AH2) in which the 97th threonine (Thr) is replaced with alanine (Ala).

(5) 2 µg each of pFU-AL-4F1, pFU-AL-6E6 and pFU-AL-14H10 obtained in Example 13 and 1 µg of pFU-AH2-2 prepared in (2) were mixed, The gene was introduced into CHO cells (DG44 strain) in the same manner as in Example 14 (1).

(6) Humanized antibody AH2-2AL4F1, AH2-2L6E6 and AH2-2L14H10 proteins were prepared from the transfected CHO cells in the same manner as in Example 14 (2) to (5).

(7) After calculating the protein concentration in the same manner as in Example 14 (6), using a sensor chip CM5 (manufactured by GE Healthcare) on which human gp130 receptors are immobilized, the same as in Example 14 (6) The affinity of humanized antibody A was measured by the method of .

As a result, the affinity of AH2-2AL4F1 is ka = 1.11 × 10 ⁵ [1/Ms], kd = 2.21 × 10 ^-4 [1/s], KD = 1.98 × 10 ^-9 [M]. and the affinity of AH2-2L6E6 is ka=2.11×10 ⁵ [1/Ms], kd=2.09×10 ⁻⁴ [1/s], KD=9.91×10 ⁻¹⁰ [M ^{[ _ _} M]. The affinities of the humanized antibodies AH2-2L4F1, AH2-2L6E6, and AH2-2L14A10 are all the KD values of the humanized antibody AH2-L13H10 prepared in Example 14 (KD = 2.27 × 10 ^-9 [M]). and the KD values of the humanized antibodies AH2-AL3 and AH2-AL4 prepared in Example 11 (AH2-AL3: 3.80 × 10 ^-9 [M], AH2-AL4: 4. It had a higher affinity than 61×10 ⁻⁹ [M].

Affinities possessed by humanized antibody A generated in Example 11 (AH2-AL3 and AH2-AL4), Example 14 (AH2-L13H10) and this example (AH2-2L4F1, AH2-2L6E6, and AH2-2L14A10) and the affinity of Antibody A (mouse antibody) before humanization (Example 8) is shown in Table 11. Both the humanized antibodies prepared in Example 14 and this example, like the humanized antibody prepared in Example 11, have an affinity approximately equal to the affinity of antibody A (mouse antibody) before humanization. It was confirmed that

Claims (2)

at least (1) amino acid residues from position 252 valine to position 262 arginine of SEQ ID NO: 1 and/or (2) 288 of SEQ ID NO: 1 of the human gp130 receptor consisting of the amino acid sequence set forth in SEQ ID NO: 1 An antagonist to an interleukin-6 family cytokine capable of binding amino acid residues from aspartic acid to arginine at position 300,
The antagonist is an antibody, and the heavy chain complementarity determining region (CDR) 1 of the antibody is SEQ ID NO: 28, the heavy chain CDR2 is SEQ ID NO: 29, the heavy chain CDR3 is SEQ ID NO: 30, and the light chain CDR1 is the sequence 33, wherein the light chain CDR2 comprises the amino acid sequences set forth in SEQ ID NO: 34 and the light chain CDR3 in SEQ ID NO: 35, respectively. The antibody heavy and light chain amino acid sequences are (B) below:
Antagonist according to claim 1 .
(B) the heavy chain contains the amino acid sequences set forth in SEQ ID NO: 26 and the light chain contains the amino acid sequences set forth in SEQ ID NO: 31

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