JP3369160B2 - IL-6 receptor derivative - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a derivative of human interleukin-6 (human IL-6) receptor, a method for producing the same by gene recombination method, an expression vector for use in this method, and the expression vector. It relates to a transformed host.

[0002]

BACKGROUND OF THE INVENTION Interleukin-6 (hereinafter abbreviated as IL-6) is a protein having various important physiological activities and widely involved in cell growth and differentiation. Further IL-
A correlation between high production of 6 and various diseases has been reported (Kis
himoto, Blood 74 pl, 1989, Hirano et al., Immuno
l.Today 11, p443, 1990).

I on the cell membrane that specifically binds to IL-6
The cDNA encoding the L-6 receptor was isolated by Yamazaki et al., And its primary structure has been reported (Science 241 , p82).
5, 1988). Further, the signal transduction mechanism of IL-6 was analyzed by Taga et al., And the IL-6 receptor on the cell membrane having the ability to bind to IL-6 or the IL-6 receptor released from the cell membrane (hereinafter referred to as soluble) was IL-
After binding with 6, it was shown to associate with the glycoprotein gp130 on the cell membrane and IL-6 signal was transduced through this gp130 (see Cell 58 , p573, 1989).

Further, this IL-6 signaling protein g
The cDNA encoding p130 has been cloned by Hibi et al. and its primary structure has been elucidated (Cell 63 , p114.
9, 1990). As a result, gp130 was ubiquitously expressed, but IL-6 receptor expression was IL-6.
It was shown to be limited to cells that can respond to. Therefore, the responsiveness of cells to IL-6 is considered to be regulated by the expression of IL-6 receptor, and I
It was found that the L-6 receptor plays a central role in the IL-6 response.

It is expected that the action of IL-6 can be modified by making the IL-6 receptor soluble. Soluble I actually created by genetic engineering
The L-6 receptor is capable of binding to IL-6 and associating with the IL-6 signaling protein gp130, thus enhancing the action of IL-6 (Yasukawa et al.
al., J. Biochem. 108 , p673, 1990).

As described above, IL-6 has various physiological activities, and it has been suggested that its high production is correlated with various diseases. In human multiple myeloma, IL-6 is responsible for the growth of its tumor cells as a self-growth factor, and it was shown that the addition of anti-IL-6 antibody to tumor cells taken from a patient suppresses their growth. Has been (Kawano eta
l., Nature 332, p83, 1988). Therefore, IL-
Inhibitors of 6 may be therapeutic agents for diseases in which IL-6 is the etiology, such as multiple myeloma.

However, as an inhibitor of IL-6,
So far, only a monoclonal antibody against IL-6 has been developed, and since it is produced by a mouse, it cannot be used as a therapeutic drug for humans as it is because of the problem of antigenicity. On the contrary, IL-
The soluble IL-6 receptor has been developed as an agent that enhances the action of IL-6, but requires a high concentration of soluble IL-6 receptor for its enhancement, and replaces the action of IL-6 by itself. You cannot do it.

On the other hand, in order to analyze the IL-6 signal transduction mechanism at the molecular level, it is essential to analyze the structure and function of IL-6, IL-6 receptor and gp130. For human IL-6, 28 amino acids from its N-terminus are not required for functional expression of IL-6, but from position 29 to 34
IL-by deletion of the last four amino acids at position and C-terminal
It has been reported that the functionality of 6 is lost (Brakenhoff
et al., J. Immunol. 143, p1175, 1989, Kruttgen et a
l., FEBS Lett. 273, p95, 1990).

Further, by NMR analysis, the tryptophan residue at the 158th position, the methionine residue at the 162nd position, and the histidine residue at the 165th position of human IL-6 were sterically close to each other, and the methionine residue at the 162th position was sterically close to each other. And the leucine residue at the 159th position adjacent to the tryptophan residue at the 158th position and the leucine residue adjacent to the histidine residue at the 165th position by introducing amino acid substitutions, thereby binding to IL-6 receptor and IL-6. It has been reported that these amino acid residues are involved in the binding to the IL-6 receptor because both activities were lost (Nishimura et al.
al., FEBS Lett. 281, p167, 1991).

Regarding the IL-6 receptor, it has been reported that its transmembrane region and intracellular region are not required for binding to IL-6 and IL-6 signaling through the signaling protein gp130 (Taga et al. al., Cell
63, p1149, 1990, Yasukawa et al., J. Biochem. 10
8, p673, 1990). The mouse IL-6 receptor also has a structure similar to that of human, but in some cancer cells, the intracellular region is completely replaced by another amino acid sequence, and the IL-6 signal is still transmitted. Have been found to have the ability to do so (see Sugita et al., J.Exp.Med.171, p2001, 1990).
. This means that the intracellular region of the IL-6 receptor is
It is shown to be dispensable for binding to IL-6 and for IL-6 signaling.

However, even though the IL-6 receptor plays the most important role in the IL-6 signal transduction mechanism as described in the preceding paragraph, it binds to IL-6 or interacts with the signal transduction protein gp130. IL-
Its structure-function analysis such as identification of amino acid residues involved in association in the presence of 6 is extremely insufficient.

[0012]

SUMMARY OF THE INVENTION Therefore, the present invention relates to IL
It is intended to provide various IL-6 receptor derivatives having different binding ability to -6 and IL-6 signal transduction ability.

[0013]

In order to solve the above-mentioned problems, the present invention produces various derivatives with deletion or substitution of IL-6 receptor amino acids, and their binding ability to IL-6 receptor and By investigating the presence or absence of IL-6 signal transduction ability, (1) IL-6 binding ability and IL-
An IL-6 receptor derivative having both 6 signal transduction ability, (2) an IL-6 receptor derivative having neither IL-6 binding ability nor IL-6 signal transduction ability,
(3) An IL-6 receptor derivative capable of binding to IL-6 but not IL-6 signal transduction ability, and (4) not having IL-6 binding ability but having IL-6 signal transduction ability The IL-6 receptor derivative having was selected.

Accordingly, the present invention provides the above various IL-6 receptor derivatives. The present invention further provides the above IL-
A DNA encoding a 6-receptor derivative, an expression vector containing the DNA, a host transformed with the expression vector, and the host, which is cultured, and the IL is extracted from the culture.
IL characterized by collecting -6 receptor derivative
A method for producing a -6 receptor derivative is provided.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION IL-6 receptor derivative From the analysis of the primary structure and the like, human IL-6 receptor is
It has an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing and is composed of a signal sequence, an extracellular region, a transmembrane region, and an intracellular region from the N-terminal side, and the extracellular region among them is an immunoglobulin-like domain. , Followed by a cytokine receptor family domain. A so-called soluble IL-6 receptor lacking a transmembrane region and an intracellular region has an ability to bind to IL-6 (IL-6 binding ability) and an ability to transmit an IL-6 signal (IL-6 signal transduction ability). Holds together,
As these soluble IL-6 receptors, those consisting of 344 amino acids at the N-terminal side of the IL-6 receptor, N
It is known that it consists of 323 amino acids on the terminal side (Taga et al, Cell 58 , p573, 1989). Therefore,
The IL-6 receptor that forms the derivative may be a transmembrane type that exists on the cell membrane such as a natural type, or a soluble type that is liberated from the cell membrane. Alternatively, it may be one that remains intracellular.

Therefore, the IL-6 receptor derivative of the present invention comprises an IL-6 receptor containing a transmembrane region and an intracellular region, or a soluble IL lacking the transmembrane region and the intracellular region.
−6 receptor in which one or more amino acid residues are deleted, one in which one or more amino acid residues are replaced by another amino acid residue, and the like.

For example, the amino acid deletion is accompanied by deletion of an immunoglobulin-like domain,
As one example, as described in Examples 1 and 2, a mode in which the immunoglobulin-like domain is deleted by deleting alanine at position 27 to threonine at position 105 of human IL-6 receptor. Stuff is
Binds to IL-6 in the same manner as the natural one, and IL-6
Was able to transmit the signal of. Therefore, this immunoglobulin-like domain is not necessary for the basic performance of these IL-6 receptors. For example, it is possible to miniaturize the soluble IL-6 receptor by omitting this domain to simplify its handling. It will be possible.

Further, for example, those involving amino acid substitution include those in which one or a plurality of amino acids are substituted from amino acid residues 112 to 321 as described in Examples 3 to 9. . When the properties of the soluble IL-6 receptor derivative accompanied by the substitution of these amino acids were analyzed, those that could not bind to IL-6 and the ability to associate with the signal transduction protein gp130 while retaining the ability to bind to IL-6 were analyzed. IL- by losing
It was found that these properties were indistinguishable from those of the soluble IL-6 receptor which did not have the ability to transduce 6 signal transduction or which had no amino acid substitution. Further, by exerting an IL-6 signal without binding to IL-6,
An IL-6 receptor derivative capable of substituting the action of IL-6 is also included in this.

In the present invention, the results of actually expressing an IL-6 receptor derivative and measuring the IL-6 binding ability and the IL-6 signal transduction ability are shown in FIGS. 1 and 2. In these figures, the protein expression level (net), IL-6 binding ability (black), and IL-6 signal transduction ability (hatched line) are IL-6 receptors (wild-type) in which no substitution mutation is introduced.
Each ability is shown as a ratio with 100.
In these figures, those marked with * indicate derivatives lacking both IL-6 binding ability and IL-6 signaling ability, and those marked with arrowheads have IL-6 binding ability but IL-6 signal. Derivatives lacking transduction ability are shown, and unmarked ones retain both abilities.

Of these, for example, 290th place, 291st place,
Those with substitutions at positions 293, 294, 297 or 298 lack both abilities. Therefore, 292nd, 29th
It is presumed that both abilities are lost even if any amino acid residue from the 290th position to the 298th position, including the substitutions at the 5th and 296th positions, is replaced by another amino acid residue. Also, 1
Since the derivatives substituted at the 25th, 126th, 129th, or 130th positions have not lost both abilities, 1
Derivatives substituted at positions 125-130, including substitutions at positions 27 and 128, are presumed to retain both abilities. Therefore, the derivatives of the present invention can be classified as follows.

(1) Human interleukin-6 (IL-
6) Lacking the receptor or transmembrane region and intracellular region I
An IL-6 receptor derivative having an IL-6 binding ability and an IL-6 signal transduction ability, in which an immunoglobulin-like domain of the L-6 receptor is deleted. For example, an IL-6 receptor derivative in which alanine at position 27 to threonine at position 105 of IL-6 receptor is deleted.

(2) Val-6 at position 112, proline at position 113, phenylalanine at position 122, phenylalanine at position 125 and valine at position 130 of the IL-6 receptor or IL-6 receptor lacking the transmembrane region and intracellular region. Until,
From glycine at position 135 to glutamic acid at position 163,
From tyrosine at position 167 to phenylalanine at position 174, aspartic acid at position 184, lysine at position 201 to isoleucine at position 213, alanine at position 220 to tryptophan at position 233, glutamic acid at position 254, glutamic acid at position 278 to 278 IL-6, wherein at least one of the amino acid residues from the valine at position 308 to the proline at position 308, the glutamic acid at position 309, and the glutamic acid at position 317 to proline at position 321 is substituted with another amino acid residue. An IL-6 receptor derivative having a receptor binding ability and an IL-6 signal transduction ability.

For example, the IL-6 receptor or the IL-6 receptor lacking the transmembrane and intracellular regions, 112
Valine at position 113, proline at position 113, phenylalanine at position 122, serine at position 125, proline at position 126, 1
29th asparagine, 130th valine, 135th glycine, 136th proline, 140th proline,
Threonine at position 143, Threonine at position 144, 145
Lysine at position 146, Alanine at position 153, Phenylalanine at position 153, Glutamic acid at position 159, Aspartic acid at position 160, Phenylalanine at position 161, Glutamine at position 162, Glutamic acid at position 163, Tyrosine at position 167, Serine at position 168, Glutamine at 172, 173
Lysine at position 174, Phenylalanine at position 174, Aspartic acid at position 184, Lysine at position 201, Phenylalanine at position 202, Lysine at position 204, Lysine at position 205, Threonine at position 205, Cysteine at position 211, Glycine at position 212, 21
Isoleucine at position 3, alanine at position 220, asparagine at position 221, arginine at position 232, tryptophan at position 233, glutamic acid at position 254, glutamic acid at position 260, arginine at position 261 and serine at position 262,
Lysine at position 263, lysine at position 271, aspartic acid at position 272, glutamine at position 274, histidine at position 275, cysteine at position 277, valine at position 278, 30
Proline at 8th position, glutamic acid at 309th position, glutamic acid at 317th position, serine at 318th position, arginine at 319th position, serine at 320th position, and proline at 321st position 1
An IL-6 receptor derivative having IL-6 binding ability and IL-6 signal transduction ability, wherein 3 amino acid residues are replaced with other amino acid residues.

(3) 121-cysteine, 123-arginine, 124-lysine, 132-cysteine-134-tryptophan of IL-6 receptor or IL-6 receptor lacking the transmembrane region and intracellular region , Cysteine at position 165, cysteine at position 176 ~ 1
Glutamic acid at position 82, cysteine at position 193, 217
Aspartic acid at position 219, proline at position 219, valine at position 236, leucine at position 251 and leucine at position 255
At least one of 57th tyrosine, 279th isoleucine, 287th arginine, 290th valine to 298th phenylalanine, 305th glutamic acid to 307th serine, 313threonine, and 314th proline. IL-6 receptor derivative having neither IL-6 binding ability nor IL-6 signal transduction ability, in which the amino acid residue of is replaced by another amino acid residue.

For example, 121 of the IL-6 receptor or the IL-6 receptor lacking the transmembrane region and intracellular region,
Cysteine at position 123, arginine at position 123, lysine at position 124, cysteine at position 132, tryptophan at position 134, cysteine at position 165, cysteine at position 176, 1
Proline 81, glutamic acid 182, cysteine 193, aspartic acid 217, proline 218, proline 219, valine 236, 2
Tryptophan at position 38, serine at position 247, phenylalanine at position 248, arginine at position 250, leucine at position 251, leucine at position 255, arginine at position 256, tyrosine at position 257, isoleucine at position 279, 2
Arginine at position 87, valine at position 290, glutamine at position 291, arginine at position 293, alanine at position 294, glutamic acid at position 297, phenylalanine at position 298, glutamic acid at position 305, tryptophan at position 306, serine at position 307, Tryptophan in 313th place,
And at least one amino acid residue of proline at position 314 is replaced with another amino acid residue, IL-
6 which has neither 6-binding ability nor IL-6 signal transduction ability
L-6 receptor derivative.

(4) Valine at position 190, histidine at position 280 to glycine at position 285, histidine at position 288, and histidine at position 289 of IL-6 receptor lacking the transmembrane region and intracellular region. At least one amino acid residue of valine is replaced by another amino acid residue, which has IL-6 binding ability but IL-6
An IL-6 receptor derivative having no signal transduction ability.

For example, the IL-6 receptor or the IL-6 receptor lacking the transmembrane and intracellular regions, 190
Valine at position 280, Histidine at position 280, Aspartic acid at position 281, Alanine at position 282, Glycine at position 285,
Histidine at position 288 and 1 or 2 amino acid residues of valine at position 289 are substituted or deleted by other amino acid residues, and have IL-6 binding ability, but IL-6 An IL-6 receptor derivative having no signal transduction ability.

(5) At least one amino acid residue of glycine at position 301 to serine at position 304 of the IL-6 receptor lacking the transmembrane region and intracellular region of the IL-6 receptor is replaced by another amino acid residue. An IL-6 receptor derivative which is substituted with a group and has no IL-6 binding ability but IL-6 signaling ability. Method for Producing IL-6 Receptor Derivative The method for producing the IL-6 receptor derivative described in the preceding paragraph includes genetically engineering to introduce a base substitution, deletion, or insertion into cDNA or genomic DNA encoding the IL-6 receptor. Then, it is convenient to insert this into an expression vector and efficiently express it in various hosts to produce a recombinant. In this case, the host to be expressed may be, for example, Escherichia coli, Bacillus subtilis, yeast, or animal cells, and the expression vector may be any as long as it is effective for expression in each host, and both are particularly preferable. It is not limited.

It may also be produced by using a cell-free protein synthesis system. Furthermore, it may be prepared by reacting an IL-6 receptor protein produced by genetic engineering or extracted and purified from tissues or cells with a chemical substance or an enzyme. Alternatively, it may be a chemically synthesized peptide itself comprising all or part of the amino acid sequence of IL-6 receptor, or may be prepared by reacting this with a chemical substance or an enzyme. Good.

CDNA encoding the IL-6 receptor
Containing the plasmid pBSF2R. 236 has already been obtained, and this cDNA portion was cloned into the commercially available plasmid pIBI.
Since the plasmid pIBIBSF2R inserted in 76 has been deposited as Microtechnology Research Institute No. 2232, DNA encoding the IL-6 receptor derivative of the present invention has been deposited.
Can be obtained from these plasmids according to a conventional method, for example, by site-specific base substitution. The plasmid pBSF2R. The nucleotide sequence of cDNA in 236 and the amino acid sequence of the coding region are shown in SEQ ID NO: 1.

[0031]

EXAMPLES Next, the present invention will be described more specifically by way of examples. Here, an example of a method for preparing an IL-6 receptor derivative in which human IL-6 receptor made soluble by a genetic engineering method is accompanied by amino acid substitution will be described. The creation method is not limited to this.

Example 1. IL-6 with deletion of amino acids
Preparation of Receptor Derivatives To prepare an IL-6 receptor derivative with amino acid deletion, a plasmid pBS containing cDNA encoding human IL-6 receptor cloned by Yamazaki et al.
F2R. 236 was used (see Science 241 , p825, 1988). This cDNA was used as a commercially available plasmid pIBI.
The plasmid pIBIBSF2R obtained by inserting it into No. 76 has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Microtechnology Research Institute No. 2232 (FERM BP-2232).

PBSF2R. 236 restriction enzyme ApaL
Digested with I, treated with S1 nuclease, and then further digested with restriction enzyme BamHI to obtain 2.5 kbp DNA.
A fragment was obtained. On the other hand, pBSF2R. 236 restriction enzyme F
Digested with spI and BamHI, 4.5 kbp D
The NA fragment was obtained. Both DNA fragments were ligated with T4 DNA ligase, and as a result, a plasmid pBSF2RΔIg encoding an IL-6 receptor derivative in which amino acids from alanine at position 27 to threonine at position 105 of human IL-6 receptor were deleted was obtained.

In addition, pBSF2R. 236 restriction enzyme A
After digesting with ccIII, Klenow fragment treatment was performed, and then further digested with restriction enzyme NotI to obtain 5.7.
A kbp DNA fragment was obtained. On the other hand, pBSF2R. 236
Was digested with restriction enzymes SspI and NotI to give 0.
A 9 kbp DNA fragment was obtained. Both DNA fragments are T4DNA
Ligase ligation, resulting in the plasmid pBSF2RΔFam, which encodes an IL-6 receptor derivative lacking the amino acids from lysine at position 124 to asparagine at position 342 of human IL-6 receptor.

The plasmid pBSF prepared as described above
2RΔIg and pBSF2RΔFam and original pB
SF2R. 236 was transfected into each COP cell by the DEAE-dextran method, and after 3 days, each cell was fluorescently stained with biotinylated IL-6 and FL-avidin and analyzed by FACS to analyze these IL-. 6 receptor and its derivatives I
L-6 binding ability was examined.

As shown in FIG. 3, cells transfected with the plasmid pBSF2RΔIg, which encodes the IL-6 receptor derivative in which amino acids 27 to 105 have been deleted, are transformed into the plasmid pBSF2R which encodes the complete IL-6 receptor. ． It was stained similarly to cells transfected with 236. Meanwhile, 34th to 34th
Cells transfected with the plasmid pBSF2RΔFam, which encodes an IL-6R derivative lacking the amino acids up to position 2, were not stained at all. The staining of these cells was specific for IL-6, as confirmed by the addition of excess non-biotinylated IL-6 which inhibited the staining. Therefore, 27 of IL-6 receptor
The amino acids from positions 105 to 105 are not required for binding to IL-6.

Example 2. Amines from 27th to 105th
Of IL-6 receptor derivative lacking no acid
Measurement of signal transduction ability A plasmid pBSF2RΔIg having a cDNA encoding a human IL-6 receptor derivative lacking amino acids 27 to 105 prepared in Example 1 and a plasmid pB encoding human IL-6 receptor
SF2R. 236 10 μg each of 1 μg of plasmid p
Mouse culture cell line M1 was transfected with SV2neo by electric pulse, and selected with G418 to select each transfectant M1IL6RΔIg.
And M1IL6R were obtained.

These transfectants and the parent strain M
1 cells were cultured with various concentrations of IL-6 and ³ H thymidine incorporation was measured after 2 days. As shown in FIG. 4, M1 cells stop growing in response to IL-6, ³
It is known that H 3 thymidine uptake is reduced. Human IL-6 receptor transfectant M
1IL6R is more sensitive to IL-6 than the parental M1 cells, and its growth is arrested at lower concentrations of IL-6.

Human I lacking amino acids 27 to 105, whose IL-6 binding ability was confirmed in Example 1, was deleted.
L-6 receptor derivative transfectant M1I
L6RΔIg has the same level of IL-activity as M1IL-6R cells.
It showed sensitivity to 6. Therefore, as shown in Example 1, the region from amino acids 27 to 105 was IL.
It is not required for binding to -6 or for transducing the IL-6 signal.

Example 3. Soluble husks with amino acid substitutions
Preparation of IL-6 receptor derivative As the cDNA encoding human IL-6 receptor, the one used in Example 1 was used. Regarding the method for preparing soluble human IL-6 receptor using this cDNA,
It has already been reported by Yasukawa et al. (Yasukawa et al.,
J. Biochem. 108, p673, 1990).

Soluble human I consisting of 344 amino acids
S containing cDNA encoding L-6 receptor precursor
The phI fragment was added to the commercially available phage vector M13.
A vector mp18-3 in which the HindIII site of mp18 was converted into an XhoI site and inserted into the SphI site.
44 was infected with Escherichia coli JM105,
Single-stranded DNA was obtained. Using this single-stranded DNA as a template,
Base substitution was introduced site-specifically using a synthetic oligonucleotide and an in vitro mutant preparation system (Amersham). The synthetic oligonucleotide used for this is

[0042] SEQ ID NO: 2 (p112; Val112 Pro113 → Gly112Thr113) SEQ ID NO: 3 (p121a; Cys121Phe122 → Ala121Gly122) SEQ ID NO: 4 (p121b; Cys121 → Ser121) SEQ ID NO: 5 (p122; Phe122 → Ala122) SEQ ID NO: 6 (p123b; Arg123Lys124 → Gln123Ser124) SEQ ID NO: 7 (p125; Ser125Pro126 → Ile125Ser126) SEQ ID NO: 8 (p129; Asn129Val130 → Lys129Leu130) SEQ ID NO: 9 (p132b; Cys132 → Ala132) SEQ ID NO: 10 (p134; Trp134 → Leu134)

SEQ ID NO: 11 (p135; Gly135Pro136 → Ser135Thr136) SEQ ID NO: 12 (p140; Pro140 → Gly140) SEQ ID NO: 13 (p143; Thr143Thr144Lys145 → Gly143Al
a144Glu145) SEQ ID NO: 14 (p145; Lys145Ala146 → Arg145Pro146) SEQ ID NO: 15 (p153; Phe153 → Leu153) SEQ ID NO: 16 (p159; Glu159Asp160 → Leu159Gly160) SEQ ID NO: 17 (p160; Asp160Phe161 → Glu160Leu161) SEQ ID NO: 18 (P162; Gln162Glu163 → Leu162Gln163) SEQ ID NO: 19 (p165; Cys165 → Leu165) SEQ ID NO: 20 (p167; Tyr167Ser168 → Ser167Thr168)

[0044] SEQ ID NO: 21 (p172; Gln172Lys173 → Leu172Gln173) SEQ ID NO: 22 (p174; Phe174 → Ile174) SEQ ID NO: 23 (p176b; Cys176 → Ala176) SEQ ID NO: 24 (p181; Pro181Glu182 → Leu181Gln182) SEQ ID NO: 25 (p184; Asp184 → Thr184) SEQ ID NO: 26 (p190; Val190 → Gly190) SEQ ID NO: 27 (p193; Cys193 → Asp193) SEQ ID NO: 28 (p201a; Lys201Phe202 → Met201Leu202) SEQ ID NO: 29 (p201b; Lys201Phe202 → Glu201Leu202) SEQ ID NO: 30 (p204; Lys204Thr205 → Arg204Ser205)

[0045] SEQ ID NO: 31 (p211; Cys211 → Ala211) SEQ ID NO: 32 (p212; Gly212Ile213 → Glu212Phe213) SEQ ID NO: 33 (p217; Asp217 → Val217) SEQ ID NO: 34 (p218; Pro218Pro219 → Ala218His219) SEQ ID NO: 35 (p220; Ala220Asn221 → Val220Asp221) SEQ ID NO: 36 (p232; Arg232 → Ser232) SEQ ID NO: 37 (p233; Trp233 → Gln233) SEQ ID NO: 38 (p236; Val236, Trp238 → Ser236, Leu238) SEQ ID NO: 39 (p247; Ser247Phe248 → Ala247Val248) SEQ ID NO: 40 (p250; Arg250Leu251 → Ser250Ile251)

SEQ ID NO: 41 (p254a; Glu254 → Ala254) SEQ ID NO: 42 (p254b; Glu254Leu255 → Val254His255) SEQ ID NO: 43 (p256; Arg256Tyr257 → Ser256Asp257) SEQ ID NO: 44 (p260; Glu260Arg261 → Ala260Gly261) SEQ ID NO: 45 (p261; Arg261Ser262Lys263 → Gly261Al
a262Gly263) SEQ ID NO: 46 (p271; Lys271Asp272 → Arg271Tyr272) SEQ ID NO: 47 (p274; Gln274His275 → Leu274Gln275) SEQ ID NO: 48 (p277; Cys277 → Asp277) SEQ ID NO: 49 (p278a; Val278Ile279 → Gly278Met279) sequence (P278b; Val278 → Asn278)

[0047] SEQ ID NO: 51 (p279; Ile279 → Asp279) SEQ ID NO: 52 (p280a; His280Asp281 → Ser280Gly281) SEQ ID NO: 53 (p280b; His280 → Ile280) SEQ ID NO: 54 (p281a; Asp281Ala282 → Val281Asp282) SEQ ID NO: 55 (p281b; Asp281 → Gly281) SEQ ID NO: 56 (p285; Gly285 → Asp285) SEQ ID NO: 57 (p287; Arg287His288 → Ala287Ile288) SEQ ID NO: 58 (p288; His288Val289 → Tyr288Leu289) SEQ ID NO: 59 (p290; Val290Gln291 → Met290His291) SEQ ID NO: 60 (p291; Gln291 → Lys291)

SEQ ID NO: 61 (p293a; Arg293 → Gly293) SEQ ID NO: 62 (p293b; Arg293Ala294 → Gly293Thr294) SEQ ID NO: 63 (p297; Glu297Phe298 → Ala297Ile298) SEQ ID NO: 64 (p301; Gly301Glu302 → Val301Asp302) SEQ ID NO: 65 (p303a; Trp303Ser304 → His303Ala304) SEQ ID NO: 66 (p304; Ser304, Ser307 → Ala304, Ala307) SEQ ID NO: 67 (p305; Glu305Trp306 → Asp305Arg306) SEQ ID NO: 68 (p308; Pro308Glu309 → Leu308Gln309) SEQ ID NO: 69 (p308; p313b; Thr313Pro314 → Lys313Leu314) SEQ ID NO: 70 (p317; Glu317Ser318Arg319 → Val317Va
l318Gly319) SEQ ID NO: 71 (p320; Ser320Pro321 → Asn320Ser321)

It is The names in parentheses are the derivative name, the type and position of the amino acid before mutation, and the type and position after the mutation. After introducing the mutation, each XhoI
-X of the commercially available vector pSVL (Pharmacia) for expression of the BamHI fragment in COS-7 cells.
It was inserted at the hoI-BamHI site. The introduced mutation is Se
The sequence was confirmed using a quenase Sequencing Kit (USB).

Example 4. Various soluble human IL-6 receptors
Of the expression amount of the ter derivative. Various expression vector DNAs having the soluble human IL-6 receptor derivative cDNA prepared in Example 3 or soluble human I to which no mutation was introduced (hereinafter referred to as wild type)
Expression vector DNA containing L-6 receptor cDNA
To the monkey-derived cultured cell line COS-7 cells, respectively.
It was introduced and expressed by the AE-dextran method. After 3 days, the cells were cultured in a medium containing [ ³⁵ S] -methionine,
Radiolabeled culture supernatant was obtained.

Each culture supernatant was treated with anti-human IL-6 receptor monoclonal antibody MT18 (Hirata et al., J. Immunol. 143,
p2900, 1989). Sediment to S
DS polyacrylamide gel was electrophoresed and detected by autoradiography. All soluble human IL-6
Receptor derivatives are immunoprecipitated in varying amounts,
It showed a mobility similar to that of the wild-type soluble human IL-6 receptor. Further, in order to know the amount of sediment, the polyacrylamide gel of the portion containing each sediment was cut out and measured with a liquid scintillator. 1 and 2
Shows the amount of each sediment in% when the amount of the wild-type sediment is 100%.

Example 5. Various soluble human IL-6 receptors
Of IL-6-binding ability of ter derivative wild-type or various soluble human IL-6 as in Example 4
The receptor derivative was expressed in COS-7 cells, and after 3 days, the culture supernatant containing various soluble human IL-6 receptor derivatives was collected. Each culture supernatant was prepared in advance with anti-human I
L-6 receptor monoclonal antibody MT18 was added 100μl per well in 96-well microtiter plates coated, washed 3 times with PBS-Tween was incubated for 2 hours at room temperature, was added IL-6 was ¹ 25I label. After incubation at room temperature for 2 hours, the plate was washed 3 times with PBS-Tween, and the residual radioactivity was measured by a gamma counter.

1 and 2, wild type soluble human IL
Radioactivity of IL-6 bound to -6 receptor is 100%
The radioactivity of IL-6 bound to each soluble human IL-6 receptor derivative is shown in%. Many soluble human IL-6 receptor derivatives showed IL-6 binding ability correlated with the expression level measured in Example 4. Further, a soluble human IL-6 receptor derivative having a significantly low binding ability or no binding ability as compared with the expression level was also obtained. Such a derivative is indicated by an asterisk (*) in FIGS. 1 and 2.
Is attached.

Example 6. Soluble human IL-6 receptor
Of IL-6 signal transduction ability of IL-6 cDNA of IL-6 signal transduction protein human gp130
Was inserted into pZipNeoSV (X) I (see Hibi et al. Cell 63 , p1149, 1990) and IL-6
The mouse myeloid Leukemia cell line NFS60, which proliferates in response to various cytokines including, was electroporated and selected with G418 to obtain transfectant NFS130.

COS into which 5 × 10 ⁴ NFS130 cells were introduced with the expression vector pSVL having a wild-type soluble human IL-6 receptor cDNA in the same manner as in Example 3.
-7% cell culture supernatant and 25% of COS-7 cell culture supernatant introduced with pSVL having no cDNA to be expressed
³ H thymidine incorporation was measured after overnight culture in the presence or absence of 400 ng / ml IL-6 in a culture medium containing.

As shown in FIG. 5, in the absence of IL-6,
Uptake of ³ H thymidine is not seen, but uptake is seen in the presence of IL-6, and wild type soluble human I
When COS-7 cell culture supernatant containing L-6 receptor was added, a marked increase in ³ H thymidine incorporation was observed as compared to the case where COS-7 cell culture supernatant containing no L-6 receptor was added. It was This increase in ³ H thymidine uptake was taken as IL-6 of the wild-type soluble human IL-6 receptor.
The signal transduction ability.

Example 7. Various soluble human IL-6 receptors
Of IL-6 Signal Transduction Ability of the Ter Derivatives IL-6 signal transduction ability of wild-type and various soluble human IL-6 receptor derivatives contained in the COS-7 cell culture supernatant obtained in Example 5 It measured using the measuring method shown in 6. ³ H by wild type soluble human IL-6 receptor in the presence of 400 ng / ml IL-6
The IL-6 signal transduction ability of various soluble human IL-6 receptor derivatives when the increase in thymidine uptake is taken as 100% of the IL-6 signal transduction ability is shown as a percentage of the increase in ³ H thymidine uptake. And shown in FIG.

The IL-6 signal transduction ability of most of the soluble human IL-6 receptor derivatives showed a correlation with the IL-6 binding ability measured in Example 5. In addition, a soluble human IL-6 receptor derivative p190, which has an IL-6-binding ability but has a significantly low IL-6 signal transduction ability or no detection at all,
p280a, p280b, p281a, p281b, p
285 and p288 were obtained. Such a derivative is shown in FIG.
2 and the arrow mark in FIG. Also, p301 and p303
Showed no IL-6 binding ability, but only IL-6 signal transduction ability.

Example 8. Preparation of Soluble Human gp130 The λu-5 and λu-7 clones harboring the cDNA fragment encoding human gp130 cloned by Hibi (Cell 63 , p1194, 1990) et al.
2.1 kbp and 1.0 kbp produced by digestion with RI
Fragment of EcoRI of M13mp18 vector
M that can be inserted into the site and encode the entire gp130 precursor
p18gp130 was obtained. Then, site-specific base substitution was carried out in the same manner as in Example 3 using the synthetic oligonucleotide 5'-GAAATTGAAGCCTGAGCTCATAGTCGTGCCT-3 'to give gp1.
A translation stop codon was inserted into codon 621 of 30.

Then, c of the prepared soluble gp130
The DNA fragment was excised by digestion with HindIII and SalI, and the expression vector pECEdh for CHO cells.
inserted into the HindIII-SalI site of fr, pEC
Edhfrsgp620 was obtained. 10 μg pECed
The hfrsgp620 DNA was transfected into the dhfr-deficient CHO cell line DXB-11 by the calcium phosphate method, and selection was performed in an αMEM medium containing 10% dialyzed FCS.

Then, the selection with methotrexate was started from 20 nM, the concentration was increased successively, and finally the selection with 200 nM was performed. As a result, a clone named G16 was selected. The G16 cell culture supernatant was concentrated by ultrafiltration, and then soluble gp130 was partially purified by DEAE-HPLC and gel filtration.
The partially purified soluble gp130 was labeled with ¹²⁵ I using Bolton Hunter reagent.

Example 9. Soluble IL-6 receptor induction
Assay of body's ability to associate with soluble gp130 The various soluble IL-6 receptors obtained in Example 5 were concentrated 30 times by ultrafiltration. To 470 μl of this concentrated solution, 30 μl of ¹²⁵ I-labeled soluble gp130 prepared in Example 8 was added, and 250 μg / ml of IL-6 was added to 2 μm.
1 and the mixture was incubated at 37 ° C. for 30 minutes. In addition to this, the monoclonal antibody MT against the human IL-6 receptor
5 μg of 18 was added, and the mixture was left standing at 4 ° C. for 1 hour, then
Protein A Sepharose of 1 was added and gently shaken at 4 ° C. for 1 hour.

This Sepharose gel was treated with 1% dichitonin-
10 mM triethanolamine (pH 7.5) -0.15
After washing with M NaCl four times, SDS-PAGE was performed, and then autoradiography was performed. As shown in FIG. 6, when a concentrated solution containing wild-type soluble IL-6 receptor is used, a band of approximately 90K corresponding to the radiolabeled soluble gp130 is observed. That is, soluble IL-
The 6 receptor can associate with soluble gp130 in the presence of IL-6.

On the other hand, in Example 6, it was shown that the ability to bind to IL-6 was maintained, but in Example 7, soluble IL-6 was shown to be unable to transduce the signal of IL-6.
Receptor derivatives p190, p280a, p280b,
When p281a, p281b, p285 and p288 were used, the 90K band was not detected. That is, these soluble IL-6 receptor derivatives are
It cannot associate with gp130 even in the presence of -6.

[0065]

[Sequence list]   SEQ ID NO: 1   Array length:   Sequence type: Nucleic acid   Topology: linear   Sequence type: cDNA   Array:   TCATGTGCGA GTGGGAAGTC GCACTGACAC TGAGCCGGGC CAGAGGGAGA 50   GGAGCCGAGC GCGGCGCGGG GCCGAGGGCC GGGCTCCTGC GGATGGGGGC 100   TGCCCCCGGG GCCTGAGCCC GCCTGCCCGC CCACCGCCCC GCCCCGCCCC 150   TGCCACCCCT GCCGCCCGGT TCCCATTAGC CTGTCCGCCT CTGCGGGACC 200   ATGGAGTGGT AGCCGAGGAG GAAGC ATG CTG GCC GTC GGC TGC GCG CTG 249                               Met Leu Ala Val Gly Cys Ala Leu                                                 Five   CTG GCT GCC CTG CTG GCC GCG CCG GGA GCG GCG CTG GCC CCA AGG 294   Leu Ala Ala Leu Leu Ala Ala Pro Gly Ala Ala Leu Ala Pro Arg        10 15 20   CGC TGC CCT GCG CAG GAG GTG GCA AGA GGC GTG CTG ACC AGT CTG 339   Arg Cys Pro Ala Gln Glu Val Ala Arg Gly Val Leu Thr Ser Leu        25 30 35   CCA GGA GAC AGC GTG ACT CTG ACC TGC CCG GGG GTA GAG CCG GAA 384   Pro Gly Asp Ser Val Thr Leu Thr Cys Pro Gly Val Glu Pro Glu        40 45 50   GAC AAT GCC ACT GTT CAC TGG GTG CTC AGG AAG CCG GCT GCA GGC 429   Asp Asn Ala Thr Val His Trp Val Leu Arg Lys Pro Ala Ala Gly        55 60 65   TCC CAC CCC AGC AGA TGG GCT GGC ATG GGA AGG AGG CTG CTG CTG 474   Ser His Pro Ser Arg Trp Ala Gly Met Gly Arg Arg Leu Leu Leu        70 75 80   AGG TCG GTG CAG CTC CAC GAC TCT GGA AAC TAT TCA TGC TAC CGG 519   Arg Ser Val Gln Leu His Asp Ser Gly Asn Tyr Ser Cys Tyr Arg        85 90 95   GCC GGC CGC CCA GCT GGG ACT GTG CAC TTG CTG GTG GAT GTT CCC 564   Ala Gly Arg Pro Ala Gly Thr Val His Leu Leu Val Asp Val Pro       100 105 110   CCC GAG GAG CCC CAG CTC TCC TGC TTC CGG AAG AGC CCC CTC AGC 609   Pro Glu Glu Pro Gln Leu Ser Cys Phe Arg Lys Ser Pro Leu Ser       115 120 125   AAT GTT GTT TGT GAG TGG GGT CCT CGG AGC ACC CCA TCC CTG ACG 654   Asn Val Val Cys Glu Trp Gly Pro Arg Ser Thr Pro Ser Leu Thr       130 135 140   ACA AAG GCT GTG CTC TTG GTG AGG AAG TTT CAG AAC AGT CCG GCC 699   Thr Lys Ala Val Leu Leu Val Arg Lys Phe Gln Asn Ser Pro Ala       145 150 155   GAA GAC TTC CAG GAG CCG TGC CAG TAT TCC CAG GAG TCC CAG AAG 744   Glu Asp Phe Gln Glu Pro Cys Gln Tyr Ser Gln Glu Ser Gln Lys       160 165 170   TTC TCC TGC CAG TTA GCA GTC CCG GAG GGA GAC AGC TCT TTC TAC 789   Phe Ser Cys Gln Leu Ala Val Pro Glu Gly Asp Ser Ser Phe Tyr       175 180 185   ATA GTG TCC ATG TGC GTC GCC AGT AGT GTC GGG AGC AAG TTC AGC 834   Ile Val Ser Met Cys Val Ala Ser Ser Val Gly Ser Lys Phe Ser       190 195 200   AAA ACT CAA ACC TTT CAG GGT TGT GGA ATC TTG CAG CCT GAT CCG 879   Lys Thr Gln Thr Phe Gln Gly Cys Gly Ile Leu Gln Pro Asp Pro       205 210 215   CCT GCC AAC ATC ACA GTC ACT GCC GTG GCC AGA AAC CCC CGC TGG 924   Pro Ala Asn Ile Thr Val Thr Ala Val Ala Arg Asn Pro Arg Trp       220 225 230   CTC AGT GTC ACC TGG CAA GAC CCC CAC TCC TGG AAC TCA TCT TTC 969   Leu Ser Val Thr Trp Gln Asp Pro His Ser Trp Asn Ser Ser Phe       235 240 245   TAC AGA CTA CGG TTT GAG CTC AGA TAT CGG GCT GAA CGG TCA AAG 1014   Tyr Arg Leu Arg Phe Glu Leu Arg Tyr Arg Ala Glu Arg Ser Lys       250 255 260   ACA TTC ACA ACA TGG ATG GTC AAG GAC CTC CAG CAT CAC TGT GTC 1059   Thr Phe Thr Thr Trp Met Val Lys Asp Leu Gln His His Cys Val       265 270 275   ATC CAC GAC GCC TGG AGC GGC CTG AGG CAC GTG GTG CAG CTT CGT 1104   Ile His Asp Ala Trp Ser Gly Leu Arg His Val Val Gln Leu Arg       280 285 290   GCC CAG GAG GAG TTC GGG CAA GGC GAG TGG AGC GAG TGG AGC CCG 1149   Ala Gln Glu Glu Phe Gly Gln Gly Glu Trp Ser Glu Trp Ser Pro       295 300 305   GAG GCC ATG GGC ACG CCT TGG ACA GAA TCC AGG AGT CCT CCA GCT 1194   Glu Ala Met Gly Thr Pro Trp Thr Glu Ser Arg Ser Pro Pro Ala       310 315 320   GAG AAC GAG GTG TCC ACC CCC ATG CAG GCA CTT ACT ACT AAT AAA 1239   Glu Asn Glu Val Ser Thr Pro Met Gln Ala Leu Thr Thr Asn Lys       325 330 335   GAC GAT GAT AAT ATT CTC TTC AGA GAT TCT GCA AAT GCG ACA AGC 1284   Asp Asp Asp Asn Ile Leu Phe Arg Asp Ser Ala Asn Ala Thr Ser       340 345 350   CTC CCA GTG CAA GAT TCT TCT TCA GTA CCA CTG CCC ACA TTC CTG 1329   Leu Pro Val Gln Asp Ser Ser Ser Val Pro Leu Pro Thr Phe Leu       355 360 365   GTT GCT GGA GGG AGC CTG GCC TTC GGA ACG CTC CTC TGC ATT GCC 1374   Val Ala Gly Gly Ser Leu Ala Phe Gly Thr Leu Leu Cys Ile Ala       370 375 380   ATT GTT CTG AGG TTC AAG AAG ACG TGG AAG CTG CGG GCT CTG AAG 1419   Ile Val Leu Arg Phe Lys Lys Thr Trp Lys Leu Arg Ala Leu Lys       385 390 395   GAA GGC AAG ACA AGC ATG CAT CCG CCG TAC TCT TTG GGG CAG CTG 1464   Glu Gly Lys Thr Ser Met His Pro Pro Tyr Ser Leu Gly Gln Leu       400 405 410   GTC CCG GAG AGG CCT CGA CCC ACC CCA GTG CTT GTT CCT CTC ATC 1509   Val Pro Glu Arg Pro Arg Pro Thr Pro Val Leu Val Pro Leu Ile       415 420 425   TCC CCA CCG GTG TCC CCC AGC AGC CTG GGG TCT GAC AAT ACC TCG 1554   Ser Pro Pro Val Ser Pro Ser Ser Leu Gly Ser Asp Asn Thr Ser       430 435 440   AGC CAC AAC CGA CCA GAT GCC AGG GAC CCA CGG AGC CCT TAT GAC 1599   Ser His Asn Arg Pro Asp Ala Arg Asp Pro Arg Ser Pro Tyr Asp       445 450 455   ATC AGC AAT ACA GAC TAC TTC TTC CCC AGA TAGCTGGCTG GGTGGCACCA 1649   Ile Ser Asn Thr Asp Tyr Phe Phe Pro Arg       460 465   GCAGCCTGGA CCCTGTGGAT GACAAAACAC AAACGGGCTC AGCAAAAGAT 1699   GCTTCTCACT GCCATGCCAG CTTATCTCAG GGGTGTGCGG CCTTTGGCTT 1749   CACGGAAGAG CCTTGCGGAA GGTTCTACGC CAGGGGAAAA TCAGCCTGCT 1799   CCAGCTGTTC AGCTGGTTGA GGTTTCAAAC CTCCCTTTCC AAATGCCCAG 1849   CTTAAAGGGG TTAGAGTGAA CTTGGGCCAC TGTGAAGAGA ACCATATCAA 1899   GACTCTTTGG ACACTCACAC GGACACTCAA AAGCTGGGCA GGTTGGTGGG 1949   GGCCTCGGTG TGGAGAAGCG GCTGGCAGCC CACCCCTCAA CACCTCTGCA 1999   CAAGCTGCAC CCTCAGGCAG GTGGGATGGA TTTCCAGCCA AAGCCTCCTC 2049   CAGCCGCCAT GCTCCTG 2066

[0066]   SEQ ID NO: 2   Sequence length: 27   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   TGCTGGTGGA TGGTACCCCC GAGGAGC 27

[0067]   SEQ ID NO: 3   Sequence length: 36   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GGAGCCCCAG CTCTCAGCCG GCCGGAAGAG CCCCCT 36

[0068]   SEQ ID NO: 4   Sequence length: 32   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GGAGCCCCAG CTCTCAAGCT TCCGGAAGAG CC 32

[0069]   SEQ ID NO: 5   Sequence length: 32   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CCCCAGCTCT CCTGCGCGCG GAAGAGCCCC CT 32

[0070]   SEQ ID NO: 6   Sequence length: 36   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CAGCTCTCCT GCTTCCAGTC TAGCCCCCTC AGCAAT 36

[0071]   SEQ ID NO: 7   Sequence length: 27   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GCTTCCGGAA GATATCCCTC AGCAATG 27

[0072]   SEQ ID NO: 8   Sequence length: 18   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CTCAGCAAGC TTGTTTGT 18

[0073]   SEQ ID NO: 9   Sequence length: 32   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CTCAGCAATG TTGTTGCTGA GTGGGGTCCT CG 32

[0074]   SEQ ID NO: 10   Sequence length: 33   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   AATGTTGTTT GTGAGCTCGG TCCTCGGAGC ACC 33

[0075]   SEQ ID NO: 11   Sequence length: 28   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GTTTGTGAGT GGAGTACTCG GAGCACCC 28

[0076]   SEQ ID NO: 12   Sequence length: 36   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   TGGGGTCCTC GGAGCACCGG ATCCCTGACG ACAAAG 36

[0077]   SEQ ID NO: 13   Sequence length: 40   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GAGCACCCCA TCCCTTGGCG CCGAGGCTGT GCTCTTGGTG 40

[0078]   SEQ ID NO: 14   Sequence length: 19   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GACGACAAGG CCTGTGCTC 19

[0079]   SEQ ID NO: 15   Sequence length: 17   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   TGAGGAAGCT TCAGAAC 17

[0080]   SEQ ID NO: 16   Sequence length: 35   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CAGAACAGTC CGGCCCTAGG CTTCCAGGAG CCGTG 35

[0081]   SEQ ID NO: 17   Sequence length: 18   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GCCGAAGAGC TCCAGGAG 18

[0082]   SEQ ID NO: 18   Sequence length: 19   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   AGACTTCCTG CAGCCGTGC 19

[0083]   SEQ ID NO: 19   Sequence length: 33   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GACTTCCAGG AGCCGCTGCA GTATTCCCAG GAG 33

[0084]   SEQ ID NO: 20   Sequence length: 36   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CAGGAGCCGT GCCAGTCGAC CCAGGAGTCC CAGAAG 36

[0085]   SEQ ID NO: 21   Sequence length: 28   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CCCAGAGGTC CCTGCAGTTC TCCTGCCA 28

[0086]   SEQ ID NO: 22   Sequence length: 17   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CCCAGAAGAT CTCCTGC 17

[0087]   SEQ ID NO: 23   Sequence length: 34   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GTCCCAGAAG TTCTCTGCGC AGTTAGCAGT CCCG 34

[0088]   SEQ ID NO: 24   Sequence length: 36   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   TGCCAGTTAG CAGTCCTGCA GGGAGACAGC TCTTTC 36

[0089]   SEQ ID NO: 25   Sequence length: 36   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   TTAGCAGTCC CGGAGGGTAC CAGCTCTTTC TACATA 36

[0090]   SEQ ID NO: 26   Sequence length: 36   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GACAGCTCTT TCTACATAGG ATCCATGTGC GTCGCC 36

[0091]   SEQ ID NO: 27   Sequence length: 32   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   TACATAGTGT CCATGGACGT CGCCAGTAGT GT 32

[0092]   SEQ ID NO: 28   Sequence length: 19   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CGGGAGCATG CTCAGCAAA 19

[0093]   SEQ ID NO: 29   Sequence length: 28   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   AGTGTCGGGA GCGAGCTCAG CAAAACTC 28

[0094]   SEQ ID NO: 30   Sequence length: 27   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GCAAGTTCAG CAGATCTCAA ACCTTTC 27

[0095]   SEQ ID NO: 31   Sequence length: 28   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   AACCTTTCAG GGCGCCGGAA TCTTGCAG 28

[0096]   SEQ ID NO: 32   Sequence length: 19   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GGGTTGTGAA TTCTTGCAG 19

[0097]   SEQ ID NO: 33   Sequence length: 34   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   TGGAATCTTG CAACCGGTAC CGCCTGCCAA CATC 34

[0098]   SEQ ID NO: 34   Sequence length: 36   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   ATCTTGCAGC CTGATGCGCA TGCCAACATC ACAGTC 36

[0099]   SEQ ID NO: 35   Sequence length: 19   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   TCCGCCTGTC GACATCACA 19

[0100]   SEQ ID NO: 36   Sequence length: 17   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GAAACCCCAG CTGGCTC 17

[0101]   SEQ ID NO: 37   Sequence length: 36   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GCCAGAAACC CCCGCCAGCT GAGTGTCACC TGGCAA 36

[0102]   SEQ ID NO: 38   Sequence length: 38   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CCCCGCTGGC TCAGTAGTAC TTTGCAAGAC CCCCACTC 38

[0103]   SEQ ID NO: 39   Sequence length: 28   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   TCCTGGAACT CAGCTGTCTA CAGACTAC 28

[0104]   SEQ ID NO: 40   Sequence length: 18   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   TTCTACAGTA TACGGTTT 18

[0105]   SEQ ID NO: 41   Sequence length: 17   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   ACGGTTTGCG CTCAGAT 17

[0106]   SEQ ID NO: 42   Sequence length: 28   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GACTACGGTT TGTGCACAGA TATCGGGC 28

[0107]   SEQ ID NO: 43   Sequence length: 36   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CTACGGTTTG AGCTCAGCGA TCGGGCTGAA CGGTCA 36

[0108]   SEQ ID NO: 44   Sequence length: 19   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   TCGGGCTGCA GGGTCAAAG 19

[0109]   SEQ ID NO: 45   Sequence length: 39   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   AGATATCGGG CTGAAGGCGC CGGTACATTC ACAACATGG 39

[0110]   SEQ ID NO: 46   Sequence length: 19   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GATGGTCAGG TACCTCCAG 19

[0111]   SEQ ID NO: 47   Sequence length: 29   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   TCAAGGACCT CCTGCAGCAC TGTGTCATC 29

[0112]   SEQ ID NO: 48   Sequence length: 33   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GACCTCCAGC ATCACGACGT CATCCACGAC GCC 33

[0113]   SEQ ID NO: 49   Sequence length: 29   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   AGCATCACTG TGGCATGCAC GACGCCTGG 29

[0114]   SEQ ID NO: 50   Sequence length: 30   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CAGCATCACT GTAATATTCA CGACGCCTGG 30

[0115]   SEQ ID NO: 51   Sequence length: 25   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CATCACTGTG TCGACCACGC GCCTG 25

[0116]   SEQ ID NO: 52   Sequence length: 35   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CATCACTGTG TCATCTCTGG CGCCTGGAGC GGCCT 35

[0117]   SEQ ID NO: 53   Sequence length: 30   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CACTGTGTCA TCATCGATGC CTGGAGCGGC 30

[0118]   SEQ ID NO: 54   Sequence length: 28   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GTGTCATCCA CGTCGACTGG AGCGGCCT 28

[0119]   SEQ ID NO: 55   Sequence length: 25   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GTGTCATCCA CGGCGCCTGG AGCGG 25

[0120]   SEQ ID NO: 56   Sequence length: 30   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CACGACGCCT GGTCAGATCT GAGGCACGTG 30

[0121]   SEQ ID NO: 57   Sequence length: 30   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CTGGAGCGGC CTCGCGATCG TGGTGCAGCT 30

[0122]   SEQ ID NO: 58   Sequence length: 28   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   AGCGGCCTGA GGTACCTGGT GCAGCTTC 28

[0123]   SEQ ID NO: 59   Sequence length: 30   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CTGAGGCACG TGATGCATCT TCGTGCCCAG 30

[0124]   SEQ ID NO: 60   Sequence length: 25   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:  AGGCACGTGG TGAAGCTTCG TGCCC 25

[0125]   SEQ ID NO: 61   Sequence length: 33   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CACGTGGTGC AGCTTGGCGC CCAGGAGGAG TTC 33

[0126]   SEQ ID NO: 62   Sequence length: 28   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GTGGTGCAGC TTGGTACCCA GGAGGAGT 28

[0127]   SEQ ID NO: 63   Sequence length: 27   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GTGCCCAGGA GGCGATCGGG CAAGGCG 27

[0128]   SEQ ID NO: 64   Sequence length: 28   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   AGTTCGGGCA AGTCGACTGG AGCGAGTG 28

[0129]   SEQ ID NO: 65   Sequence length: 35   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   TTCGGGCAAG GCGAGCATGC CGAGTGGAGC CCGGA 35

[0130]   SEQ ID NO: 66   Sequence length: 42   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GGGCAAGGCG AGTGGGCTGA GTGGGCTCCG GAGGCCATGG GC 42

[0131]   SEQ ID NO: 67   Sequence length: 28   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GGCGAGTGGA GCGATCGGAG CCCGGAGG 28

[0132]   SEQ ID NO: 68   Sequence length: 27   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GCGAGTGGAG CCTGCAGGCC ATGGGCA 27

[0133]   SEQ ID NO: 69   Sequence length: 30   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GAGGCCATGG GCAAGCTTTG GACAGAATCC 30

[0134]   SEQ ID NO: 70   Sequence length: 41   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   GCACGCCTTG GACAGTTGTA GGATCCCCTC CAGCTGAGAA C 41

[0135]   SEQ ID NO: 71   Sequence length: 27   Sequence type: Nucleic acid   Number of chains: Single chain   Topology: linear   Sequence type: Synthetic DNA   Array:   CAGAATCCAG GAATTCTCCA GCTGAGA 27

[Brief description of drawings]

FIG. 1 shows the properties of various soluble IL-6R derivatives having amino acid substitutions. The shaded area represents the expression level, the black line represents the IL-6 binding ability, and the shaded area represents the IL-6 signal transduction ability.
In all cases, wild type soluble IL-6R was expressed as 100%. In addition, each number is the position of the substituted amino acid residue, and the type of the amino acid is shown in parentheses in one letter.

FIG. 2 is similar to FIG.

FIG. 3: IL-6 of COP cells transfected with a plasmid encoding IL-6R or a derivative thereof.
The result of having analyzed the binding ability by FACS is shown. The transfected plasmid is a; pBSF2R. 236,
b; pBSF2RΔIg, c; pBSF2RΔFam.

FIG. 4 M1 cells, M1IL6 expressing IL-6R
The response to IL-6 of R cells and M1IL6RΔIg cells expressing an IL-6R derivative lacking amino acids 27 to 105 is shown.

FIG. 5 shows the results of measuring the signal transduction ability of soluble IL-6R using NFS130 cells.

FIG. 6 shows the soluble gp130 association ability of various soluble IL-6R derivatives in the presence of IL-6.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI C12N 5/10 C12N 15/00 ZNAA C12P 21/02 5/00 A (72) Inventor Kiyo Hogawa 7-Sagamiono, Sagamihara-shi, Kanagawa 37-17 (56) References JP-A-3-133390 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/00 C07K 14/715 JISST file (JOIS)

Claims (6)

(57) [Claims] 1. An IL-6 receptor having the amino acid sequence shown in SEQ ID NO: 1 or lacking a transmembrane region and intracellular region
In the IL-6 receptor, (1) Val at position 190 is changed to Gly; (2) His-Asp at position 280-281 is changed to Ser-Gly; (3) His at position 280 is changed (4) Asp-Ala at position 281-282 has changed to Val-Asp; (5) Asp at position 281 has changed to Gly; (6) Gly at position 285 Is changed to Asp; or (7) His-Val at positions 288-289 is changed to Tyr-Leu; and it has the ability to bind IL-6 but not the ability to signal IL-6 IL-6 receptor derivative. 2. An IL-6 receptor having the amino acid sequence shown in SEQ ID NO: 1 or lacking the transmembrane region and intracellular region.
In the IL-6 receptor, (a) Gly-Glu at position 301-302 is changed to Val-Asp; or (b) Trp-Ser at position 303-304 is changed to His-Ala; , An IL-6 receptor derivative having no IL-6 binding ability but having IL-6 signal transduction ability. 3. A DNA encoding the IL-6 receptor derivative according to claim 1 or 2. 4. An expression vector containing the DNA according to claim 3. 5. A host transformed with the expression vector according to claim 4. 6. The method for producing an IL-6 receptor derivative according to claim 1 or 2, wherein a host transformed with a DNA encoding the IL-6 receptor derivative or an expression vector containing the DNA is cultured. , And the IL from the culture
-6 A method of collecting a receptor derivative.