JPH0899902A - Immature type myeloma cell therapeutic agent containing IL-6 receptor antibody as an active ingredient - Google Patents

Abstract

PURPOSE: To obtain an immature type myeloma cell treating agent inhibiting survival of immature type myeloma cell which becomes a main body of a cell group exhibiting resistance to a chemotherapeutant. CONSTITUTION: This immature type myeloma cell-treating agent contains an interleukin-6 receptor (IL-6R) antibody. A monoclonal antibody derived from mammal is preferably used as the IL-6R antibody and the antibody includes PM-1 antibody, AU 12-20 antibody, AUK146-15 antibody, especially preferably human PM-1 antibody. IL-6R antibody is bound to IL-6R. Thereby, the bond of IL-6 with IL-6R is inhibited and a signal transmission of IL-6 is blocked and biological activity of IL-6 is inhibited and survival of immature type myeloma cell in which survival is kept by IL-6 is inhibited and eradicated.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a chemotherapeutic drug-resistant immature myeloma cell therapeutic agent containing an interleukin 6 receptor antibody as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Myeloma is a tumor in which plasma cells have become malignant.
It is a tumor that develops at multiple sites using the bone marrow as a growth site. As a major growth factor of myeloma cells, interleukin 6 (IL-6) is considered as a strong candidate (Kawano et al., Nature, 332, 83, 19).
88: Klcin et al., Blood, 73, 517, 19
89).

IL-6 is a cytokine called B cell stimulating factor 2 or interferon β2.
IL-6 was discovered as a differentiation factor involved in the activation of B lymphoid cells (Hirano et al., Nature, 3).
24, 73, 1986), and then became a multifunctional cytokine that affects the functions of various cells (Akira, et al., Adv, in Immunolo).
gy, 54, 1, 1993).

IL-6 transmits its biological activity on cells via two proteins. One is IL-6
Is an IL-6 receptor (IL-6R), which is a ligand binding protein having a molecular weight of about 80 KD. IL-
6R is a soluble IL-6R mainly composed of its extracellular region in addition to the membrane-bound type which is expressed on the cell membrane by penetrating the cell membrane.
Exists as (sIL-6R). The other is gp130, which has a molecular weight of about 130 KD and is involved in non-ligand binding signal transduction. IL-6 and IL-6R are IL-6 /
The biological activity of IL-6 is transferred to cells by forming the IL-6R complex and then binding to another membrane protein, gp130 (Taga et al., J. Ex.
p. Med. 196: 967, 1987).

[0005] Kawano et al., VLA (very l
ate activation antigen) -5
Alternatively, MPC (mature plasma cell)
l) -1 (Huang et al., Blood 82,372.
1,1993, the surface antigens on myeloma cells Hei 6-86688) and the like, myeloma cells VLA-5 negative ^(-) MPC-1 - immature myeloma cells, VLA-5
^- MPC-1 intermediate myeloma cells and VL of positive ⁽⁺⁾
A-5 ⁺ MPC-1 ⁺ mature myeloma cells were reported to be divided (Blood, 82, 564, 199.
3).

[0006] Of these, immature myeloma cells are known to be the main constituents of cell populations that are resistant to chemotherapeutic agents (Kawano et al., 56th Annual Meeting of the Japanese Society of Hematology, 261). P. 641, 1994). So far, the addition of IL-6R antibody generally suppresses the growth of myeloma cells (Goto et al., Biot.
therapy 7,655,1993) and IL-6.
Stimulates the growth of immature myeloma cells (Kawan
O., Blood, 82, 564, 1993).

[0007] However, these findings use the proliferation of myeloma cells as an index, and suggest the eradication of immature myeloma cells which are considered to be important in the treatment of myeloma and which are the main constituents of chemotherapeutic drug resistance. It wasn't something. Further, until now, there is no data on whether the IL-6R antibody is useful as a fundamental therapeutic agent for immature myeloma cells, and it is still unclear whether it is directly involved in the survival itself of immature myeloma cells. It was unknown.

[0008]

[Problems to be Solved by the Invention] For immature myeloma cells, which are the main constituents of chemotherapeutic drug resistance, no drug effective until now has been found to be the fundamental treatment thereof. The emergence of drugs that have the effect of eradicating cells has been awaited. Therefore, the present invention provides a therapeutic agent for immature myeloma cells.

[0009]

Means for Solving the Problems As a result of intensive studies aimed at achieving the above-mentioned object, the present inventors have shown that the IL-6R antibody inhibits the survival of immature myeloma cells which are the main constituents of chemotherapeutic drug resistance. It was found that the present invention has been completed. That is,
The present invention provides a novel therapeutic agent for immature myeloma cells, which fundamentally treats immature myeloma cells. More specifically, the present invention provides a therapeutic agent for immature myeloma cells, which comprises an IL-6R antibody as an active ingredient and has a survival inhibitory effect on immature myeloma cells. More specifically, the invention relates to IL-6
Provided is a survival inhibitor of immature myeloma cells containing R antibody as an active ingredient.

[0010]

DETAILED DESCRIPTION The IL-6 receptor antibody used in the present invention is any antibody that blocks IL-6 signal transduction of immature myeloma cells and inhibits the biological activity of IL-6. The origin and type (monoclonal or polyclonal) thereof are not particularly limited, but a mammalian-derived monoclonal antibody is particularly preferable. This antibody is an antibody that inhibits the binding of IL-6 and IL-6R by binding to IL-6R, blocks the signal transduction of IL-6, and inhibits the biological activity of IL-6. .

The animal species of the cell producing the monoclonal antibody is not particularly limited as long as it is a mammal, and may be a human antibody or a mammal other than human. As the monoclonal antibody derived from mammals other than humans, a rabbit or rodent-derived monoclonal antibody is preferable because of its ease of preparation. The rodent is not particularly limited, but mice, rats, hamsters and the like are preferably exemplified.

Examples of such IL-6 receptor antibody include PM-1 antibody (Hirata et al., J. Immuno).
l. 143: 2900-2906, 1989), AUK.
12-20 antibody, AUK64-7 antibody or AUK1
46-15 antibody (International patent application publication number WO92-19
759) and the like. Monoclonal antibodies can be prepared basically as follows using known techniques. That is, using IL-6R as a sensitizing antigen,
It can be prepared by immunizing this with a usual immunization method, fusing the obtained immune cells with a known parent cell by a usual cell fusion method, and screening a monoclonal antibody-producing cell by a usual screening method.

More specifically, the following method may be used to prepare a monoclonal antibody. For example, the sensitizing antigen can be obtained by using the gene sequence of human IL-6R disclosed in European Patent Application Publication No. EP325474. After inserting the human IL-6R gene sequence into a known expression vector system to transform an appropriate host cell, the IL-6R protein of interest is purified from the host cell or the culture supernatant. Purified IL-6R
The protein may be used as a sensitizing antigen.

IL-6R is expressed on the cell membrane as well as possibly released from the cell membrane (sIL-R).
6R) can be used as an antigen. sIL-6R is mainly composed of the extracellular region of IL-6R bound to the cell membrane, and is a membrane-bound type IL-6R in that it lacks the transmembrane region or the transmembrane region and the intracellular region. Is different. The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion, generally, mouse, rat, Hamsters, rabbits, etc. are used.

Immunization of animals with the sensitizing antigen is carried out according to known methods. For example, as a general method,
It is performed by injecting a sensitizing antigen into a mammal intraperitoneally or subcutaneously. Specifically, the sensitizing antigen is PBS (P
phosphate-Buffered Saline)
It is preferable to administer an appropriate amount of a suspension obtained by diluting or suspending in an appropriate amount with saline or physiological saline or the like, if desired, in combination with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, to a mammal several times every 4 to 21 days. In addition, a suitable carrier can be used during immunization with the sensitizing antigen.

After immunizing as described above and confirming that the desired antibody level in serum is increased, immune cells are taken out from the mammal and subjected to cell fusion. Preferred immune cells are particularly spleen cells. Cells. Mammalian myeloma cells as the other parent cell fused with said immune cells are already known various cell lines such as P3.
(P3x63Ag8.653) (J. Immunol. 1
23: 1548, 1978), p3-U1 (Curre).
nt Topics in Micro-biolog
y and Immunology 81: 1-7,1
978), NS-1 (Eur. J. Immunol.
6: 511-519, 1976), MPC-11 (Ce
11, 8: 405-415, 1976): SP2 / 0.
(Nature, 276: 269-270, 197.
8), FO (J. Immunol. Meth. 35: 1.
-21, 1980), S194 (J. Exp. Med.
148: 313-323, 1978), R210 (Na
True, 277: 131-133, 1979) and the like are preferably used.

The cell fusion between the immune cell and the myeloma cell is basically a known method, for example, the method of Milstein et al. (Milstein et al., Methods Enz.
ymol. 73: 3-46, 1981) and the like. More specifically, the cell fusion is carried out, for example, in the usual nutrient culture in the presence of a cell fusion promoter. As the fusion accelerator, for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used, and if desired, an auxiliary agent such as dimethyl sulfoxide can be added and used to enhance the fusion efficiency.

The ratio of immune cells to myeloma cells used is, for example, 1 to 1 of immune cells to myeloma cells.
It is preferably 0 times. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for the growth of the myeloma cell line, MEM culture medium, and other common culture medium used for this type of cell culture can be used. Alternatively, serum supplement such as fetal calf serum (FCS) can be used together.

In the cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture medium, and
PEG solution heated to about ℃, for example, average molecular weight 10
PEG of about 00-6000 Normally, 30-60 in the culture solution
The desired fused cells (hybridomas) are formed by adding at a concentration of% (w / v) and mixing. Then, by sequentially adding an appropriate culture medium and centrifuging to remove the supernatant, a cell fusion agent or the like which is unfavorable for the growth of the hybridoma can be removed.

The hybridoma is selected by culturing in an ordinary selective culture medium, for example, HAT culture medium (culture medium containing hypoxanthine, aminobuterin and thymidine). Culturing in the HAT medium is continued for a sufficient period of time to kill cells (non-fused cells) other than the target hybridoma, usually several days to several weeks. Then, the usual limiting dilution method is carried out to screen and hybridize the hybridomas producing the desired antibody.

The hybridoma producing the monoclonal antibody thus produced can be subcultured in an ordinary culture medium and can be stored in liquid nitrogen for a long period of time. To obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a usual method and obtained as a culture supernatant, or the hybridoma is transplanted into a mammal compatible therewith and proliferated. The method of obtaining as. The former method is suitable for obtaining high-purity antibody, while the latter method is suitable for mass production of antibody.

Further, the monoclonal antibody obtained by the above method can be purified to a high degree of purity by using a conventional purification means such as a salting out method, a gel filtration method and an affinity chromatography method. In this way, the monoclonal antibody thus prepared is subjected to radioimmunoassay (RI
A), enzyme-linked immunosorbent assay (EIA, ELISA), fluorescent antibody method (Immunofluorescence Ana)
It can be confirmed that the antigen is recognized with high sensitivity and high accuracy by a usual immunological means such as lysis).

The monoclonal antibody used in the present invention is not limited to the monoclonal antibody produced by a hybridoma, and may be artificially modified for the purpose of reducing heterologous antigenicity to humans. For example, a mammal other than human, for example, a chimeric antibody consisting of a variable region of a mouse monoclonal antibody and a constant region of a human antibody can be used, and such a chimeric antibody is a known method for producing a chimeric antibody, In particular, it can be produced using a genetic recombination technique.

Furthermore, reshaped human antibodies can be used in the present invention. This is a mammal other than human, for example, the complementarity determining region of a human antibody is replaced by the complementarity determining region of a mouse antibody,
The general gene recombination method is also known. The reconstituted human antibody useful in the present invention can be obtained using the known method.

If necessary, amino acids in the framework (FR) region of the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen-binding site (Sato). Et al., Cancer Res.
53: 1-6, 1993). A humanized PM-1 (hPM-1) antibody is preferably exemplified as such a reshaped human antibody (International Patent Application Publication No. WO92-1975).
9).

Further, as long as it binds to an antigen and inhibits the activity of IL-6, a fragment of an antibody such as Fab or F
A gene encoding a single chain Fv (scFv) in which Fvs of v, H chain and L chain are ligated with an appropriate linker is constructed, and the gene is expressed in an appropriate host cell and can be used for the above-mentioned purpose. . (For example, Bird et al., TI
BTECH, 9: 132-137, 1991; Hust
on et al., Proc. Natl. Acad. Sci. US
A, 85, 5879-5883, 1988).

The immature myeloma cell therapeutic agent containing the IL-6 receptor antibody of the present invention as an active ingredient blocks IL-6 signal transduction of immature myeloma cells, and the survival is maintained by IL-6. As long as the survival of immature myeloma cells is inhibited, it is effective in eradicating those immature myeloma cells. The immature myeloma cell therapeutic agent of the present invention is preferably parenterally, for example, intravenous injection, intramuscular injection, intraperitoneal injection,
It can be administered systemically or locally by subcutaneous injection. Furthermore, it can take the form of a pharmaceutical composition or kit together with at least one pharmaceutical carrier or diluent.

The dose of the therapeutic agent for immature myeloma cells of the present invention to human varies depending on the disease state, age or administration method of the patient, but it is necessary to select an appropriate amount as appropriate. For example, up to 4 divided doses can be selected in the range of approximately 1-1000 mg / patient. However, the therapeutic agent for immature myeloma cells of the present invention is not limited to these doses.

The therapeutic agent for immature myeloma cells of the present invention can be formulated according to a conventional method. For example, an injectable formulation may include purified IL-6R antibody in a solvent, such as
It is dissolved in physiological saline, buffer, etc., and added with an anti-adsorption agent such as Tween 80, gelatin, human serum albumin (HSA), or
It may be lyophilized for reconstitution before use. As the excipient for lyophilization, sugar alcohols and sugars such as mannitol and glucose can be used.

[0030]

EXAMPLES The present invention will be specifically described below with reference to Reference Examples, Experimental Examples and Examples, but the present invention is not limited to these. Reference Example 1 Preparation of human IL-6 receptor antibody PM-1
Manufactured by Hirata et al. (J. Immunol., 14
3: 2900-2906, 1989), the sepharose 4B (Pharmacia Fine C) obtained by activating the anti-IL-6R antibody MT18 by CNBr.
Chemicals, Piscataway, NJ) and IL-6R (Yam
asaki et al., Science 241: 825-82.
8, 1988).

That is, human myeloma cell line U266
1% digitonin (Wako Chemicals
Solubilized with 1 mM p-paraaminophenylmethanesulfonyl fluoride fluoride (manufactured by Wako Chemicals) containing 10 mM triethanolamine (pH 7.8) and 0.15 M NaCl (manufactured by Digitonin buffer) and bound to Sepharose 4B beads. Mixed with MT18 antibody. Then, the beads were washed 6 times with digitonin buffer, and partially purified IL-6R used for immunization.
And

3 × 10 ⁹ U2 of BALB / c mice
The above partially purified IL-6R obtained from 66 cells was immunized four times every 10 days, and then a hybridoma was prepared by a conventional method. The hybridoma culture supernatant from the growth positive wells was examined for IL-6R binding activity by the following method. 5x
10 ⁷ U266 cells were treated with 35S-methionine (2.5
It was labeled with mCi) and solubilized with the digitonin buffer.

Solubilized U266 cells were mixed with 0.04 ml volume of Sepharose 4B beads coupled to MT18 antibody, followed by 6 washes with digitonin buffer and 0.
³⁵ S-methionine-labeled IL-6R was flushed out with 25 ml of digitonin buffer (pH 3.4), and 0.025 ml of 1
It was neutralized with MTris (pH 7.4). 0.05 ml of hybridoma culture supernatant was mixed with 0.01 ml of Protein G Sepharose (Pharmacia).

After washing, Sepharose was incubated with 0.005 ml of the ³⁵ S-labeled IL-6R solution prepared above. The immunoprecipitated material was analyzed by SDS-PAGE to examine the hybridoma culture supernatant that reacted with IL-6R. As a result, the reaction-positive hybridoma clone P
M-1 was established. The IL-6R antibody PM-1 produced from the hybridoma PM-1 has an IgG1κ type subtype.

The IL-6 binding inhibitory activity of the antibody produced by Hybidomas PM-1 to human IL-6R was examined using human myeloma cell line U266. Human recombinant IL-6 was prepared from E. coli (Hirano et al., Immm
unol. Lett. , 17:41, 1988), Bolton-Hunter reagent (New England Nuc).
Lear, Boston. ¹²⁵ I by MA) (Taga et al., J. Exp. Med. 166: 967,
1987).

4 × 10 ⁵ U266 cells were cultured in the presence of 100-fold excess of unlabeled IL-6 at room temperature for 1 hour on 70% (v / v) hybridoma PM-1. Clear and incubated with 14000 CPM of ¹²⁵ I-labeled IL-6. 70 μl sample in 400 μl microfuge polyethylene tube 300 μl F
Radioactivity on cells was measured after overlaying on CS and centrifugation. As a result, it was revealed that the antibody produced by hybridoma PM-1 inhibits the binding of IL-6 to IL-6R.

Reference Example 2. Preparation of Humanized Antibody hPM-1 The humanized antibody hPM-1 was prepared as International Patent Application Publication No. WO9.
It was obtained by the method described in 2-19759. Total RNA was prepared from the hybridoma PM-1 prepared in Reference Example 1 by a conventional method, and single-stranded cDNA was synthesized therefrom. The DNA in the V region of mouse PM-1 was amplified by the polymerase chain reaction (PCR) method. The primers used in the PCR method are S. T. Jones et al., Bio / Techno
The one described in LOGY, 9, 88, 1991 was used.

The DNA fragment amplified by the PCR method was purified to obtain a DNA fragment containing a gene encoding a mouse kappa type L chain V region and a DNA fragment containing a gene encoding a mouse gamma type H chain variable region. These DNA
The fragment was ligated into plasmid pUC19 and transformed into E. coli DH5α
E. coli transformants were obtained by introducing into Escherichia coli. The above plasmid was obtained from this transformant, the base sequence of the cDNA coding region in the plasmid was determined according to a conventional method, and the complementarity determining region (CDR) of each V region was determined.
It was determined.

In order to prepare a vector expressing the chimeric PM-1 antibody, cDNAs encoding the V regions of mouse PM-1 κ L chain and H chain were inserted into the HCMV expression vector. In order to prepare humanized human PM-1 antibody, V region CDR of mouse PM-1 by CDR grafting method
Were transplanted into human antibody. Amino acids in the variable region framework (FR) region of the antibody were replaced so that the CDRs of the humanized antibody formed appropriate antigen-binding sites.

Humanized PM-1 thus prepared
In order to express the antibody light chain and heavy chain genes in mammalian cells, human elongation factor Iα (H
Each was introduced into a vector containing the EF-1α) promoter to prepare a vector expressing the humanized PM-1 antibody L chain and H chain. CH these two expression vectors
Humanized PM-1 by simultaneous insertion into O cells
A cell line producing (hPM-1) was established. The binding ability of the obtained humanized antibody to human IL-6R was determined by ELISA.
Confirmed in. Furthermore, hPM-1 inhibited the binding of human IL-6 to human IL-6R, similar to mouse and chimeric antibodies.

Experimental Example (1) Classification of myeloma cells Myeloma cells obtained from myeloma patients were identified and classified based on the surface antigens by flow cytometry. Bone marrow fluid, lymphocyte separation fluid Separa
te-L (Muto Pure Chemicals
Co. ) Was centrifuged to separate mononuclear cells.

These bone marrow cells were treated with 200 μg / ml BS.
5 × 10 in PBS containing A and 0.01% NaN ₃
^5/20 [mu] l and then suspended at each 20 Introduction
μl of anti-CD19 monoclonal antibody (mAB) (Im
anti-CD56mAB (Coult)
er), anti-VLA-5mAB (Immunotech
Company) or anti-MPC-1 mAB (JP-A-6-8668)
8) was added and reacted at 4 ° C. for 30 minutes, and then 20
mM Sodium Phosphate and 0.25
The cells were washed twice with PBS containing M NaCl (pH 7.2).

Then, 40 μl of PE diluted 100 times
(Phycoerythrin) conjugated goat anti-mouse Ig
Staining with G (manufactured by Immunotech) (4 ° C., 30
After washing twice with PBS, 15 μl of mouse serum (Chemicon) was added, incubated at 4 ° C. for 20 minutes, and further 5 μl of FITC (fluorescein isot) in 20 μl of PBS.
hiocianate) conjugated anti-CD38 mAB (Imm
(manufactured by unotech) was added and stained (4 ° C., 30 minutes), and washed twice with the PBS.

These double-stained bone marrow cells were analyzed by a flow citometer (EPICS ELITE, Coult).
er) and analyzed by measuring fluorescence. As a result of analyzing the surface antigens of cells appearing in the CD38 strongly positive fraction, which is a characteristic of myeloma cells, the myeloma cells were found to be VLA-
5 ⁺ MPC-1 ⁺ mature form, VLA-5 ⁻ MPC-1 ⁺
Intermediate type and ^{VLA-5 - MPC-1} - divided into immature type (Kawano et al., Blood, 82,56
4, 1993).

(2) Induction of apoptosis of myeloma cells The determination of the survival rate of myeloma cells and the apoptosis of dead cells was carried out by FDA (fluorescein diaceta).
te, Aldrich Chem. Co. Made) and P
I (propridium iodide, Sigma)
Type staining flow cytometry method (Can)
cer Res. , 49, 3776, 1989), and the distribution of apoptosis-induced cells is shown mechanically in FIG.

Human myeloma cell line KMS-5 (Ohtsu
ki et al., Acto. Haematol. Jpn. , 5
1,1052,1988) was prepared in a culture medium containing 10% FCS-supplemented RPMI at a concentration of (1 × 10 ⁶ ) cells / ml to induce apoptosis.
One (manufactured by Sigma) was added so as to have a ^{concentration} of 1 × 10 ⁻⁷ M. After culturing at 37 ° C. for 24 hours, KMS-5 cells were subjected to F
Double staining was performed with DA / PI, and fluorescence was measured with a flow cytometer. As a result, as shown in FIG.
Live cell fraction (FDA ⁺ P
I ^-) was added, the fraction of apoptosis was induced cell (FDA ^- PI ^-) appeared.

[0047] FDA ⁺ PI ^- fractions and FDA ^- PI ^-
Fractions of cells were collected by flow cytometry, and Br.
J. Haematol. , 71, 343, 1988, and DNA was extracted. The DNA thus prepared was analyzed by 1.2% agarose gel electrophoresis. As a result, as shown in FIG. 3, the cell-derived DNA of the FDA ⁺ PI ⁻ fraction was not decomposed, but FDA ⁻ P ⁻
Cells of the I ^- fraction showed a clear degradation of DNA and ladder-like bands characteristic of apoptosis.

[0048] was studied the change of survival in an in vitro patient myeloma cells by FDA / PI double staining flow cytometry as described in Example Experimental Example. The PE-conjugated anti-VLA-5 antibody and the PE-conjugated anti-MPC were prepared in the same manner as in the above Examples.
Myeloma cells were stained with the -1 antibody, and the following cell groups were collected. Fractionated VLA-5 ⁺ MPC-1 ⁺ mature form, VLA-5 ⁻ MPC-1 ⁺ intermediate form and VLA-
5 ^- MPC-1 ^- 1 of immature myeloma cells × 10 ⁶ cells / ml and made as 10% FCS and 1 × 10 ^-5 M 2
-Suspended in RPMI culture medium containing mercaptoethanol and 35 × 10 mm tissue culture
Dispensed in ish (Falcon).

These cells were treated with 20 U / ml of recombinant human IL-6 (rIL-6; Hirano et al., Nature.
e, 324, 73, 1986), 50 μg / ml of humanized anti-hIL-6 receptor antibody (hPM-1) was incubated in the presence or absence at 37 ° C. for 3 days, and then FDA /
Flow cytometer (EPICS EL
Fluorescence was measured by ITE (Coulter) to determine the ratio of viable cells. The control is rIL-6.
And cultured in the absence of hPM-1. The results are shown in Table 1.
Shown in.

[0050]

[Table 1]

From these results, the effect of rIL-6 on the survival rate of mature, intermediate and immature myeloma cells is shown in FIG. ^{VLA-5 - MPC-1} - the action of hPM-1 antibody rIL-6 reactive immature myeloma cells thereto having a surface antigen are shown in FIG. 5. V
LA-5 ⁺ MPC-1 ⁺ mature myeloma cells (Table 1,
The patient-derived myeloma cells 1-9) maintained a relatively high survival rate even with the culture medium alone. These mature myeloma cells have rIL-
It hardly reacted with 6 and the survival inhibitory effect by the hPM-1 antibody was not observed. On the other hand, VLA-5 ^- MPC-1
^- immature myeloma cells (in Table 1, patient-derived myeloma cells 1
It was shown that 3-18) could not maintain the survival only with the culture medium and was prone to apoptosis.

These cells were highly reactive to IL-6, and the survival rate was increased by rIL-6. At this time, r
The effect of maintaining the survival of immature myeloma cells by IL-6 is h
It was clearly inhibited by PM-1 antibody (Fig. 5). Immature myeloma cells in the presence of control, rIL-6, hPM-1 and rIL-6 and hPM-1 (Table 1
The FDA / PI double-stained image of the patient-derived myeloma cell 17) is shown in FIG.

In the rIL-6-added group, the intensity of the image inducing apoptosis (lower left, D) was lower and the ratio of living cells (lower right, E) was higher than that in the control. On the other hand, in the presence of rIL-6 and hPM-1, the intensity of the cell fraction that induced apoptosis (lower left, D) was high. Intermediate-type myeloma cells displaying VLA-5 ^- MPC-1 ⁺ also showed a similar increase in survival in response to rIL-6 and this effect was hPM-1
It was shown to be inhibited by

[0054]

[Effect of the Invention] forming a main body of indicating treatment resistant often myeloma cell populations, surface antigens ^{VLA-5 -} MPC-1 -
IL-6 is deeply involved in the survival and maintenance of immature myeloma cells. Since the inhibitory effect on the survival of immature myeloma cells by the IL-6 receptor antibody was strongly recognized in VLA-5 ^- MPC-1 ^- immature myeloma cells, the IL of the present invention
It was suggested that the -6 receptor antibody is useful as a therapeutic agent for immature myeloma cells.

[Brief description of drawings]

FIG. 1 schematically shows the distribution of apoptosis-induced myeloma cells by a double-staining flow cytometry method. E is an image of living cells (FDA ⁺ PI ⁻ ), D is apoptosis-inducing cells (FDA ⁻ PI ⁻ ), and C is an image of dead cells having no apoptotic characteristic.

FIG. 2 is a flow cytometric image of myeloma cell line KMS-5 in which apoptosis was induced by dexamethasone treatment.

FIG. 3 shows FDA ⁺ PI ⁻ (E) and FDA ^−.
FIG. 7 is a DNA agarose gel electropherogram of cells of the PI ⁻ (D) fraction. Lane 1 is molecular weight marker, lane 2 is F
DA ⁺ PI ^- (E) fraction, lane 3 is FDA ^- PI
^- (D) Fraction. Lane 3 shows a ladder-like band characteristic of DNA degradation and apoptosis.

FIG. 4 shows the effect of IL-6 on the viability of myeloma cells. In the presence of IL-6, heat-sensitive myeloma cells (VLA-5 ⁺ MPC-1 ⁺ ) are almost unaffected by their presence (about 1.1 times). On the other hand, intermediate type (VLA-
5 ^- MPC-1 ⁺ ) and immature myeloma cells (VLA-
5 ^- MPC-1 ^-) are each about 2.4 times, the increase in the number of 3.5 times of viable cells is observed.

[Fig. 5] Fig. 5 shows the reactivity of VLA-5 ^- MPC-1 ^-- unheated myeloma cells with respect to IL-6 and the h response thereto.
3 shows the action of PM-1 antibody. ◯ is the patient-derived myeloma cell 13 in Table 1, □ is the same as 14, Δ is the same as 15, ● is the same as 16, and ×
Indicates the same 17 and ▼ indicates the same 18. The survival of these immature myeloma cells is supported by IL-6, with hPM-1 antibody clearly inhibiting its survival supporting effect.

Figure ^{6, VLA-5 - MPC-1} - Control of immature myeloma cells (in Table 1, patient-derived myeloma cells 17), rIL-6, hPM -1 and rIL-6 and h
The FDA / PI double-stained image in each presence of PM-1 is shown.

Claims (9)

[Claims] 1. A therapeutic agent for immature myeloma cells, which comprises an interleukin-6 receptor antibody as an active ingredient. 2. The therapeutic agent for immature myeloma cells according to claim 1, wherein the immature myeloma cells are resistant to chemotherapeutic agents. 3. The immature myeloma cell is VLA-5 ^−.
MPC-1 ^- immature myeloma cell therapy according to claim 1, characterized in that it comprises a surface antigen. 4. The therapeutic agent for immature myeloma cells according to claim 1, wherein the interleukin-6 receptor is a human interleukin-6 receptor. 5. The therapeutic agent for immature myeloma cells according to claim 1, wherein the interleukin-6 receptor antibody is a monoclonal antibody. 6. The therapeutic agent for immature myeloma cells according to claim 1, wherein the interleukin-6 receptor antibody is a humanized antibody. 7. The therapeutic agent for immature myeloma cells according to claim 1, wherein the interleukin-6 receptor antibody is PM-1 antibody. 8. The therapeutic agent for immature myeloma cells according to claim 1, wherein the interleukin-6 receptor antibody is a humanized PM-1 antibody. 9. An immature myeloma cell survival inhibitor comprising an interleukin-6 receptor antibody as an active ingredient.