JPH05213775A - Bfa antibody - Google Patents

Abstract

PURPOSE:To obtain a BFA antibody effective for treating cancers, especially malignant tumors such as human mammary cancer. CONSTITUTION:A complex antibody wherein one antibody constituting the complex antibody is human c-erbB-2 specific antibody and the other is human lymphocyte antibody. Especially the complex antibody wherein an antibody recognizing surface antigen of target cell is anti-human c-erbB-2 antibody and an antibody which is bonded to an effector cell and transmits a signal is an anti-human lymphocyte antibody. A therapeutic agent for cancer comprising the complex antibody as an essential component.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a composite antibody effective for treating cancer, and more particularly to a composite antibody effective for treating malignant tumors such as human breast cancer and a therapeutic agent for cancer.

[0002]

2. Description of the Related Art Conventionally, in cancer immunotherapy, a monoclonal antibody has an antibody-dependent cellular cytotoxicity (ADCC;
tibody-dependent celluar cytotoxicity) [Martin, J.
S., et al., Blood, 73 , 1431-1439 (1989)] or complement-dependent cytolysis.
[Irie, RF, et al., Proc.Natl.Acad.USA, 83 , 8649
-8698 (1986)] has been widely clinically tested by passive immunization. However, the monoclonal antibody that induces the efficacious cytotoxic immune response required for cancer treatment was exceptional. Their clinical results have shown the limit of research on cancer treatment with monoclonal antibodies.

As a promising method for overcoming the problems caused by the above-mentioned monoclonal antibody, an attempt was made to dissociate two different kinds of antibody molecules and re-bond them to obtain a hybrid antibody, which was a rabbit polyclonal antibody in the old days. [Nisonoff, A. and MMRivers, A
rch.biochem.Biophys., 93 , 460 (1961)], but it was not widely known until the technology for producing monoclonal antibodies was developed. [Koehler, G. and C. Milstein,
Nature, 256, 495 (1975)].

Many methods have been reported for producing a hybrid antibody (monomer type) by chemically binding each half of these two types of monoclonal antibodies.
In general, they are produced using F (ab ') ₂ molecules as a material. For example, Brennan et al. Reduced the F (ab ') ₂ molecule with a reducing agent of DTT (dithiothreitol) to reduce the SH group of one Fab' to 5,5'-.
Fab'-SNB was protected with dithiobis (2-nitrobenzoic acid) (DTNB) and mixed with the other Fab'-SH to successfully prepare a monomer-type hybrid antibody in high yield. [Brennan, M.,
PFDavison and H. Pavlus, Science, 229, 81-83 (198
5); Nitta, T., Et al., Eur.J.Immunol., 19 , 1437-14
41 (1989)].

Another method is to use two different antibody molecules as a cross-linking agent SPDP [N-succinimidyl-3].
-(1-Vyridyldithio) propionate] to produce a dimer-type hybrid antibody
[Staerz, UD, et al., Nature, 314, 628-631 (1985)]
Alternatively, a method for producing a dimer type hybrid antibody in which two types of antibody F (ab ′) ₂ fragments are similarly bound using a crosslinking agent SPDP [Nitta, T., et al.,
Eur.J.Immunol., 19 , 1437-1441 (1989)].

[0006] As another means for producing a hybrid antibody, there is a method utilizing a cell fusion method. For example, a method in which an antibody-producing hybridoma for one antigen is fused with splenocytes of an animal immunized with another antigen to obtain a hybrid antibody-producing hybridoma, and the hybrid antibody of interest is obtained by culturing the hybridoma [Mil
stein, C. and ACCuello, Nature, 305 , 537 (1983)]
Alternatively, a method of obtaining hybrid antibody-producing hybridomas by fusing different antibody-producing hybridomas with each other and obtaining a desired antibody therefrom [Staerz, UD and MJBeva
n, Proc.Natl.Acad.Sci, USA, 83, 1453 (1986); L
anzavecchia, A. and D.Scheidegger, Eur.J.Immunol., 1
7 , 105 (1987)] etc.

Further, as another type of hybrid antibody, a variable region (V) region having an antigen-binding activity is derived from a mouse hybridoma, and a constant region (C) region having an immunological activity is derived from a human. Are known. The method for producing the antibody has been attempted using gene recombination technology.
In addition to Morrison of 984, a method for producing this kind of hybrid antibody having various specificities is exemplified [Morrison SL et al., Proc. Natl. Acad. Sci. USA, 8
1 , 6851 (1984): Sharon J. et al., Nature, 309, 364
(1984): Neuberger, MS, et al., Nature, 312 , 604
(1984): Boulianne, GL, et al., Nature, 312 , 634.
(1984) etc.]. Further, as a new type of hybrid antibody, a mouse derived only from the complementarity determining region (CDR) of V region (Reshaped antibody) can be exemplified [Jone
s, PT, et al., Nature, 321 , 552 (1986): Riechman
n L., et al., Nature, 332 , 323 (1988)].

The above-mentioned hybrid antibody is applied to immunoassay systems in addition to cancer treatment described below [Suresh, M.
R., et al., Proc.Natl.Acad.Sci. USA, 83 , 7989-7
993 (1986)], use in immunohistochemistry [Milsten, C. and
ACCuello, Nature, 305, 537 (1983)] has been reported. In addition, a hybrid antibody [complex antibody (hereinafter, referred to as BFA; Bifunctional an
BFA in which one antibody (abbreviated as tibody) recognizes an antigen associated with cancer cells and the other antibody recognizes the T cell antigen of T lymphocytes. The B
Based on the structure of FA, BFA capable of binding to a target cancer cell can have cytotoxic activity against the target cancer cell. This mechanism has led to expectations for cancer treatment. Subsequently, the activity of BFA was studied in vitro in some tumor cells [Mezzanica, D., et al., I.
nt.J.Cancer, 41 , 609-615 (1988); Mansfield, PF, e
t al., Cancer Immunol. Immunon, 33 , 247-254 (199
1); Oshimi, K., et al., Blood, 77 , 1044-1049 (1991)
Nitta, T., et al., EJImmunol., 19 , 1437-1441 (1
989)].

[0009] Furthermore, B can be used for glioma, ovarian cancer, and lung cancer.
Clinical study of FA undertaken [Nitta, T., et al., Lance
t, 335 , 368-371 (1990); de Leij, L., et al., Fonda
tionNationale de Transfusion Sanguine, Les Ulis Fr
ance, 249-253 (1990)].

On the other hand, in recent years, due to remarkable progress in cell biology, attention has been paid to the relationship between growth factor receptors and carcinogenesis in cells. Among the numerous oncogene products, among the receptor tyrosine kinases (tyrosine residue-specific protein kinases), epidermal growth factor (epide
rmal growth factor (EGF) and a c-erbB-2 gene found as a related oncogene encoding a protein that closely resembles the receptor of EGF [Yamamoto,
T., et al., Nature, 319, 230-234 (1986)], the oncogene product p185 gene, has been observed to be amplified and highly expressed oncogene product in various human malignancies. , Especially in breast cancer, gastric cancer, lung cancer, pancreatic cancer
-Amplification of the erbB-2 gene has been observed. Regarding the adenocarcinoma, especially breast cancer, amplification of the c-erbB-2 gene was observed in human breast cancer and cultured cells derived from breast cancer [King, C.
R., et al., Science, 229 , 974-976 (1985); Yamamot
o, T., et al., Nature, 319, 230-234 (1986)], c-
It has already been found that the degree of amplification of the erbB-2 gene has a strong correlation with the prognosis of breast cancer [Slamon, DJ, et.
al., Science, 235 , 177-182 (1987)]. However, expression of the gene product is rarely observed in normal adult tissues [Natali, PG, et al., Int. J. Cancer.
r, 45 , 457-461 (1990)].

Antibodies against the c-erbB-2 protein and monoclonal antibodies have already been developed, and not only pathological materials but also methods for immediately examining surgical materials by immunostaining are being developed [Masuko, T., et. al., Jpn.J.Canc
er Res., 80 , 10-14 (1989)]; Yamada, Y., et al., Jpn.
J. Cancer Res., 80 , 1192-1198 (1989)], c-erb
As an antibody that recognizes the B-2 gene product, for example, c-
A polyclonal antibody that recognizes the C-terminal region of the erbB-2 gene, pAb1 (T4881) [Triton Bioscience Inc. (Alameda, CA)], or a polyclonal antibody Ab- that recognizes the kinase domain.
1 [Oncogene Science Inc.; Ma
nhasset, NY)], or a monoclonal antibody that recognizes the extracellular domain of c-erbB-2, SV2-61γ [Nichiren Co., Ltd. (see Japanese Patent Laid-Open No. 2-150293)].
And the like, and the present inventors have previously provided a human breast cancer cell line SK-BR for the purpose of providing a diagnostic agent and therapeutic agent for adenocarcinoma, particularly breast cancer.
-3 (ATCC deposit number; ATCC HTB30) produced by a hybridoma formed by fusion of immune cells of a mammal immunized with the culture supernatant of ATCC HTB30 and bone marrow cells of the mammal, c
-An anti-ce specifically reacting with erbB-2-related protein
rbB-2 monoclonal antibody, GFD-OA-p18
5-1 was prepared [Ouzge, Alper, et al., Cell Growth
& Differentation, 1, 591-599 (1990)]. GFD-
The OA-p185-1 antibody is a human cancer cell line SK-BR-3 expressing the c-erbB-2 gene and A-549.
In vitro proliferation of (human lung cancer cell line; ATCC CCL185) was significantly suppressed (Japanese Patent Application No. 3-229835). Each of the above antibodies reacts with the c-erbB-2 protein, and is useful for diagnosing diseases associated with the expression of the c-erbB-2 gene product, particularly adenocarcinoma, especially breast cancer (Japanese Patent Laid-Open No. 3-191865). As described above, these antibodies are monoclonal antibodies as therapeutic agents for the above-mentioned malignant tumors, and a good therapeutic effect is not necessarily expected. We have BFA for more common epithelial cancers
C-erbB-2 for developing an immunotherapy method by
Develop BFA targeting those related to the gene product and evaluate the anti-tumor activity of BFA in vitro,
The present invention has been completed here.

It should be noted that there has been no report on BFA related to c-erbB-2, and the present invention provides a composite antibody in which one antibody constituting BFA is an anti-human c-erbB-2 antibody. It became possible for the first time.

[0013]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention
Cells expressing the B-2 gene, especially breast cancer, lung cancer, gastric cancer,
Target cells such as malignant tumor cells such as pancreatic cancer and cytotoxic T
A new BFA (complex antibody) capable of specifically binding to effector cells such as lymphocyte cells (Cytotoxic TLymphocytes), and further enhancing the cytotoxic activity of the effector cells against the target cells, and The purpose of the present invention is to provide a clinical therapeutic agent for malignant tumors and the like.

[0014]

According to the present invention, a BFA
One antibody constituting (complex antibody) is human c-erbB
-2 antibody, and the other is a human lymphocyte antibody, BFA, in particular, the antibody that recognizes the surface antigen of the target cell is an anti-human c-erbB-2 antibody, which binds to effector cells. The above BFA and anti-human c-erbB- in which the antibody that carries out signal transduction is an anti-human lymphocyte antibody.
2 The above BF in which the antibody is GDF-OA-p185-1
A, and the above BFA that specifically reacts with the human c-erbB-2 related protein, wherein the anti-human lymphocyte antibody is an anti-CD3 antibody. Further, according to the present invention, there is provided a cancer therapeutic agent comprising the above BFA as an essential component.

The antibody of the present invention has a strong specific binding ability to antigens (target cells and effector cells), and its use can activate or enhance the cytotoxic activity of effector cells such as CTL against target cells. However, it does not activate the effector cells to attack normal cells, and further avoids the possibility of adverse effects due to ADCC or the like via Fc receptors. Therefore, the antibody of the present invention is particularly effective for clinical treatment of malignant tumors and the like.

Hereinafter, the antibodies of the present invention, particularly one of the antibodies is the Fab 'portion of the anti-human c-erbB-2 antibody, and the other antibody is the Fab' portion of the anti-human lymphocyte antibody. The method for producing the antibody of the present invention will be described in detail.

The antibody used as a material for producing the antibody of the present invention, that is, a monoclonal antibody is, for example, anti-ce
Antibodies such as rbB-2 antibody or antitumor antibody, and anti-CD3
Anti-human lymphocyte antibodies such as antibodies can be used. Anti-c-erbB-2 belonging to the above anti-c-erbB-2 antibody
The antibody recognizes the c-erbB-2 gene product expressed on the surface of cancer cells, which includes GFD-OA-
p185-1 [Alper, O., et al., Cell Growth & Differ
entiation, 1, 591-599 (1990)], a recombinant DNA obtained by incorporating the human c-erbB-2 gene into an appropriate expression vector using a gene recombination technique is introduced into an appropriate animal cell, and Transforming animal cells into human c-erbB
SV2-61 in which cells expressing the C-2 gene were selected, and the cells expressing on the cell surface were prepared as an immunizing antigen for preparing a c-erbB-2 monoclonal antibody
Antibody, SV2-61γ antibody (JP-A-2-150293), TAb251 antibody, TAb255 antibody, TAb
256 antibody, TAb258 antibody, TAb259 antibody (P
CT published patent W091-202062), 9G6 antibody [Van de Vijver MJ, et al., New Eng. J. Med., 31 .
9 , 1239 (1988)] etc. are included.

Further, anti-C belonging to the above-mentioned anti-human lymphocyte antibody
Specific examples of the D3 antibody include UCHT1 antibody [anti-CD3
ε antibody: mouse IgG ₁ : Beverley, PCL and Callar
d, RE, Eur.J.Immunol., 11 , 329-334 (1981): Imperial Cancer Research Foundation (Imper
ial Cancer Reseach Foundation, UK)], OKT3 antibody [anti-CD3 antibody: mouse IgG _2a : Kung, PC, et al.,
Science, 206 , 347-349 (1979)] and the like. The U
CHT1 antibody is human C expressed on the cell surface of T lymphocytes.
It is known to be an antibody that recognizes the D3ε antigen.

For each antibody, a monoclonal antibody-producing hybridoma is newly prepared, or a known monoclonal antibody-producing hybridoma is used, and a known method [eg, Koehler, G. and C. Milstein, Nature, 2
56 , 495 (1975), etc.].
As a general method, first, a hybridoma producing a monoclonal antibody is first treated with an appropriate culture medium, for example, 10% FC.
Culture is performed using RPMI-1640 medium (Nissui Pharmaceutical Co., Ltd.) containing S (fetal bovine serum). This cultured hybridoma was suspended in PBS (phosphate buffered saline) or RPMI-1640 medium containing no FCS, and the suspension was preliminarily injected with pristane (2,6,10,14-tetramethylpentadecane) intraperitoneally. It is intraperitoneally administered to the administered BALB / c nude mouse. Approximately 7 to 14 days after the above administration, when the abdomen of the mouse swells, ascites is drained from the abdominal cavity of the mouse, and then the ascites is centrifuged to remove cells to obtain ascites. From this ascites, a general method such as Protein A [Forsgren, A. and J. Sjoequist, J. Immunol., 97 , 82
2 (1966)], the target monoclonal antibody can be collected and purified by performing affinity chromatography or the like. More specifically, the above purification is carried out by adding 2 to 4 ml of binding buffer (1.5 M to 1 ml of ascites fluid).
Glycine, 3M NaCl, pH 8.9) was added to the mixture, for example, Protein A Cephaloin (manufactured by Chisso Corporation) column, and the antibody was adsorbed to the column, followed by 0.1M sodium citrate buffer ( pH 3.0 ~
It can be carried out by eluting with 5.0).

The preparation of F (ab ') ₂ from the monoclonal antibody obtained as described above can be carried out by a method already reported [eg Parham, P., J. Immunol., 131 , 2895-2902].
(1983)], followed by digestion with pepsin. The following method can be illustrated as a specific example of this preparation. That is, 5-10 mg
0.1M sodium citrate solution (pH)
4.1 to 4.5), 1/10 to 1/100 times the weight of the antibody by weight of pepsin (manufactured by Sigma) is added and mixed, and the digestion reaction is performed in a 37 ° C. thermostat for 1 to 8 hours. 1M in the reaction solution
The reaction was stopped by adding Tris solution to pH 8.0 and adding 20 mM Tris hydrochloric acid, 2 mM EDTA and 150 mM.
Solution consisting of mM NaCl (pH 8.0:
"S buffer") equilibrated with TSK-SW-G30
Gel filtration is performed using a 00 (Tosoh) column, and the first peak is pooled. Then add 2 to this reaction mixture.
Approximately 4 times the volume of binding buffer is added and applied to a Protein A column to collect a flow-through fraction, and complete antibody molecules undigested with pepsin are adsorbed to the column and removed. The flow-through fraction thus obtained is subjected to extraocular filtration using a membrane having a molecular weight of 10,000 to obtain concentrated F (ab ') ₂
Fractions are obtained. Yield varies depending on the antibody, but usually 10
About 4 to 6 mg of F (ab ′) ₂ is obtained from mg of antibody.

The F (ab ') ₂ fragment obtained as described above is used to bind the antibody of the present invention, that is, Fab' [monomer] of the antibody to the target cells and Fab '[monomer] of the antibody to effector cells. BFA
For example, the method of Nitta et al. [Nitta, T., et al., Eur.J.Im
munol., 19 , 1437-1441 (1989)].

In the above method, first, an antibody recognizing the surface antigen of the target cell, for example, F (a) of anti-c-erbB-2 antibody is used.
b ′) To a TES buffer containing ₂ fragments, an aqueous solution of DTT (manufactured by Wako Pure Chemical Industries, Ltd.) was added so that the final concentration was 1-2 mM, and the reaction was carried out at room temperature for 30 minutes at pH 7.5. An aqueous solution of DTNB (manufactured by Wako Pure Chemical Industries, Ltd.) was added so that the final concentration was 5 mM, and the reaction was further performed at room temperature for 30 minutes, and the reaction solution was equilibrated with a TES buffer solution (Sephadex G25 column, manufactured by Pharmacia). The antibody Fab'-SNB against the target cells can be obtained by carrying out gel filtration using the above method. Apart from
To a TES buffer containing an antibody that binds to effector cells and performs signal transduction, for example, an F (ab ′) ₂ fragment of anti-CD3 antibody, a DTT aqueous solution is added so that the final concentration is 1-2 mM, and the pH is 7.5. At room temperature at 30
The reaction was carried out for a minute, and the resulting reaction solution was subjected to gel filtration using a Sephadex G25 column equilibrated with TES buffer to give antibody F against effector cells.
Ab'-SH can be obtained. Fab′-obtained above
Fab'-SNB, an antibody that recognizes the target cells obtained above, was added to a TES buffer containing SH at a molar ratio of about 1: 1 and concentrated with an ultrafiltration membrane, followed by about 4 hours at room temperature. By reacting, Fab ′ of the antibody against the target cell
[Monomer] and antibody Fab ′ against effector cells
A desired BFA bound with [monomer] can be obtained.

In the above, also the antibody Fab'-SN
It is also possible to make B for antibodies to effector cells, and to make antibody Fab'-SH for antibodies to target cells, which also results in desired B
FA can be acquired.

The BFA thus obtained was mixed with TSK-Gel G.
By subjecting it to HPLC (high performance liquid chromatography) using 3000 SW (manufactured by Tosoh Corporation), the unreacted Fab′-thiol derivative (molecular weight: about 55 kd) can be separated, and BFA having a molecular weight of about 110 kd can be purified. it can.

7.5, under non-reducing conditions, in the presence of 0.1% SDS (sodium dodecyl sulfate: Sigma).
By performing a% PAGE (polyacrylamide gel electrophoresis) analysis, it can be confirmed that the BFA purified above has the expected molecular weight.

Further, it is possible to prepare a BFA in which the antibody F (ab ') _n [polymer] for the target cells and the antibody F (ab') _n [polymer] for the effector cells are bound according to the above method. The F (ab ') _n
A method for producing a BFA in which [polymer] and F (ab ′) _n [polymer] are bound is described in the method for producing a composite antibody previously filed by the present inventors (Japanese Patent Application No. Hei.
-229835). By using a plurality of arms for a target cell (using F (ab ′) _n [polymer]) according to the method, the BFA obtained has a higher binding property to the target cell, which is desirable for the present invention. It is considered that the therapeutic effect can be further enhanced.

By using the BFA of the present invention obtained as described above, the toxic activity of effector cells such as CTL against target cells can be enhanced. For example, anti-c-
By using the BFA of the present invention prepared by using the erbB-2 antibody and the anti-CD3 antibody, cancer cells expressing the c-erbB-2 gene product on the cell surface can be effectively injured or necrotic. These facts are based on the already reported test method [Staerz, UDJ, as shown in the test example below.
W. Yewdell and MJ Bevan, Eur. J. Immunol., 17 , 571-5
74 (1987); Nitta, T., Et al., Eur.J.Immunol., 19 , 1
437-1441 (1989)].

In the above, there is no restriction that lymphocytes having a histocompatibility-matching antigen type must be used as effector cells. For example, cloned CT
L, etc. may be used, or peripheral blood lymphocytes (PBL) of a healthy person may be used, and further, clonal CTL or PBL is cultured for 3 days or more in the presence of interleukin-2. LAK (lymphokin
It may be used by inducing e activated killer cells).
Preparation of peripheral blood lymphocytes of a healthy person can be performed as follows according to the specific gravity separation method. That is, 10 ml of blood of a healthy person who had heparinized blood was first added to 2-3 times the weight of PBS.
Add buffer and mix. Separately, after overlaying the whole volume of the diluted blood on a centrifuge tube to which a specific gravity separation liquid (for example, Ficoll Pack: manufactured by Pharmacia Co., Ltd.) of the diluted blood is added, 400 × g, at room temperature for 30 to 40 minutes. Density gradient centrifugation is performed, and after centrifugation, the white turbid layer formed between the plasma in the centrifuge tube and the specific gravity separation liquid is absorbed, and 10% FC
Wash with S-containing RPMI-1640 medium. Thus, 1 to 2 × 10 ⁷ peripheral blood lymphocytes can be obtained. LAK is induced by suspending the obtained PBLs in RPMI-1640 medium containing 10% FCS at 10 ⁶ cells / ml and culturing by adding 10 to 1000 units of interleukin-2 per 1 ml of the medium. be able to.

The labeling of the target cells with sodium chromate (Na ₂ ⁵¹ CrO ₄ ) containing the radioisotope ⁵¹ Cr is carried out by first pelleting the target cells (1-2 × 10 ⁶ cells).
In addition, 3.7 MBq / ml of the above sodium chromate (NE
N, manufactured by Daiichi Pure Chemicals Co., Ltd.) and added at 37 ° C, 5% C
It can be carried out by culturing in the presence of O ₂ for 60 to 90 minutes, followed by thorough washing with 10% FCS-containing RPMI-1640 medium to remove excess unreacted chromium ( ⁵¹ Cr). .. Thus, target cells containing chromium ( ⁵¹ Cr) can be obtained.

Target cells labeled with chromium ( ⁵¹ Cr),
More specifically, the cytotoxicity test using the effector cells and the hybrid antibody should be conducted at an appropriate concentration (usually 0.0
1~3μg / ml) and 100 [mu] l effector cells 10 ^6/100 [mu] l of diluted BFA were mixed, at room temperature for 3
The reaction was allowed to proceed for 0 minutes, and the reaction solution was added with RPMI containing 10% FCS
Wash with 1640 and collect the antibody-bound effector cells as a precipitate (unreacted BFA by this procedure).
Can be excluded). Next, 150 μl of each effector cell (the number of effector cells: 2 × 10 ⁵ ) to which the antibody of each concentration was bound and 50 μl of the target cell (the number of cells: 10 ⁴ ) were respectively added to a 96-well U-bottom culture plate ( Corning), at 37 ℃, 5%
The cells are cultured in the presence of CO ₂ to carry out a specific injury reaction of target cells. After the reaction, the radioactivity of each well is counted.
Degree (%) of specific damage of target cells that occurs when effector antibody and target cells are bound via BFA
Can be obtained according to the following calculation formula (1).

Specific injury% = [(a−b) / (c−d)] × 100 (1) [a is a measured value showing specific ⁵¹ Cr release of target cells by BFA-mediated effector cells, b shows the radioactivity (spontaneous release) in the well when only the target cells were cultured,
c indicates the radioactivity (maximum release) in the reaction solution when the target cells were lysed using a 1% NP-40 (manufactured by Sigma) solution. ] According to the above cytotoxicity test, the BFA of the present invention, particularly anti-c-
Fab ′ of erbB-2 antibody and Fab ′ of anti-CD3 antibody
The BFA of the present invention produced by using and was observed to have a specific target cell cytotoxicity of about 25% at a dose of 100 ng / ml, which confirmed that it has a high target cell cytotoxicity (Fig. 1 described later). -See A). On the other hand, anti-c-er
F (ab ') ₂ fragment of bB-2 antibody and anti-CD3
No significant cytotoxicity was observed even when the concentration was raised to 1000 ng / ml in the mixed solution in which the antibody F (ab ') ₂ fragment was simply mixed at a ratio of 1: 1 (Fig. 1-B described later). reference). Also, the F (ab ') ₂ fragment of each parent did not show significant cytotoxicity even when the concentration was increased to 1000 ng / ml.

Thus, the Fa of the antibody against the target cell is
b'fragment and F of antibody to effector cells
The BFA of the present invention bound to the ab ′ fragment shows a specific cytotoxic activity of target cells from a very small amount of 10 ng / ml (see FIG. 1-A described later).
-It is useful as a therapeutic agent for malignant tumors caused by cancer cells expressing the erbB-2 gene product on the cell surface, particularly as a therapeutic agent for breast cancer.

In addition to the above-mentioned therapeutic agents, the BFA of the present invention applies the ability to maintain high binding power to target cells to
For example, it can be used as various diagnostic agents in place of conventional monoclonal antibodies.

As described above, the antibody of the present invention is effective as a therapeutic agent for malignant tumor, and therefore also provides a therapeutic agent for malignant tumor according to the present invention. This therapeutic agent for malignant tumors of the present invention is based on the fact that it contains the above-mentioned BFA as an essential component thereof, and can be prepared according to other conventional formulation techniques or technical means commonly used in immunotherapy using this type of BFA. it can. That is, the therapeutic agent is usually prepared in the form of a pharmaceutical composition by mixing a suitable pharmaceutical carrier with the antibody of the present invention. As the carrier to be used, various kinds of carriers usually used for the preparation of a formulation depending on the use form, for example, a filler,
Any of excipients or diluents such as a bulking agent, a binder, a surface active agent, a buffer solution, a stabilizer, etc. may be used, and the preparation form to be prepared is such that it effectively contains the therapeutic agent component of the present invention. It may be a solid preparation such as tablets and powders, but it is usually in the form of injections such as solutions, suspensions and emulsions. In addition, this may be in the form of a dried product which can be made into a liquid form by adding an appropriate carrier before use, and any form can be prepared by a conventional method. In addition, the preparations of each form are administered by an appropriate administration route depending on the form, for example, intravenous, intramuscular, subcutaneous, intradermal, and intraperitoneal injections are given for preparations in the form of injections, and oral or enteral administrations for preparations in the solid form To be done. Of course, it is also a useful method to react the antibody of the present invention with lymphocytes or the like in vitro in advance and then administer the antibody to intravenous, intraperitoneal, infectious lesion or the like.

The dose of the therapeutic agent of the present invention is appropriately determined depending on the administration method, dosage form, purpose of use, patient to be applied, etc. of the preparation and is not constant, but generally the amount of the antibody of the present invention is about 0.
It is preferable to contain about 0,0001 to 80% by weight, and this preparation contains about 0.01 µg to 1 per adult per day.
It is preferably applied in the range of about 0 mg. Thus, the administration of the therapeutic agent of the present invention enhances the cytotoxicity of lymphocytes in the body of the patient to whom the therapeutic agent is administered, thus exerting the desired therapeutic effect.

In the immunotherapy using the therapeutic agent of the present invention, adoptive immunotherapy, that is, lymphocytes once collected from the patient's body are activated by some means and then returned to the patient's body for treatment. The method to be performed can be implemented as follows, for example. That is, lymphocytes were separated from about 100 ml of peripheral blood of a patient and cultured in a serum-free medium supplemented with about 100 U / ml of IL-2 for about 1 week to obtain about 1 × of LAK cells (lymphokine-activated killer cells) obtained. 10 ⁸ BFA of the present invention
It is carried out by injecting into the patient's body together with about 100 to 1000 μg, preferably about 100 μg, and this treatment can be usually divided into several times a week depending on the medical condition, age and the like of the patient.

[0037]

INDUSTRIAL APPLICABILITY The antibody of the present invention has a strong specific binding power to antigens (target cells and effector cells), and its use can activate or enhance the cytotoxic activity of effector cells such as CTL against target cells. have.

[0038]

EXAMPLES The BFA of the present invention and a therapeutic agent for malignant tumor using the same will be described in more detail below, but the present invention is not limited thereto.

[0039]

Example 1 Production of BFA 1) Preparation of UCHT1 ascites UCHT1 cells [anti-CD3ε antibody: mouse IgG ₁ : Bev
erley, PCL and Callard, RE, Eur.J.Immunol., 11 ,
329-334 (1981): Imperial Cancer Research Foundation
n, UK)] using RPMI-1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% FCS (manufactured by Flow Co.).
Hybridomas were obtained by culturing at 7 ° C. in the presence of 5% CO ₂ .

Separately, 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane, manufactured by Wako Pure Chemical Industries, Ltd.) was administered intraperitoneally to BALB / c (produced by Charles River Japan). The animals were raised for 1-2 weeks.

Next, the UCHT1 cells obtained above were treated with PB
The cells were suspended in S, and 1 × 10 ⁷ cells / 0.5 ml was intraperitoneally administered to each mouse. At the time when the abdominal cavity of the mouse was swollen 2 to 3 weeks after the above administration, ascites was drained from the abdominal cavity of the mouse, and the ascites was 10,000 rpm using Hitachi Centrifuge 05PR-22 (manufactured by Hitachi, Ltd.) for 30 minutes, After centrifugation at 4 ° C., ascites supernatant containing UCHT1 antibody was obtained.

2) Preparation of UCHT1-F (ab ') ₂ fragment To the UCHT1 ascites supernatant obtained in 1) above was added a buffer (pH 8.9) consisting of 2 volumes of 1.5 M glycine and 3 M NaCl. And mixed, and the mixture was applied to an immobilized protein A (Immobilized-Protein A (Repligen)) column to adsorb the UCHT1 antibody to the column. After sufficiently washing with a buffer solution (pH 8.9) consisting of 1.5 M glycine and 3 M NaCl, the adsorbed U
The CHT1 antibody was eluted with 0.1 M sodium citrate buffer (pH 3.0 to 5.0) to obtain an IgG solution.

2 ml of the IgG solution obtained above (IgG
10 mg) in 0.1 M sodium citrate buffer (p
It was dialyzed against H4.1) overnight at 4 ° C. After collecting the dialysate, 1/10 to 1/25 amount (w / w) of the antibody, pepsin (manufactured by Sigma), was added and mixed, and the digestion reaction was performed in a thermostat at 37 ° C for 6 to 8 hours. It was After the reaction, add 1 to the reaction mixture.
Stop the reaction by adding 1/2 volume of M Tris solution,
20 mM phosphate buffer containing 0 Mm NaCl (pH 7.
Gel filtration was performed using a TSK-G3000SW (manufactured by Tosoh Corporation) column (21.5 mm ID × 60 cm) equilibrated with 0) to pool F (ab ′) ₂ fractions.

Next, a buffer solution (pH 8.9) consisting of 1.5 M glycine and 3 M NaCl was added to the collected fractions, and the mixture was applied to a protein A column to collect a flow-through fraction, UHC undigested with pepsin on the column
The L1 antibody was adsorbed and removed. The thus-obtained flow-through section was subjected to ultrafiltration using a membrane having a molecular weight of 10,000 (Amicon, Diaflow membrane YM10, 4).
3 mm membrane was used) and the buffer was exchanged with a solution consisting of 20 mM Tris-hydrochloric acid, 2 mM EDTA and 150 mM NaCl (pH 8.0: hereinafter referred to as “TES buffer”), and the F (ab ′) ₂ fragment of UCHT1 was replaced. 4 mg was obtained.

3) Preparation of GFD-OA-p185-1 ascites First, the GFD-OA-p185-1 antibody as an antibody recognizing the human c-erbB-2 gene product was prepared as follows. That is, the hybridoma GFD-OA-p185-1 (GFD-OA-p185-1 antibody-producing hybridoma GFD-OA-p185-1) was cultured in the same manner as in 1) above, and then the cells were intraperitoneally injected into mice. The ascites supernatant containing the GFD-OA-p185-1 antibody was obtained from the ascites.

4) GFD-OA-p185-1-F
Preparation of (ab ') ₂ fragment GFD-OA-p obtained in 3) in the same manner as in 2) above
The 185-1 ascites supernatant was mixed with 2 volumes of a buffer (pH 8.9) consisting of 1.5 M glycine and 3 M NaCl, and the mixture was applied to a protein A column to give GFD-
The OA-p185-1 antibody was adsorbed on the column. After washing with the same buffer, adsorbed GFD-OA-p18
5-1 antibody was added to 0.1 M sodium citrate buffer (p
H3.0-5.0) was eluted to obtain a solution containing IgG.

2 ml of the solution containing IgG obtained above
(IgG 10 mg) was dialyzed against 0.1 M sodium citrate buffer (pH 4.1) at 4 ° C. overnight. After collecting the dialysate, 1/10 to 1/25 the amount of the antibody (w /
w) pepsin was added and mixed, and the mixture was mixed in a thermostat at 37 ° C for 4 to
The digestion reaction was performed for 8 hours. After the reaction, 1/2 volume of 1 M Tris solution was added to the reaction solution to stop the reaction, and 150 Mm
Gel filtration was performed using a TSK-G3000SW column equilibrated with 20 mM phosphate buffer (pH 7.0) containing NaCl, and F (ab ′) ₂ fractions were pooled.

Next, 1.5 M glycine and 3 were added to this fraction.
A buffer solution (pH 8.9) consisting of M NaCl was added, the mixture solution was applied to a protein A column to collect a flow-through fraction, and pepsin-undigested GFD-OA- was applied to the column.
The p185-1 antibody was adsorbed and removed. The flow-through fraction thus obtained was subjected to ultrafiltration using a membrane having a molecular weight of 10,000, and the buffer was exchanged with a TES buffer solution to obtain 5 mg of F (ab ') ₂ fragment of GFD-OA-p185-1. Obtained.

5) GFD-OA-p185-1Fa
Preparation of BFA of b ′ and UCHT1-Fab ′ 1.0 ml T containing 3 mg of F (ab ′) ₂ fragment of GFD-OA-p185-1 antibody prepared in 4) above
20 μl of 100 mM dithiothreitol (DTT; manufactured by Wako Pure Chemical Industries) aqueous solution was added to the ES buffer to adjust the pH to 7.
5. After reacting for 30 minutes at room temperature, continue to 10
A 1-2 times amount of mM DTNB aqueous solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the reaction was further performed at room temperature for 30 minutes. The reaction solution was subjected to gel filtration using a Sephadex G25 column equilibrated with a TES buffer solution substituted with nitrogen gas, and the first peak was pooled to give GFD-OA-p185-1-Fa.
b'mixed nitrophenyl disulfide derivative (GFD
-OA-p185-1-Fab'-SNB) was obtained.

Separately, 1.0 ml containing 3 mg of F (ab ') ₂ fragment of UCHT1 antibody prepared in 2) above.
To the TES buffer solution, add 20 μl of DTT aqueous solution and p
After reacting for 30 minutes at room temperature under H7.5, the obtained reaction solution was subjected to gel filtration using a Sephadex G25 column equilibrated with a TES buffer solution substituted with nitrogen gas, and the first peak was pooled. UCHT1-Fab'-SH
Was obtained.

GFD-OA-p185-1-Fab'-SN prepared above was added to the UCHT1-Fab'-SH.
After adding B and concentrating with an ultrafiltration membrane (YM10), at room temperature, 4
After reacting for a period of time and then continuing the reaction at 4 ° C., GFD-OA-p185-1-Fab ′ and UCT were obtained.
BFA in which monomers of H1-Fab ′ were bound to each other was obtained.

The TES buffer solution containing BFA obtained above was subjected to HPLC (high performance liquid chromatography) using TSK gel G3000SW (manufactured by Tosoh Corp.), ion exchange chromatography or hydroxyapatite column chromatography to unreact. Fa
The target BFA was separated and purified from the b'-thiol derivative (molecular weight about 55 kd).

The purified BFA was added to 0.1% under non-reducing conditions.
Purified BFA by performing 7.5% PAGE analysis in the presence of% SDS (sodium dodecyl sulfate, manufactured by Sigma).
The molecular weight of was confirmed.

As a result of the above SDS-PAGE, the molecular weight was about 1
The band was confirmed at the position of 10 kd, and from this, the BFA
Was confirmed to have the expected molecular weight of about 110 kd.

[0055]

Example 2 BFA and GFD of the present invention obtained in Example 1
-OA-p185-1-F (ab ') ₂ fragment and UCHT1-F (ab') ₂ fragment were immunoreactive with peripheral blood lymphocytes prepared from seven human cancer cell lines and healthy human volunteers. Tested as follows using (PBL).

In this test, the following seven human cancer cell lines (all were obtained from American Type Culture Collection, ATCC; Rockville, Md. USA) were used.

ZR-75-1 (human breast cancer cell line: ATC
C CRL 1500) SK-BR-3 (human breast adenocarcinoma cell line: ATCC HTB
30) MCF-7 (human breast adenocarcinoma cell line: ATCC HTB2
2) PANC-1 (human pancreas, epithelial adenocarcinoma cell line: ATCC
CRL1469) MIAPaCa-2 (human pancreatic cancer cell line: ATCC C
RL1420) ASPC-1 (human metastatic pancreatic cancer cell line: ATCC C
RL1682) BxPC-3 (primary human pancreatic cancer cell line: ATCC C
RL1687) The above 7 human cancer cell lines and PBL of healthy volunteers
Were washed with PBS buffer containing 0.1% BSA and 0.1% NaN ₃ , respectively, and then washed with 10% PBS buffer.
μg of each antibody was added and reacted on ice for 30 minutes. After the reaction, the cells were washed twice, FITC-conjugated anti-mouse IgG (Tago Inc., USA) was added, and the mixture was further reacted on ice for 30 minutes. After the reaction, the plate was washed twice, and then the reaction activity of each antibody was measured by Ortho Spectrum III (Ortho Spectrum III).
It was measured by flow cytometry using Diagnostic Inc., USA. Positive cells were quantified as possessing logarithmic display fluorescence intensity.

The results are shown in Table 1.

[0059]

[Table 1]

In the table, the numbers indicate the percentage (%) of multicolor defense reacted with each antibody. The control represents only the second antibody, and NT represents untested.

From the table, it can be seen that the anti-c-erbB-2 gene product F (ab ') ₂ fragment was associated with two breast cancer cell lines ZR.
Reacted strongly with -75-1 and SK-BR-3.
These two cancer cell lines are rich in c-erbB-2mR
A cancer cell line known to express NA [Kr
aus, MH, et al., EMBO J., 6 , 605-610 (1987)]. In addition, this F (ab ') ₂ fragment was used as a cancer cell line MCF.
-7, PANC-1, MIAPaCa-2 and ASPC
It showed a weak reactivity with -1 (compared to the previous two cancer cell lines) and little reactivity with the cancer cell line BxPC-3. Furthermore, it did not react with PBL.

The anti-CD3εF (ab ') ₂ fragment reacted only with PBL and not with the cancer cell line. The BFA of the present invention comprises two human breast cancer cell lines ZR-75.
-1 and SK-BR-3 reacted strongly with PBL. Further, the reaction activity of the BFA is the above anti-c-erbB-2 gene product F (ab ') against other human cancer cell lines.
It was similar to the ₂ segment.

From the above test results, it was revealed that the BFA of the present invention possesses the expected immunological characteristics.

[0064]

Example 3 The cytotoxic activity of the BFA of the present invention obtained in Example 1 was measured using PBL as effector cells and ZR-75-1 and SK-BR-3 as target cells, respectively. Using the specific lysis of the target cells, which occurs when the respective effector cells and the target cells are bound to each other, as an index, the following procedure was performed.

Labeling of target cells with ⁵¹ Cr Using RPMI-1640 medium containing 10% FCS,
ZR-75-1 cells and SK-BR-3 cells were incubated at 37 ° C.
Were cultured in the presence of 5% CO ₂ . Then, the culture supernatant was removed, and after washing with 25 ml of PBS ^(-) , 0.05% trypsin-0.05% EDTA-PBS was added to each cultured cell.
Add 2.5 ml of the solution and incubate at 37 ° C. for 1 minute to detach the cells and remove RPMI-16 containing 10% FCS.
Suspend in 20 ml of 40 medium to neutralize, 1000 rp
Centrifugation with the same medium was performed twice at room temperature for 8 minutes.

Then, the resulting pellets of each cell (5 × 10 5
⁶ cells) with 3.7 MBq of sodium chromate (Na ₂
⁵¹ CrO ₄ : NEN) was added and 5% CO ₂ was added at 37 ° C.
Incubate for 60 minutes in the presence of 10% FCS
Excess sodium chromate was removed by washing twice with the RPMI-1640 medium containing the cells, and after counting the number of cells with a hemocytometer, the concentration of the culture solution was adjusted to 2 × 10 ⁵ cells / ml. It was diluted with RPMI-1640 medium containing% FCS.

Preparation of effector cells (PBL) PBS buffer 5 was added to 50 ml of heparin-collected blood of a healthy person.
Add 0 ml and mix. Separately, add 15 ml of specific gravity separation liquid (Lymphosite Separation Medium: Flow (Flow Laboratorues)) to 4 50 ml centrifuge tubes, and add 25 ml of the above blood to each tube. Overlaid. After that, 1500 rpm using Hitachi Centrifuge O5PR-22
Density gradient centrifugation was performed at room temperature for 30 minutes.

After centrifugation, the white turbid layer formed between the plasma and the specific gravity separated liquid in the four centrifuge tubes was sucked up using a Pasteur pipette, and this was transferred to a new 50 ml centrifuge tube.
ml of RPMI-1640 medium containing 10% FCS was added and mixed, and centrifuged at 1000 rpm for 5 minutes to recover PBL as a precipitate. RPMI-164 containing 10% FCS
After the washing operation with 0 medium was repeated 3 times, the obtained precipitate was suspended in 10% FCS-containing RPMI-1640 medium.

Preparation of each antibody GFD-OA-p185-of the present invention obtained in Example 1
1-Fab'-UCHT1-Fab 'linked BFA, GFD-OA-p used as a starting material for the preparation of this BFA
185-1-F (ab ') ₂ fragment and UCHT1
1: 1 mixture with -F (ab ') ₂ fragment, GF
Each of the D-OA-p185-1-F (ab ′) ₂ fragment and the UCHT1-F (ab ′) ₂ fragment was subjected to RPMI-1640 medium containing 10% FCS, and each of them was subjected to Mylex GV filter 0.22 μ.
After aseptic filtration using M (manufactured by Japan Millipore), 2
ng / ml, 20 ng / ml, 200 ng / ml and 2
Dilution was prepared to each concentration of 000 ng / ml.

Cytotoxic activity test 100 μl of various concentrations of antibody prepared above and 50 μl of effector cells (cell number = 5 × 10 ⁴ , Effecto)
r; E) and 50 μl of target cells (1 × 10 ⁴ cells, Taget; T)
) Is added to each well of a 96-well U-bottom culture plate (Falcon) (Effector / Taget).
= E / T = 5), and the cells were cultured at 37 ° C. in the presence of 5% CO ₂ for 4 hours to cause a specific damaging reaction of target cells. Next, the culture supernatant was collected by a Supernatant Collection System (manufactured by Dainippon Pharmaceutical Co., Ltd.), put in a measurement tube, and the radioactivity contained in the tube was counted. Further, each antibody was reacted under the same conditions as described above, except that the ratio (E / T) of effector cells to target cells was changed to 0, 10 and 20.

The degree (%) of the specific cytotoxic activity of the target cells, which occurs when the effector cells and the target cells are bound via each antibody, was determined according to the above calculation formula (1).

The obtained results are shown in FIGS. 1 and 2.

FIG. 1 shows the results of using the BFA of the present invention on ZR-75-1 cells, and FIG. 2 shows ZR-75-1.
GFD-OA-p185-1-F (a
The results are shown using a 1: 1 mixture of the b ') ₂ fragment and the UCHT1-F (ab') ₂ fragment.

In each figure, the horizontal axis represents the absolute amount of antibody used (ng / ml), and the vertical axis represents the specific injury% (%) of target cells.
Specific Cytotoxicity), where (1) is E / T
= 0, (2) shows E / T = 5, (3) shows E / T = 10, and (4) shows E / T = 20.

From the above FIG. 1, it was confirmed that the BFA of the present invention bound to PBL possesses significant cytotoxic activity in the case of ZR-75-1 cells. The concentration of BFA required for expression of this cytotoxic activity is 10 ng / ml.
Above, the E / T ratio was 5 or more. In addition, the BFA of the present invention can observe specific target cell damage of up to about 25% at a dose of 100 ng / ml and an E / T ratio of 20,
From this, it was confirmed to have a high target cytotoxic activity. On the other hand, GFD-OA-p185-1-
F (ab ') ₂ fragment and UCHT1-F (a
When a 1: 1 mixture with b ′) ₂ fragment was used, no significant cytotoxic activity was observed even when the concentration was increased to 1000 ng / ml (see FIG. 2).

Furthermore, GFD-OA-p185-1-F
(Ab ') ₂ fragment and UCHT1-F (a
b ') The same test was carried out using each of the ₂ fragments, and as a result, each of these fragments was 1000 ng / m 2.
No significant cytotoxic activity was observed even when the concentration was increased to 1.

From the above results, the BFA of the present invention is 10 ng
A therapeutic agent for malignant tumors, particularly a breast cancer therapeutic agent, which is composed of cancer cells expressing a c-erbB-2 gene product on the cell surface, which exhibits a specific cytotoxic activity of target cells from an extremely small amount of 1 / ml. It turns out to be useful as

In addition to the above therapeutic agents desired in the present invention, for example, the BFA of the present invention can be used as various diagnostic agents in place of conventional monoclonal antibodies in addition to the above therapeutic agents desired in the present invention. Can be expected.

[Brief description of drawings]

FIG. 1 is a graph showing the cytotoxic activity of the BFA of the present invention.

FIG. 2 is a graph showing the cytotoxic activity of the antibody mixture used as a control in comparison with FIG. 1.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Kyohei Exit 1-9-703 Suehiro, Tokushima City, Tokushima Prefecture (72) Inventor Kenichi Imagawa 1-83 Kitahari, Kitakijima, Itano-gun, Tokushima Prefecture (72) ) Inventor Mikio Kikuchi 4-29-14 Imaizumidai, Kamakura City, Kanagawa Prefecture

Claims (6)

[Claims] 1. One of the antibodies constituting the composite antibody is human c
-A composite antibody characterized in that it is an erbB-2 specific antibody and the other is a human lymphocyte antibody. 2. The antibody recognizing the surface antigen of a target cell is an anti-human c-erbB-2 antibody, and the antibody that binds to effector cells to perform signal transduction is an anti-human lymphocyte antibody. Complex antibody. 3. The anti-human c-erbB-2 antibody is GFD-
The composite antibody according to claim 1 or 2, which is OA-p185-1. 4. The composite antibody according to claim 1, wherein the anti-human lymphocyte antibody is an anti-CD3 antibody. 5. The composite antibody according to claim 1 or 2, which specifically reacts with a human c-erbB-2 related protein. 6. A therapeutic agent for cancer, which comprises the complex antibody according to claim 3 as an essential component.