JP2564412B2 - Chimeric antibody against drug-resistant cancer and production thereof - Google Patents

Description

Description: BACKGROUND OF THE INVENTION Technical Field The present invention relates to a chimeric antibody against drug-resistant cancer and its production. More specifically, the present invention relates to a chimeric antibody comprising a variable (V) region of a mouse-derived monoclonal antibody and a constant (C) region of a human-derived immunoglobulin and a method for producing the same.

<Prior Art> Tumor resistance to various chemotherapeutic agents has become an important issue in the treatment of cancer. Tumor cells can be treated with single-agent doxorubicin (adriamycin),
It is possible to acquire resistance to drugs such as vinca alkaloids and actinomycin D (references 1 and 2 below). The gene that causes multidrug resistance, mdr, encodes a membrane sugar protein (P-sugar protein) that acts as a pump that transports various cytotoxic drugs from cells (3).
This P-sugar protein has been shown to be an ATPase (6, 7) that binds to anti-cancer agents (4,5) and is localized to the plasma membrane of resistant cells (8, 9). Transfection of cloned mdr sequences confers multidrug resistance to susceptible cells (10-12).

The amount of P-sugar protein expression is measured in tumor samples and is elevated in intrinsically drug-resistant colon, kidney and adrenal cancers, and some types of tumors that have acquired drug resistance after chemotherapy Was found (13-15). Since P-sugar proteins appear to be associated with both acquired multidrug resistance and intrinsic drug resistance in human cancers, selective killing of tumor cells expressing P-sugar proteins is a treatment of cancer. Is extremely important to. In the study of effective treatment for human drug-resistant cancer, we developed a monoclonal antibody reactive with multidrug transporter P-sugar protein (16). Intrapulsation of this monoclonal antibody effectively prevented tumor development in athymic mice subcutaneously inoculated with drug-resistant human ovarian cancer cells (17). Treatment with one of the monoclonal antibodies, MRK16, resulted in rapid regression of established subcutaneous tumors and healing in certain animals. These monoclonal antibodies have potential as therapeutic tools for multidrug-resistant human tumors with P-sugar proteins (17). The present inventors have already applied for a monoclonal antibody relating to drug-resistant cancer including this monoclonal antibody (see Japanese Patent Application No. 60-20144 and Japanese Patent Application Laid-Open No. 62-61596).

However, murine antibodies as xenoproteins can provoke a reactive immune response that destroys their effects, and can also cause an allergic reaction in patients (18, 19).

The use of monoclonal antibodies against the surface of tumor cells for cancer therapy has long been expected (36). In some cases, monoclonal antibodies alone can inhibit the growth of tumor cells (17, 37-39), while in other systems tumor growth inhibition is associated with antibodies and various toxic agents. Achieved by complex (40-42). Monoclonal antibodies can be used to eradicate residual malignant cells in vitro (43). While there are many difficulties in such treatment (36), several new successful cases have been reported (44), and immunotherapy will have important clinical value in the future.

A major limitation of the clinical use of murine-derived monoclonal antibodies is the immune response elicited against the heterologous protein, which can render the antibody ineffective or bear the patient (18, 19, 36). In addition, mouse monoclonal antibodies may be less efficient at interacting with human effector cells that mediate tumor destruction. Although treatments with human monoclonal antibodies are under study, human hybridoma cell lines are largely unavailable, and, if present, are usually unstable and produce low amounts of immunoglobulin (45 ).

[Outline of Invention]

It is an object of the present invention to solve the above problems and provide an antibody that is specific to multidrug-resistant human cancer and has low immunogenicity.

One way to prevent the antigenicity of murine monoclonal antibodies in humans is to construct murine V / human C chimeric immunoglobulins. Since the antigenicity of most immunoglobulins is in the C region, the generation of murine V / human C chimeric immunoglobulins has the specificity of murine monoclonal antibodies but does not induce human immune responses (20, 46, 47). Antibodies are obtained. Furthermore, because such chimeric proteins interact more effectively with the human cellular immune system because of their human C (constant) regions, they may provide a more advantageous therapy than the corresponding murine antibody (48). . Mouse-human chimeric antibodies have been shown to retain the ability to react with hapten antigens (49-51) and some cancer-associated antigens (52-55). Several attempts to use these chimeric antibodies in cancer immunotherapy are currently in progress (56).

<Summary> The present inventors have prepared a recombinant chimeric antibody in which the antigen recognition variable (V) region of the monoclonal antibody MRK16 is bound to the constant (C) region of the human antibody (20, 21) to produce human effectors. When the ability to kill drug-resistant tumor cells using cells was measured by an antibody-dependent cytotoxicity test, it was found that this chimeric antibody was more effective than mouse MRK16, and the present invention was based on this finding. It came to completion.

That is, a chimeric antibody against drug-resistant cancer according to the present invention was prepared by fusing splenocytes and mouse myeloma cells obtained from mice immunized with adriamycin-resistant strain K562 / ADM of human myeloid leukemia cell line K562. A variable region having an amino acid sequence substantially homologous to a variable region in a mouse monoclonal antibody produced by hybridoma MRK16 or MRK17, and a constant region having an amino acid sequence substantially homologous to a constant region in human immunoglobulin, It has a binding specificity with the multidrug resistant cell line K562 / ADM.

The method for producing a chimeric antibody against drug-resistant cancer according to the present invention is characterized by including the following steps (a) to (f).

(A) A constant region in the H chain of human immunoglobulin and a constant region in the H chain of a human immunoglobulin are located downstream of the gene encoding an amino acid sequence substantially homologous to the variable region in the H chain of MRK16 or MRK17 which is a mouse monoclonal antibody against drug-resistant cancer To prepare a DNA chain having a nucleotide sequence encoding a chimeric H difference by ligating the transcription upstream side of a gene encoding a homologous amino acid sequence.

(B) a constant region in the L chain of human immunoglobulin and a constant region downstream of the transcription of a gene encoding an amino acid sequence substantially homologous to the variable region in the L chain of MRK16 or MRK17 which is a mouse monoclonal antibody against drug-resistant cancer To prepare a DNA difference having a nucleotide sequence encoding a chimeric L chain by linking the transcription upstream side of a gene encoding a homologous amino acid sequence.

(C) Inserting each of the DNA chains obtained in steps (a) and (b) into the same or different expression vector in a state where the genetic information can be expressed to prepare a recombinant DNA.

(D) Transforming a host cell with the recombinant DNA obtained in step (c) to prepare a transformant.

(E) Culturing the transformant obtained in step (d) to produce in the culture a chimeric antibody against a drug-resistant cancer having a binding specificity with the multidrug-resistant cell type K562 / ADM.

(F) If necessary, recovering the chimeric antibody produced in the culture in step (e).

<Effect> The chimeric antibody according to the present invention has the ability to selectively inhibit the growth of cancer cells showing multidrug resistance or enhance the sensitivity to the drug, and the constant region is a human-derived C region. Therefore, it has an extremely low immunogenicity, that is, it is difficult to induce a human immune reaction.

Therefore, the chimeric antibody according to the present invention can be an excellent solution to the important problem of establishing a drug or method effective for cancer cells showing extremely few side effects, high selectivity and multidrug resistance. .

[Specific Description of the Invention]

<Chimeric antibody> The chimeric antibody according to the present invention is substantially composed of a variable (V) region having an amino acid sequence substantially homologous to a variable region in a mouse monoclonal antibody against drug-resistant cancer and a constant (C) region in human immunoglobulin. It is as described above that it is characterized by consisting of a constant region having an amino acid sequence homologous to, and basically belongs to immunoglobulin IgG, that is, each homologous consisting of a variable region and a constant region. And two H (heavy) chains and an L (light) chain each having an S-S bond.

Here, the monoclonal antibody against the drug-resistant cancer from which the variable region is derived is specifically described in (a) to
A monoclonal antibody against the P-sugar protein defined by (d), which is described in the patent application by the present inventors-Japanese Patent Application No. 60-201445.

(A) It is produced by a hybridoma prepared by fusing spleen cells obtained from a mouse immunized with an adriamycin-resistant strain K562 / ADM of a human myeloid leukemia cell line K562 with mouse myeloma cells. .

(B) The ability to specifically identify adriamycin resistant strains.

(C) The ability to inhibit cell growth of an adriamycin-resistant strain or to enhance the sensitivity of the cells to vincristine or actinomycin D.

(D) Being of the IgG isotype.

Specific examples of such monoclonal antibodies include MRK16 having an isotype of IgG _2a and MRK17 having an isotype of IgG ₁ .

The hybridomas MRK16 and MRK17 that produce the monoclonal antibodies MRK16 and MRK17 are
No. 00 (FERM BP-2200) and No. 2201 (FERM BP-22
It has been deposited as 01).

Monoclonal antibodies MRK16 and MRK17 have selective growth inhibitory action or drug susceptibility increasing action on drug-resistant human cancer cells (see Japanese Patent Application No. 60-201445).

The amino acid sequences of the H chain and L chain variable regions of the monoclonal antibody MRK16 and the nucleotide sequences encoding them are shown in FIG. 4 (a to b) and FIG. 5 (a to b).

The variable region in the present invention means, in addition to the variable region of the mouse monoclonal antibody as described above, the polypeptide of the variable region has an amino acid sequence It also includes variable regions with some modifications such as substitutions, deletions and additions.

The human immunoglobulin from which the above-mentioned constant region is derived is produced by human immunoreaction, and two H (heavy) chains and two L (light) chains each having a homologous basic unit of its structure are connected by an SS bond. It is defined as being a polypeptide molecule. The gene sequence and amino acid sequence of the constant region of the H chain of human immunoglobulin have already been published (IgM (6
2), IgD (63), IgG ₁ (64), IgG ₂ (65), IgG ₃ (66), IgG
₄ (67), IgE (68) and IgA (69)). L chain (κ
Type) constant region gene and amino acid sequences are also known (70).

The constant region in the present invention means, in addition to such a constant region of human immunoglobulin, as long as the polypeptide of the constant region has a physiological function as a constant region of human immunoglobulin (for example, complement fixing ability). It also includes constant regions having substitutions, deletions, additions and the like in some of the amino acid sequences of the polypeptides.

Therefore, examples of the chimeric antibody according to the present invention include human immunoglobulin in the variable region having an amino acid sequence substantially homologous to the variable region in mouse monoclonal antibody MRK16.
Human immunoglobulin Ig in which a constant region having an amino acid sequence substantially homologous to the constant region in IgG ₁ is linked, and a variable region having an amino acid sequence substantially homologous to the variable region in mouse monoclonal antibody MRK17
It is a combination of constant regions having an amino acid sequence substantially homologous to the constant region in G ₁ .

<Production of Chimeric Antibody> The method for producing a chimeric antibody according to the present invention is characterized by including the following steps (a) to (f), as described above, and as described above. A chimeric antibody of the present invention is produced.

(A) Amino acids substantially homologous to the constant region in the H chain of human immunoglobulin on the downstream side of transcription of a gene encoding an amino acid sequence substantially homologous to the variable region in the H chain of a mouse monoclonal antibody against drug-resistant cancer To prepare a DNA chain having a nucleotide sequence encoding a chimeric H chain by linking the transcription upstream side of a gene encoding the sequence.

(B) Amino acids that are substantially homologous to the constant region in the L chain of human immunoglobulin on the downstream side of the transcription of the gene encoding the amino acid sequence that is substantially homologous to the variable region in the L chain of mouse monoclonal antibody against drug-resistant cancer To prepare a DNA chain having a nucleotide sequence encoding a chimeric L chain by linking the transcription upstream side of a gene encoding the sequence.

(C) Inserting each of the DNA chains obtained in steps (a) and (b) into the same or different expression vector in a state in which the genetic information can be expressed to prepare a recombinant DNA.

(D) Transforming a host cell with the recombinant DNA obtained in step (c) to prepare a transformant.

(E) Culturing the transformant obtained in step (d) to produce a chimeric antibody against drug-resistant cancer in the culture.

(F) If necessary, recovering the chimeric antibody produced in the culture in step (e), the chimeric antibody of the present invention basically comprises variable H chain and L chain of mouse monoclonal antibody. Recover the gene encoding the region from a suitable mouse-derived gene source.
Genes obtained by cloning and the use of suitable regulatory enzymes, while coding for the constant regions in the H and L chains of human immunoglobulin were likewise obtained from a suitable human-derived gene source and described above. Chimera H
Chain and L chain genes are prepared, both genes are introduced into a host cell in the state of being linked to separate or the same appropriate expression vector to prepare a transformant, and the transformant is cultured. It can be manufactured.

Such a chimeric antibody or chimeric protein or recombinant protein can be obtained by using a recombinant technology known in the art, for example, Maniati.
s) et al., Molecular Cloning: Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory (1989) (Molecular Cloning: A Loboratory man
ual, 2nd Ed. (1989) Cold Spring Harbor Laboratory)
Etc., and can be manufactured.

In the present invention, steps (a) and (c), and (b) and (c) are separately performed (that is, chimeric H chain and L chain genes are prepared and then these genes are inserted into an expression vector). However, in a more preferable embodiment, in steps (a) and (b), one of the two region genes, for example, the gene encoding the constant region of human immunoglobulin is previously added to the expression vector of step (c). It is to use the connected ones. In this case, since the recombinant DNA is constructed in the steps (a) and (b), the step (c) of inserting the recombinant DNA into separate source vectors to prepare the recombinant DNA is unnecessary. .

The following will further explain the method for producing the chimeric antibody according to the present invention based on the preferred embodiment.

1) Construction of chimeric H chain gene and recombinant DNA (i) Gene of variable region The gene encoding the variable region is specifically a cell producing a mouse monoclonal antibody against P-sugar protein, such as the above-mentioned monoclonal antibody. From the library of chromosomal genes of hybridoma MRK16 producing MRK16 and hybridoma MRK17 producing monoclonal antibody MRK17 (both of which have been deposited as described above), a method commonly used in the field of genetic engineering, for example, by an appropriate probe is used. It can be obtained by the hybridization method. As a preferable probe, for example, a fragment containing a mouse J _H gene (a gene encoding a J region in the H chain variable region of mouse IgG) (J _H probe,
(26)).

(Ii) Gene of constant region The gene encoding the constant region can be obtained by a gene library prepared from human placenta DNA and a method commonly used in the field of genetic engineering, for example, a hybridization method using an appropriate probe. it can.

If necessary, at least a part of the chain length of the genes (i) and (ii) can be chemically synthesized according to an ordinary nucleic acid synthesis method. These genes include degenerate isomers that differ only in degenerate codons, as well as changes (substitutions, deletions, additions, etc.) in the amino acid sequence of the polypeptide in each region of the variable and constant regions It may have a corresponding base sequence or an isomer thereof.

By actually connecting the genes of (i) and (ii), the chimeric H
When constructing a chain gene, it is more convenient to prepare a recombinant DNA as described above when a constant region gene is ligated to an expression vector in advance.

The expression vector may be any one as long as it can express the desired gene contained therein in a host cell, and is generally in the form of a plasmid. In addition, it is necessary to have a marker gene for selection of transformed cells (for example, those relating to drug resistance, auxotrophy, etc.).

Specific preferred examples of the expression vector in which the constant region (human-derived) gene is ligated in advance include, for example, pSV2-HG1-gpt having a human-derived Cγ1 region (see (24)).
Is raised.

Transcriptional downstream side (3 'side) of the variable region gene of (i) obtained from a gene library using an appropriate restriction enzyme
To the transcriptional upstream side (5 'side) of the constant region gene of the expression vector to which the gene of (ii) is ligated, the chimeric H chain gene is constructed and the recombinant DNA
Is built. In order to secure a correct base sequence, the connecting side end of the gene of (i) or (ii) may be deleted by an appropriate length or an appropriate base sequence may be added, if necessary. Human-derived Cγ1 as the expression vector
The region-containing pSV2-HG1-gpt was used, to which MRK-16
Expression vector pSV2-VH16 for chimeric antibody heavy chain by ligating a gene encoding the variable region of
The preparation of HG1gpt is shown in Fig. 1A (see Experimental Example [2] below).

The expression vector is for expressing the genetic information of the above chimeric H chain gene in a host cell, that is, its DNA.
It is necessary to have an appropriate promoter for transcribing the RNA into mRNA. In order to express a larger amount of antibody, it is necessary to include an enhancer.

When a gene encoding the H chain variable region of a mouse monoclonal antibody against drug-resistant cancer is collected from genomic DNA, it can be excised as a fragment containing a promoter upstream of the gene and an enhancer downstream thereof. It is convenient to use such a fragment because it is not necessary to separately incorporate a promoter and an enhancer.

2) Construction of chimeric L chain gene and recombinant DNA (i) Gene of variable region The gene encoding the variable region is as described above in 1), for example, hybridomas MRK16 and MRK.
It can be obtained from a library of 17 chromosomal genes by a hybridization method using an appropriate probe. As a preferable probe, for example, a fragment containing a mouse Jκ gene (a gene encoding the J region in the variable region of the κ type L chain of mouse IgG) (Jκ probe, (2
5)).

(Ii) Gene of constant region The gene encoding the constant region of L chain can be obtained from the library of human placental chromosome genes as in the case of H chain.

In the above (i) and (ii), at least a part of the chain length of this gene can be chemically synthesized in the same manner as in the case of the variable region of 1). It may have a base sequence corresponding to a change (substitution, deletion, addition, etc.) in the amino acid sequence in each of the region and the constant region, or an isomer thereof.

By actually connecting the genes of (i) and (ii), the chimeric L
In order to construct a chain gene, it is convenient to use a gene in which the constant region gene is linked to an expression vector in advance, as in the case of the chimeric H chain gene. A preferred specific example of a vector in which a constant region (human-derived) gene is previously ligated is, for example, pS having a human-derived Ck region.
_{V2-HC κ -neo ((24} ) see), and the like.

Transcriptional downstream side (3 'side) of the variable region gene of (i) obtained from a gene library using an appropriate restriction enzyme
To the transcriptional upstream side (5 'side) of the constant region gene of the expression vector to which the gene of (ii) is ligated, the chimeric L chain gene is constructed and the recombinant DNA
Is built. In order to secure a correct base sequence, the connecting side end of the gene of (i) or (ii) may be deleted by an appropriate length or an appropriate base sequence may be added, if necessary. As the expression vector, pSV2-HC _κ- neo having a human-derived Cκ region is used, and a gene encoding a variable region derived from MRK-16 is ligated to the expression vector pSV2- V _κ 16
-HC _κ neo was shown in Fig. 1B (Experimental Example [2] below.
reference).

The expression vector needs to have an appropriate promoter for expressing the genetic information of the above chimeric L chain gene in a host cell. In order to express a larger amount of antibody, it is necessary to include an enhancer.

When the gene encoding the L chain variable region of a mouse monoclonal antibody against drug-resistant cancer is collected from genomic DNA, it is convenient to excise it as a fragment containing a promoter and an enhancer and use it. It is also possible to use an enhancer located upstream of the gene encoding the constant region of human L chain.

3) Transformation Chimeric H chain gene and L obtained as described above
The chain gene can be introduced into an appropriate host cell by a general biotechnology technique to prepare a transformant, and the expression product of both chimeric chain genes, that is, the chimeric antibody according to the present invention can be produced in this host cell. .

The host cell for transformation is preferably a myeloma that is a B cell line tumor for the purpose of producing a large amount of antibody. As an example of this, a mutant strain derived from myeloma such as NS1, P3U1, SP2 / 0, etc., which does not already produce an antibody, is most effective.

Transformation of a host cell with an expression vector into which the above-mentioned chimeric chain gene has been inserted, that is, a recombinant DNA can be performed by any purposeful method commonly used in the field of genetic engineering. Preferred examples of such a method include a transfection method using calcium phosphate or calcium chloride, a transfection method by electroporation, or a lipofection method.

The transformant is derived from a new trait introduced by both of the above chimeric chain genes (ie, the ability to produce an IgG chimeric chain) and a trait derived from the used vector, as well as from the used vector at the time of gene recombination that may possibly occur. Except for some missing genetic information, it is the same as the host cell used in its genotype or phenotype or mycological properties.

4) Expression of chimeric chain gene / production of chimeric antibody By culturing the transformant clone obtained as described above, a chimeric antibody (IgG) is produced in the culture (intracellular and / or medium). To be done.

The culture conditions for transformants are essentially the same as for the original host cells used.

5) Isolation of chimeric antibody Isolation of the chimeric antibody from the above-mentioned culture can be performed according to a conventional method for protein or antibody isolation. A preferred example of such a method is a method using affinity chromatography with a protein A-Sepharose column (see Reference (33)).

From the transformant expressing the chimeric antibody once prepared, m-RNA encoding this chimeric antibody can be separated and purified by a conventional method. From this mRNA, a cDNA is further prepared by a conventional method to encode the obtained chimeric antibody.
By incorporating an appropriate promoter or enhancer region upstream of the cDNA, host cells other than myeloma cells,
For example, yeast, silkworm, or plant can express and produce this chimeric response.

As shown in the experimental examples below, chimeric mouse V / human C
Transfection of mouse myeloma cells with an expression vector containing the immunoglobulin gene produced functional chimeric IgG with the same affinity and binding proficiency as the original hybridoma antibody. This chimeric antibody is much less immunogenic than the mouse antibody. Moreover, the human C region of the chimeric antibody can perform human effector functions more effectively.

[Practical example]

[1] Materials and Experimental Method (a) Vector, clone, probe and cell: As a gene source encoding the variable region of a mouse monoclonal antibody against drug resistant cancer, a hybridoma MRK16 (FERM-BP-2200) producing MRK16 is used. Using. Eco
Λ phage λgt10 (22) was used as a subcloning vector excised by RI and containing the variable region of the H chain of a mouse monoclonal antibody. Further, λEMBL3 (23) was used as a subcloning vector containing the variable region of the L chain of a mouse monoclonal antibody excised with BamHI.

As a probe of the variable region of the L chain of the mouse monoclonal antibody, a mouse Jκ gene-containing fragment (Jκ probe) (25) isolated from clone Ig146 was used. A mouse J _H probe (26) isolated from MEP203 was used as a probe of the variable region of the H chain of the mouse monoclonal antibody.

PSV2HG1gpt was used as an expression vector in which a gene encoding the constant region of the H chain of human IgG was linked, and human
PSV2HCκneo (24) was used as an expression vector to which a gene encoding the constant region of IgG L chain was linked.

ATCC (Rockville,
Mouse myeloma Sp2 / 0 (Sp2 / 0-Ag14) obtained from Maryland (Rockville, MD) was used.

Human drug-resistant cell lines (K562 / ADM and 2780 ^AD ) and their patient drug-sensitive cell lines (K562 and A2780, respectively) used in the antibody-dependent cytotoxicity assay were retained as previously reported (27).

(B) Cloning of size-fractionated DNA: The fragment containing the gene encoding the variable region of the H chain of mouse monoclonal antibody was identified by Southern analysis of genomic DNA digested with EcoRI with a _JH probe. The fragment containing the gene encoding the variable region of the L chain was identified by Southern analysis of BamHI digested genomic DNA with an Iκ probe. It was confirmed that these genes were rearranged.

Each fragment was eluted from the identified portion on an agarose gel, ligated with the λgt10 arm and λEMBL and packaged into λ phage.

Plaque hybridization is based on Bent
on) -performed by the method of Davis (2
9).

(C) DNA transfection of mouse Sp2 / 0 myeloma cells: plasmids pSV2-VH16-HG1gpt and pSV2-Vκ16-HCκ
200 μg of each neo (see FIG. 1) was co-transfected into 10 ⁷ mouse Sp2 / 0 cells (CRL1581, ATCC) by electroporation (30,31). Transformants were 10% fetal bovine serum and 0.8 mg / ml G418 (GIBCO, Grand Island, NY (Gra
nd Island NY)) and selected in RPMI 1640 medium. Chimeric antibodies in the growth medium were detected by enzyme-linked immunosorbent assay (ELISA) method (16,27),
Antibody-producing cells were collected.

(D) Isolation of chimeric antibody: The antibody-producing cells were protein A-sepharose CL-4B.
Protein A bound bovine immunoglobulin was pre-removed by affinity chromatography using (Pharmacia, Uppsala, Sweden) 1.0
Proliferated in RPMI 1640 medium supplemented with% fetal bovine serum (32). Purification of the antibody is done by Protein A-Sepharose CL-
Affinity chromatography on 4B was used as previously reported (33).

(E) SDS-PAGE: Denaturing gel electrophoresis was performed by the method of Laemmli (34) using a 4-20% polyacrylamide linear gradient gel, and the gel was stained with 0.05% Coomassie Brilliant Blue.

(F) Antibody-dependent cytotoxicity test (Antibody-dependen
Cell-mediated Cytolysis): Mononuclear cells from peripheral blood of normal volunteers were used as a source of effector cells. Target cells were labeled with ⁵¹ Cr as previously reported (17). Cell suspensions (100 μl) containing 10 ⁴ labeled target 2780 ^AD cells were incubated with various concentrations of monoclonal antibody in 96-well microculture plates for 30 minutes at 37 ° C. 100 μl of cell suspension containing effector mononuclear cells was then added to each well. The plates were incubated for 6 hours at 37 ° C in a humidified 5% CO ₂ atmosphere. Undissolved 2780 ^AD was removed by centrifugation and the supernatant 100
Radioactivity in μl was counted with a gamma counter. The measurement was performed 3 times. Percentage specific cell lysis was calculated from ⁵¹ Cr release of test and control samples as follows. %
Specific release amount = [(E−S) / (M−S)] × 100, where E
= Experimental release (cpm of supernatant from target cells incubated with effector cells and experimental antibody), S =
Spontaneous release (cpm of supernatant from target cells incubated in medium only), and M = maximum release (cpm released from target cells lysed with 1% Triton X-100).

[2] Experimental Example Using the materials and methods of [1], a chimeric antibody was prepared and a confirmation test of its properties was performed below.

1) Preparation of chimeric antibody (a) Construction of chimeric H chain gene: The variable region gene of mouse H chain was excised from MRK16 producing hybridoma cells as a 3.0-Kb fragment by EcoRI and subcloned into λgt10. This was identified by hybridizing with a mouse J _H probe. The variable region gene was rearranged in J3 segment (VDI). The mouse variable region gene thus obtained was used as an EcoR I fragment for constructing a chimeric H chain gene (1A
Figure). The mouse variable region gene was ligated to the 5'site of the human constant region in the same transcription direction to construct pSV2-VH16-HG1gpt (Fig. 1A).

(B) Construction of chimeric L chain gene: The L chain variable region gene was excised from the genomic DNA of the MRK16 producing hybridoma as a 11-kb BamHI fragment and subcloned into λEMBL3. This was identified by hybridizing with a mouse _Jκ probe. The variable region gene was rearranged in the L1 segment. pBluescriptSKM
13 ⁺ (Stratagene, La Joya
lla), California) and multiple cloning sites were cut out and inserted into the HindIII site of pSV2HCκneo (Fig. 1B). Then, the mouse variable region gene of 7-kb
It was trimmed to a BamHI-XbaI fragment and subcloned into the BamHI-XbaI site of pBluescripsSKM13 ⁺ . The resulting 7-kb fragment of the mouse variable region was cleaved by Not I / Sal I digestion to generate pSV2HC
It was cloned into the multiple cloning site of κneo. Thus, the mouse L chain variable region gene was ligated in the same transcriptional direction to the 5'site of the human constant region to obtain pSV2-Vκ16-HCκ.
neo was constructed (Fig. 1B).

(C) Quantitative transformation of mouse myeloma cells: SP2 / 0, a non-producer mouse myeloma cell line, was cotransfected with the chimeric H chain and L chain genes using the electroporation method (30, 31).
Transformed cells were selected with G418. The resulting stable transformants, obtained about 2 weeks after electroporation, were screened to select clones producing antibodies specific for the multidrug resistant cell line K562 / ADM. Screening was performed by enzyme-linked immunosorbant assay using parental K562 cells (adriamycin sensitive strain) as a negative control (16). Two types of positive clones producing chimeric antibody were established from 1 × 10 ⁷ cells. Of the recloned transformants, the product of the clone with higher production was named MH162, and it was used for further analysis.

SP2 / 0 transformants produced a sufficient amount of chimeric antibody (1% of protein A-binding immunoglobulin removed)
5-10 μg / ml in medium supplemented with serum). Chimeric antibody (MH
162), the apparent affinity for K562 / ADM cell antigen is
The affinity was similar to that of the mouse antibody (MRK16) as measured by the enzyme-linked immunosorbent assay method.

2) Properties of chimeric antibody (a) Test for antibody specificity: The specificity of the antibody (MH162) was tested by enzyme-linked immunosorbent assay (data not shown). The multidrug resistant cell lines K562 / ADM and 2780 ^AD conjugated to the chimeric antibody almost to the same extent as the original mouse MRK16. On the other hand, the parent drug-sensitive system 562 and A2780 both did not bind. Therefore, this MH162 is MRK16 (16,35)
It has the same binding specificity as.

(B) SDS-PAGE analysis of chimeric antibody: The chimeric antibody MH162 was purified by one-step affinity chromatography using protein A-Sepharose until it became apparently homogeneous (Fig. 2). 0.5 μg of each of the monoclonal antibodies MRK16 (lane 1) and MH162 (lane 2) was subjected to SDS-PAGE analysis (A) with or without mercaptoethanol treatment (B). Molecular weight markers were obtained from Amersham, Japanese.

(C) Antibody-dependent cytotoxicity test using chimeric antibody: Chimera MH162 was used in the antibody-dependent cytotoxicity test (ADSS) analysis method using human mononuclear cells as effectors. FIG. 3 shows the findings of the experiment in which 2780 ^AD cells were exposed to various field doses of antibody 1 μg / ml and human effector cells. Chimera MH162 has a ratio of effector to labeled cells of 1
Although 0: 1 caused significant cytotoxicity, the level of ADCC activity of mouse MRK16 monoclonal antibody was insignificant. Cells from the parental A2780 line are chimeric MH162 or mouse
It was not lysed by MRK16 (data not shown). Here, ADCC is 1 μg / ml MH162 (●), 1 μg
Performed as described in Materials and Methods with / ml MRK16 (▲) or without monoclonal antibody (■). The measurement was performed 3 times.

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[Brief description of drawings]

FIG. 1 shows plasmids pSV2-VH16-HG1gpt (A) and pSV2-Vκ16-HCκneo (B).
It is an explanatory view showing the construction of, and the abbreviations in the figure are as follows. P = promoter; En = enhancer; Ori = pBR322 ori; Amp = β-lactamase; SV40 = SV40 promoter; polyA = polyA attachment signal; MCS = multicloning site; VDJ3 = mouse heavy chain variable region-encoding gene J4 = gene encoding the J4 region of mouse H chain; VJ1 = gene encoding the variable region of mouse L chain; J2-5 = gene encoding the J2-5 region of mouse L chain; Cκ = mouse or human Gene encoding the constant region of the L chain; Cγ1 = gene encoding the constant region of the human H chain; Ecogpt = gene encoding the xanthine-guanine phosphoribosyl transferase of E. coli; neo = gene encoding Tn5 neomycin resistance; MCS ( pBluecript SK +) = plasmid pBluecript SK +
Restriction enzyme: E = EcoR I; B = BamHI; H = Hind III: X = Xba I. FIG. 2 is a copy of the migration pattern showing the results of SDS-PAGE analysis of the mouse-human chimeric antibody MH162. FIG. 3 is a graph showing the activity of MH162 or MRK16 antibody-dependent cellular cytotoxicity test on 2780 ^AD cells. Bars represent standard deviation (SD). FIG. 4 is an explanatory diagram showing the amino acid sequence (a to b) of the variable region of the MRK16-derived H chain and the base sequence encoding the same. FIG. 5 is an explanatory diagram showing the amino acid sequence (a to b) of the variable region of the L chain derived from MRK16 and the nucleotide sequence encoding the same.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // G01N 33/574 9162-4B C12N 15/00 ZNAC 33/577 9281-4B 5/00 B ( (72) Inventor Hirofumi Hamada 3-46-5-206 Takinogawa, Kita-ku, Tokyo (72) Inventor Yoshikazu Kurosawa 1-39 Ogimachi, Meito-ku, Nagoya, Aichi (56) References JP-A-62-61596 (JP, A) JP-A-61-47500 (JP, A)

Claims (2)

(57) [Claims] 1. A hybridoma MRK16 or MRK17 produced by fusing splenocytes obtained from a mouse immunized with an adriamycin-resistant strain K562 / ADM of human myeloid leukemia cell line K562 with mouse myeloma cells. A multi-drug resistant cell line K562 / ADM consisting of a variable region having an amino acid sequence substantially homologous to a variable region in a mouse monoclonal antibody and a constant region having an amino acid sequence substantially homologous to a constant region in human immunoglobulin. A chimeric antibody against drug-resistant cancer, which has a binding specificity with. 2. The method for producing a chimeric antibody according to claim 1, which comprises the following steps (a) to (f). (A) A constant region in the H chain of human immunoglobulin and a constant region in the H chain of a human immunoglobulin are located downstream of the gene encoding an amino acid sequence substantially homologous to the variable region in the H chain of MRK16 or MRK17, which is a mouse monoclonal antibody against drug-resistant cancer. To prepare a DNA chain having a nucleotide sequence encoding a chimeric H chain by linking the transcriptional upstream side of a gene encoding a homologous amino acid sequence. (B) a constant region in the L chain of human immunoglobulin and a constant region downstream of the transcription of a gene encoding an amino acid sequence substantially homologous to the variable region in the L chain of MRK16 or MRK17 which is a mouse monoclonal antibody against drug-resistant cancer To prepare a DNA chain having a nucleotide sequence encoding a chimeric L chain by ligating the transcription upstream side of a gene encoding a homologous amino acid sequence. (C) Inserting each of the DNA chains obtained in steps (a) and (b) into the same or different expression vector in a state in which the genetic information can be expressed to prepare a recombinant DNA. (D) Transforming a host cell with the recombinant DNA obtained in step (c) to prepare a transformant. (E) Culturing the transformant obtained in step (d) to produce in the culture a chimeric antibody against a drug-resistant cancer having a binding specificity with the multidrug-resistant cell line K562 / ADM. (F) If necessary, recovering the chimeric antibody produced in the culture in step (e).