CA2025695A1 - Chimeric antibody against drug-resistant cancers and process for production thereof - Google Patents
Chimeric antibody against drug-resistant cancers and process for production thereofInfo
- Publication number
- CA2025695A1 CA2025695A1 CA002025695A CA2025695A CA2025695A1 CA 2025695 A1 CA2025695 A1 CA 2025695A1 CA 002025695 A CA002025695 A CA 002025695A CA 2025695 A CA2025695 A CA 2025695A CA 2025695 A1 CA2025695 A1 CA 2025695A1
- Authority
- CA
- Canada
- Prior art keywords
- chain
- human
- chimeric
- chimeric antibody
- mouse
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Abstract A monoclonal antibody, MRK16, reactive to the multidrug transporter P-glycoprotein, has been generated in an effort to devise an effective treatment for human drug-resistant cancers.
The monoclonal antibody inhibited the growth of human drug-resistant tumor cells in a xenograft model, suggesting its potential usefulness in the immunotherapy of drug-resistant cancers. A recombinant chimeric antibody has been developed by joining the antigen-recognizing variable regions of MRK16 to the constant regions of human antibodies. When human effector cells were used, the chimeric antibody MH162 was more effective in killing drug-resistant tumor cells than the all-mouse monoclonal MRK16. The chimeric antibody against the multidrug transporter P-glycoprotein will be a useful agent in immunotherapy of human drug-resistant cancers.
The monoclonal antibody inhibited the growth of human drug-resistant tumor cells in a xenograft model, suggesting its potential usefulness in the immunotherapy of drug-resistant cancers. A recombinant chimeric antibody has been developed by joining the antigen-recognizing variable regions of MRK16 to the constant regions of human antibodies. When human effector cells were used, the chimeric antibody MH162 was more effective in killing drug-resistant tumor cells than the all-mouse monoclonal MRK16. The chimeric antibody against the multidrug transporter P-glycoprotein will be a useful agent in immunotherapy of human drug-resistant cancers.
Description
~ HOE 90/5 013 ~2~
Chimeric antibody agains~ drug-resistant cancers and process for production thereof (1) Applied fields in industry:
This invention relates to a chimeric antibody against drug-resistant cancers and a process for its produ~ion. More s~ecifically, the invention relates to a chimeric antibody comorising variable (V) regions of murine monoclonal antibody and constant (C) regions of human immunoglobulin, and a process for producin~ the chimeric antibody.
(2J Technological bac~ground:
The resistance of tumors to a vari~ty of chemotherapeutic agents presents a major problem in the treatment of cancer.
Tumor cells can acqui~e resistance to such agent~ d3 doxorubicin (adriamycin), vinc~ alkaloids and actinomyci~ D following treatment with a single drug (1,2). The yen~ responslb1e for multidrug resi~tanc~, termed mdr, encodes a membrane glycoprotein (P-glycoprotain) that acts as a pump to t~an~port various cytotoxic drug out of the c~ 3). The P-glycoprotein has been shown to bind alticancer~dru~s (4,5), and to b- an ~TPase (6,7J
localized at the pl sma membrane of resista~t c~lls 18,9~. The transfection of cloned mdr sequences confe~rs mu1tld~ug resistance on sensitive cells (10-12).
~ he amount o~ P-glycoprotein expressed hdS b-en~ measur~d Ln :
..
- : . . . ~
æ~%~
eumor samples, and was found to be increased in intrinsically d~ug-resistant cancers of the colon, kidney and adrenal gland as well as in some tum~rs that acquired drug resistance following chemotherapy (13-15). Sinee P-glycoprotein appears to be involved in both acquired multidrug resistance and intrinsic drug resistance in human cancer, the selective killing of tumor cells that express P-glycoprotein could be very important for cancer therapy.
In an effort to devise an effective treatment for human drug-resistant cancers, the present inventors developed monoclonal antibodies reactive to ~he multidrug transporter P-glycoprotein (16). The monoclonal antibodies, given intravenously, effectively prevented tumor development in athymic mice inoculated subcutaneously with drug-resi~tant human ovarian cancer cells (17). Treatment with one of the monoclonal antibodies, MRK16, induced rapid regression of established subcutaneous tumors and produced cuxes in some animals. These monoclonal antibodies may have potential as treatment tools against multidrug-resistant human tumors that posse~s P-glycoprotein (17). A patent application ha~ been filed by the present inventors for monoclonal antibodies against drug-resistant cancers, including th~se monoclonal antibodies (see Japanese Patent Application No. 201445/1985, Japanese Laid-Open ~ ~
Patent Publication No. 61596/1987). ~ .
The mouse antibodies as foreign proteins, howevex, may 0voke j:
cou~teracting immune reactions that could destroy their effectiveness, and may also cause allergic reactions in the patients (18,19).
It has long been hoped that monoclonal aneibodies directed . 2 .
.
: " ',- ' '~' ~ , ' ~ ' , , ~ ~ 2 ~
~gainst ~umor cell surfaces could be employed in cancer therapy (36). In some cases, monoclonal antibodies alone can inhibit the growth o~ tumor cells (17,37-39), whereas in other systems, inhibit~on of tumor growth can be achieved by the antibodies com~lexed with various toxic substances (40-42). Monoclonal antibodies can also be used in vitro to eradicate residual malignant cells (43). Although such treatments have encountered numerous difficulties (36), some recent successes have been reported (44), and immunotherapy will be of great clinical value in the future.
A major limitation on the clinical use of murine-derived monoclonal antibodies is an immune response elicited against foreign protein, which may render the antibody ineffective and also harm the patient (18,19,36). Furthermore, mouse monoclonal antibodies may interact less efficiently with human effector cells that mediate tumor destruction. Although treatment with human monoclonal antibodies is under investigation, human hybridoma cell lines are largely unavailable, and where they do exist, are usually uns~able and produce sma}l amounts of immunoglobulin (45J.
(3) Outline of the invention:
This invention has been accomplished to solve the above-mentio~ed problems, and aims to provide an antibody sp~cific for multidrug-reslstant human cancers and possessing low immunogenicity.
' ~ ~
~ 3 ~ ~
' , .
Chimeric antibody agains~ drug-resistant cancers and process for production thereof (1) Applied fields in industry:
This invention relates to a chimeric antibody against drug-resistant cancers and a process for its produ~ion. More s~ecifically, the invention relates to a chimeric antibody comorising variable (V) regions of murine monoclonal antibody and constant (C) regions of human immunoglobulin, and a process for producin~ the chimeric antibody.
(2J Technological bac~ground:
The resistance of tumors to a vari~ty of chemotherapeutic agents presents a major problem in the treatment of cancer.
Tumor cells can acqui~e resistance to such agent~ d3 doxorubicin (adriamycin), vinc~ alkaloids and actinomyci~ D following treatment with a single drug (1,2). The yen~ responslb1e for multidrug resi~tanc~, termed mdr, encodes a membrane glycoprotein (P-glycoprotain) that acts as a pump to t~an~port various cytotoxic drug out of the c~ 3). The P-glycoprotein has been shown to bind alticancer~dru~s (4,5), and to b- an ~TPase (6,7J
localized at the pl sma membrane of resista~t c~lls 18,9~. The transfection of cloned mdr sequences confe~rs mu1tld~ug resistance on sensitive cells (10-12).
~ he amount o~ P-glycoprotein expressed hdS b-en~ measur~d Ln :
..
- : . . . ~
æ~%~
eumor samples, and was found to be increased in intrinsically d~ug-resistant cancers of the colon, kidney and adrenal gland as well as in some tum~rs that acquired drug resistance following chemotherapy (13-15). Sinee P-glycoprotein appears to be involved in both acquired multidrug resistance and intrinsic drug resistance in human cancer, the selective killing of tumor cells that express P-glycoprotein could be very important for cancer therapy.
In an effort to devise an effective treatment for human drug-resistant cancers, the present inventors developed monoclonal antibodies reactive to ~he multidrug transporter P-glycoprotein (16). The monoclonal antibodies, given intravenously, effectively prevented tumor development in athymic mice inoculated subcutaneously with drug-resi~tant human ovarian cancer cells (17). Treatment with one of the monoclonal antibodies, MRK16, induced rapid regression of established subcutaneous tumors and produced cuxes in some animals. These monoclonal antibodies may have potential as treatment tools against multidrug-resistant human tumors that posse~s P-glycoprotein (17). A patent application ha~ been filed by the present inventors for monoclonal antibodies against drug-resistant cancers, including th~se monoclonal antibodies (see Japanese Patent Application No. 201445/1985, Japanese Laid-Open ~ ~
Patent Publication No. 61596/1987). ~ .
The mouse antibodies as foreign proteins, howevex, may 0voke j:
cou~teracting immune reactions that could destroy their effectiveness, and may also cause allergic reactions in the patients (18,19).
It has long been hoped that monoclonal aneibodies directed . 2 .
.
: " ',- ' '~' ~ , ' ~ ' , , ~ ~ 2 ~
~gainst ~umor cell surfaces could be employed in cancer therapy (36). In some cases, monoclonal antibodies alone can inhibit the growth o~ tumor cells (17,37-39), whereas in other systems, inhibit~on of tumor growth can be achieved by the antibodies com~lexed with various toxic substances (40-42). Monoclonal antibodies can also be used in vitro to eradicate residual malignant cells (43). Although such treatments have encountered numerous difficulties (36), some recent successes have been reported (44), and immunotherapy will be of great clinical value in the future.
A major limitation on the clinical use of murine-derived monoclonal antibodies is an immune response elicited against foreign protein, which may render the antibody ineffective and also harm the patient (18,19,36). Furthermore, mouse monoclonal antibodies may interact less efficiently with human effector cells that mediate tumor destruction. Although treatment with human monoclonal antibodies is under investigation, human hybridoma cell lines are largely unavailable, and where they do exist, are usually uns~able and produce sma}l amounts of immunoglobulin (45J.
(3) Outline of the invention:
This invention has been accomplished to solve the above-mentio~ed problems, and aims to provide an antibody sp~cific for multidrug-reslstant human cancers and possessing low immunogenicity.
' ~ ~
~ 3 ~ ~
' , .
2~3~
The invention will no~ be described with particular reference to the drawing~, in which:
F~g. l is an explanatory view showins the construction of plasmid DS~2-VHl6-HGlgpt (A) and pSV2-V~16-HCkneo ~3), in which the abbreviations are as follows:
P = promoter, En = enhancer, Ori - pBR322 ori, ~mp = B-lac~amase, SV40 = SV40 promoter, PolyA = poly A sequence addition signal, MCS = multicloning site, V~J3 = gene coding for the ~ariable region of mouse H
chain, J4 ~ gene coding ~or the J4 region of mouse H chain, VJl = gene coding for the variable region of mouse L
chain, J2-5 ~ gene coding ~or the J2-5 region of mouse L chain, Ck ~ gene coding for the constan~ region of mouse or human L chain, Crl ~ gene coding for the oonstant region o~ human H
chain, Ecogpt 8 gene coding for th~ xanthine guanine phosphoribosyl trans~erase o~ . eoli, n~o ~ gene coding for Tn5 neomycin resistanc~, MCS(pBluecript SK~) ~ multicloning ~ite of plasmid pBluecript SX~
.
., . .
- ~ , Restrictlon enzymes : E = EcoRI
B = BamHI
H = HindIII
X = XbaI
Fig. 2 illustrates the SDS-PAGE patterns of mouse-human chimeric antibody MH162 in comparison with those of monoclonal antibody MRK16.
Fig. 3 is a graph showing the antibody-dependent cell-mediated cytotoxicity activity of MH162 or MRK16 against 2780AD cells. The bars in Fig. 3 represent standard deviations ~SD).
Fig. 4 is an explanatory view showing the amino acid sequence ~from a to b) of MRK16 derived H chain variable region, and the base sequence coding for it.
Fig. 5 is an explanatory view showins the amino acid sequence (from a to b) of MRK16-derived L chain variable region, and the base sequence coding for it. ~ -.
One approach to circumvent the antigenicity of a murine monoclonal antibody in humans is to COnstruGt murine V~human C
chimeric immunoglobulins. Since most immunoglobulin antigenicity :: :
,: . - . ,: ~ . - i.
resides in the C Jomain, creation of murine V/human C chimeric immunoglobulin should result in an antibody that has the specificity of a murine monoclonal antibody, but does not ellcit a human immune response (20, 46, 47). Furthermore, such chimeric proteins may interact more e~fectively with the human cellular i.~mune system by virtue of their human C domain, and thus provide more beneficiaL therapy ~han would the corresponding murine antibody (48)~ Mouse-human chimeric antibodies were shown to retain their ability ~o react with hapten antigens (49-51) as well as some carcinoma-assoclaeed antigens (52-55)~ Some ~rials in cancer immunotherapy using these chimeric antibodies are now in progress (56).
Recombinant chimeric antibodias in which th~ antigen-recognizing variabl~ (V) region~ of the monoclonal antibody MRK16 arQ ~oined to the con~tant (C) region~ of human antibodie~
(20,21) were con~tructed. When human effector cell~ were used~
the chimeric antibody was much more effective in killing drug-resistant tumor cell~ than the all-mous~ NRR16, as determined by antibody-dependent cell-medLated cytotoxicity. Ba~ed on thi~
~ind~ng, the pre~ent invention ~a~ accomplished.
In detail, the chimeric antibody against drug-resistant cancers in accordance with this inventio~ comprises varia~le regions having amino acid sequen~es sub tantially homologous with the variable regions of mouse monoclonal antibody agains~
drug-resistant cancers, and constant regions having amino acid sequ~nces substanti~lly homologous with the con~ nt regions of human immunoglobulin.
The process for preparing the chimeric antibody against drug-resistant c~ncers in accordance wi~h thi~ in~e~tion : . . :
~ 3 comprises the following steps (a) to (f):
(a) joining an upstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the constant region of the H chain of human immunoglobulin, to a downstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the variable region of the H chain of mouse monoclonal antibody against drug-resistant cancers, to prepare a DNA chain having a base sequence codin~ for a chimeric ~ chain, (b) joining an upstream site of transcription of a gene coding for an amino acid seguence substantially homologous with the constant region of the L chain of human immunoglobulin, to a downstream site of transcription of a gene coding for an amino acid sequence substantially homologous with ~he variable region o~ the L chain of mouse monoclonal antibody against drug-resistant cancers,..to prepare a DNA chain having a base sequence coding for a chimeric L chain, (c) incorporating each of the DNA chains obtained in the steps (a) and (b)~into the same or different expression vector(s) capable of expressing the relevant genetir info~mation, thereby to co~5truct r~combinant DNAs, (d) transforming a host cell with the recombinant DNAs obtained in the step (c~, to prepare a tr~nsformant, ~e) culturing the transformant obtained in the st~p (d), to produce a chimeric antibody a~ainst drug-resistant cancers in the cultured medium, and (f) collecting, if desir*d, th* chim*ric antibody produced C~7J~
in the cultured medium in the step (e).
(4) Effects of the invention:
The chimeric antibody in accordance with this in~ention is capable of selectively inhibiting the growth of cancer cells showing multidrug resistance, or has the ability to enhance their sen~itivity to drugs. The chimeric antibody is also characterized in that it has very low immunogenicity since its constant region is a C region of human origin, meaning that it minimally elicits a human immune response.
The chimeric antibody in accordance with this invention, therefore, can be an excellent means to attain the important goal of establishing a drug or method which produces few adverse reactions, has high selectivity, and is effective against cancer cells with multidrug resistance.
~5) Chimeric antibody:
The chimeric antibody in accordance with this invention, as mentioned previously, comprises variable (V) regions having amino acid sequences substantially homologous with the variable regions of mouse monoclonal antibody against drug~resistant cancers, and constant (C) regions ha~ing amino acid sequences ~ubstantially homologous with the constant regions o human immunoglobulin.
Basically, the chimeric antibody belongs to IgG, and has a structure in which two homologous H (hea~y) chains and two homologous L (lightl chains, each chain composed of a variable region and a constant region, are linked together by~disulfide bonds.
In the description herein, the monoclonal antibody agaInst 6 : ~ ;
:
~ . , , . . . , .. .... : .. . :: . . . ~
drug-resistant cancers, a source of the variable regio~ r~ ers specifically to that monoclonal antibody against the aforementioned P-glycoprotein which ful~ills ~he following requirements (a) to (d) and which is described in the specification of Japanese Patent Application No. 201445/19a5:
~a) To be produced by a hybridoma formed by ~using a mouse myeloma cell with a spleen cell from a mouse immunized with the adriamycin-resistant strain K562~ADM of humen myelogenous leukemia cell line ~552:
(b) To ha~e the ability t~ recognize an adriamycin-resistant strain specifically, (c) To be capable of inhibiting the cell growth of an adriamycin-resistant strain o~ enhancin~ i~s sensitivity eo vincristine or actinomyoin D; and (d) To belong to the IgG isotype.
Examples o~ such monoclonal antibody are MRX16 wi~h IgG2a as isotype, and MRK17 with IgGl as isotype.
Hybridoma MRK16 and MRK17 that produce monoclonal antibody MRK16 and MRX17 were deposited with the Ferment tion Research Institute, Agency of Industrial Science and Technology as FERM
BP-2200 and FERM BP-2201.
Monoclonal antibody MRK16 and MRX17 havo a seloctive aotion to inhibit the multiplication o~ drug-resistant hum~n c~ncer cells and a selective action to enhanc~ the drug ~ensitivlty of those cell~ (see tho aforementioned Japanese P~t~nt Applic~tion No. 201445/1985).
The amino acid sequences o~ the H- and ~-chain va~iable reg~ons o~ monoclonal antibody MRK16, and the b~g~ seguences - . . - - .
.:
2 ~
coding for them are shown in Figure 4 (from a to b) and Figure S
(from a to b), respectively.
The variable region referred to in ~his invention includes not only the above-mentioned variable region of mouse monoclonal antibody, but a variable region whose polypeptide is modified (subctituted, deleted or added) at some of the amino acid sequence, as far as the polypeptide has a specific antigen-binding capacity as mouse monoclonal antibody.
The human immunoglobulin, the source of the above constant region, arises upon a human immune response, and its fundamental units are polypeptide molecules comprising two homologous H
chains and two homologous L chains joined together by disulfide bonds. The gene sequence and amino acid sequence for the H chain constant region of human immunoglobulin have alraady been published (see IgM (62), IgD (63), IgGl (64), IgG2 (65), IgG3 (66), IgG4 (67), IgE (68) and IgA (69))~ The gene sequence and amino acid sequence of the constant region of the L chain (kappa type) are also of prior public knowledge (70).
The constant region referred to in this invention includes not only the abo~e-mentioned constant region of human immunoglobulin, but a constant region whose polypept~de is substituted, deleted or added at some of the amino acid sequence, as far as the poIypeptide has physiological ~unction (e.g.
complement-binding capacity) as the conistant region o~ human immunoglobulin.
Hence, examples of the chimeric antibody in accordance with this invention are an antibody prepared by joining constant regions having amino acid sequences substantially homologous with the constant regions of human IgGl, to variable regions having R
. ~ . . .. . . . .
~ ~ 2 ~
~mino acid sequences substantially homologous with the variable regionS o~ mouse monoclonal antibody MRK16; and an antibody prepared by joining constant regions having amino acid sequences substantially homologous with the constant r~gions of human IgGl, to variable regions having amino acid sequences substantially homologous with the variable regicns of mouse monoclonal antibody MRK17.
(6) Production of chimeric antibody:
The process for producing the chimeric antibody in accordance with this invention is characterized by including the following steps (a) through (f), as has been described hereinbe ore:
(a) joining an upstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the constan~ region of ~he H chain of human immunoglobulin, to a downstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the variable region of the H chain of mouse monoclonal antibody against drug-resistant cancers, to prepare a DNA chain ha~ing a ba e sequence coding ~or a chimeric H chain, (b) joining an upstream site of transcription of a gene coding for an amino acid sequence substantiall~
homologous with the constant region of tha L chain of human immunoglobulin, to a downstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the variable .~egion of the L chain of mouse monoclonal antibody :
..
- . :
.
, J''~ J''.~
against drug-resistant cancers, to prepare a DNA chain having a base sequence coding for a chimeric L chain, (c) incorporating each of the DNA chains obtained in the steps (a) and (b) into the same or different expression vector(s) capable of expressing the relevant genetic information, thereby to construct recombinant DNAs, td) transforming a host cell with the recombinant DNAs obtained in the step (c) to prepare a transformant, (e) culturing the transformant obtained in the step (d), to produce a chimeric antibody against drug-resistant cancers in the cultured medium, and ~f] collecting, if desired, the chimeric antibody produced in the cultured medium in the step (e).
ThP chimeric antibody of this invention can basically be produced by obtaining genes, which code ~or the variable regivns of the H and L chains of mouse monoclonal antibody, from a suitable gene source of murine origin by collection, cloning and the use of a suitable restriction enzyme; obtaining genes, which ~-code for the constant regions of the H and L chains of human immunoglobulin, from a suitable gene source of human origin in a similar manner; joining together both gene fragments for the variable regions and constant region~ by means of a ligase to :~
construct chimerlc H and L chain genes; introducing both~genes into a host cell, with the genes connected to the same or di.ferent suitable expression vector(s), to form a transformant;
and culturing the trans~ormant.
~` Such chimeric antibody, chimeric protein or recombinant protein can be produced by reference to literature on k~own recombination techniques i~ the releva~nt fieLds, such:as Maniatls 1 0: : ::
:
.
~ 3 t al., Molecular Cloning: A Laboratory manual, 2nd Ed. (1989) Co~d Spring Harbor Laboratory.
In this invention, the steps (a) and (c), or the steps (b) and (c), may be performed separately (namely, after a chimeric H
chain gene and a chimeric L chain gene are construc~ed, these genes may be inserted into expression vectors). A more preferred embodiment, however, would be ~o use in the steps (a) and (b) one of the two region genes, e~g. the gene coding for the constant region of human immunoglobulin, said gene having been connected with the expression vector of the ~tep (c). Since this procedure would result in the construction of recombinant DNAs in the steps ta) and (b), it would become unnecessary to carry out the step (c) in which the genes are inserted in different expression vectors to construct recombinant DNAs.
The process for production of the chimeric antibody in accordance with this invention will be described in greater detail below on the basis of preferred embodiments.
1) Construction of chimeric H chain gene and recombinant DNA
(i) Gene ~or variable region A gene coding for the variable region can be obtained by a customary method in gene engineering, e~g~, hybridization with a suitable probe, from the chromogene library of cells producing mouse monoclonal antibody against P glycoprotein, such as hybridoma MRK16 producing the aforementioned monoclonal antibody MRK16 or hybridoma MRK17 producing the monoclonal antibody MRK17 (both hybridomas deposited with the Fer~entation Research Institute as described horeinabove). A p~oferred probe is a ' , ,,, : ' , . .: - - : . . ~ , , mouse JH gene (a gene coding for the J region of the H chain variable region of mouse IgG) containing fragment (JH probe (26)).
(ii) Gene for constant region A gene coding for the constant règion can be obtained by preparing a gene library from human placental DNA, and performing a customary method in the field of gene engineering, such as hybridization with a suitable probe.
At least part of the chain length of the genes (i) and (ii) can be cnemically synthesized, if necessary, in accordance with the ordinary method of nucleic acid synthesis. These genes may be not only degenerate isomers different only in degenerative codons, but also genes having a bas~ sequence corresponding to the alteration (substitution, deletion ox addition) of the amino acid sequence of the polypeptide in each of the variable and constant regions; or their isomers.
When the gene (i) and the gene (ii) are joined together to construct a chimeric H chain gene, the use of the gene for the constant region connected with an expression vector is convenient for the preparation of a recombinant DNA as described previously.
The expression vector may be one capable of expressing the desired gen~s contained therein in a hos~ cel~, and gen~rally it is in the form of a plasmid. It should also hav~ a marker gene for the selection of a transformant (e.g. one concerned with drug resistance, auxotrophic properties, etc.).
A preferred example of the expression vector having the gene for the constant region of human origin joined thereto;~is ~ pSV2-HGl-gpt ~see the reference 24) whlch~has the Crl region of 12 ;
:
. .:, . - -, . - ~
~, ~ . , -: , : : . . , :
2 ~
uman origin.
~ A chimeric H chain gene and a recombinant DNA are constructed by joining an upstream site of transcription (5' site) of the expression vector-connected constant region gene (ii), to a downstream site of transcription (3' site) of the variable region gene (i) obtained from a gene library by use of a suitable restriction enzyme. It is possible to delete a suitable length from the connection end of the gene (i) or (ii), or add a suitable base sequence to that end, if this is necessary to secure the right base sequence. Fig. lA shows the preparation of the vector pSV2-VH16-HGlgpt for a chimeric antibody H chain by using pSV2-HGl-gpt having the Crl region of human origin as the above-mentioned expression vector, and joining a variabLe region gene of MR~-16 origin to the expression vec~or (see the Experimental Example ~2~
The expression vector should have a suitable promoter for expressing the genatic inormation of the chimeric H chain gene in host cells, namely, for transcribing its DNA to mRNA. In order to express a larger amount of antibody, the expression vector should also contain an enhancer.
When the gene coding for the H chain variable region of mouse monoclonal antibody against drug-resistant cancers is to be collected from genomic DNA, the gene may be cut out as a fragment containing a promoter residing upstream of the gene and an enhancer downstream of the gene. The use of such a ~ragment is convenient, since it eliminates the need to incorporate the promoter and the enhancer sepaFately.
, . ~
, ~
~)J ~ ~,J ,'~
2) Construction of a chimeric L chain gene and a recombinant DNA
(i) Gene for variable region Specifically, the gene coding for the variable region can be obtained, for example, by hybridization wlth a suitable probe from the chromogene library of hybridoma MRK16 or MRK17, as described earlier. A preferred example of ~he probe is a mouse JK gene (a gene coding for the J region of the kappa type L chain variable region of mouse IgG) containing fragment (JK probe (25)).
(ii) Gene for constant region The gene coding for the L chain constant region can be obtained from the chromogene library of human placenta, as in the case of the H chain constant region.
At least part of the chain length of the genes (i) and (ii) can be chemically synthesized, as can that of the H chain gene 1) as aforementioned. These gen~s may be not only degenerate isomers, but also genes having a base seguence corresponding to the alteration ~substitution, deletion or addition) of the amino acid sequence of the polypeptide in each of the variable and constant regions; or their isomers.
When the gene (i) and the gene (ii) are joined toge~her to construct, a chimeric L chain gene, the use of the gene for the constant region connected with an expression vector is convenisn~
as in the case of a chimeric H chain gene.
A preferred example of the expre~ion vector having the gene ~or the constant region of human ori~in joined th~reto is pSV2-HCk-neo (see ~he reference 24) which has the Ck region of .
':
~:
.
2 ~ J '~
uman origin.
A chimeric L chain gene and a recombinant DNA are constructed by joining an upstream site of transcription (5' site) of the expression vector-connected constant region gene (ii), ~o a downstream site of transcription (3' site) of the ~ariable region gene (i) obtained from a gene library by use of a suitable restriction enzyme. A suitable length may be deleted from, or a suitablP base sequence added to, the connection end of the gene ~i) or (ii), if this is necessary to secure the right base sequence. Fig. lB shows the preparation of the vector pSV2-Vkl6-HCkneo for a chimeric antibody L chain by using pSV2-HCk-neo having the Ck region of human origin as the above-mentioned expression vector, and joining a variable region gene of MRK-16 origin to the expression vector (see the Experimental Example ~2~).
The expression Yector should have a suitable promoter for expressing the genetic information of the chimeric L chain gene in host cells. In order to express a lar~er amount of antibody the expression ~ector should also contain an enhancer.
When the gene coding for the L chain variable region of mouse monoclonal antibody against drug-resistant cancers is to be collected ~rom genomic DNA, it is convenient to cut out the gene as a fragment containing a promoter and an enhancer and use it.
It is also possible to use the enhancer located upstream of the gene coding for the human L chain constant regio~l.
The invention will no~ be described with particular reference to the drawing~, in which:
F~g. l is an explanatory view showins the construction of plasmid DS~2-VHl6-HGlgpt (A) and pSV2-V~16-HCkneo ~3), in which the abbreviations are as follows:
P = promoter, En = enhancer, Ori - pBR322 ori, ~mp = B-lac~amase, SV40 = SV40 promoter, PolyA = poly A sequence addition signal, MCS = multicloning site, V~J3 = gene coding for the ~ariable region of mouse H
chain, J4 ~ gene coding ~or the J4 region of mouse H chain, VJl = gene coding for the variable region of mouse L
chain, J2-5 ~ gene coding ~or the J2-5 region of mouse L chain, Ck ~ gene coding for the constan~ region of mouse or human L chain, Crl ~ gene coding for the oonstant region o~ human H
chain, Ecogpt 8 gene coding for th~ xanthine guanine phosphoribosyl trans~erase o~ . eoli, n~o ~ gene coding for Tn5 neomycin resistanc~, MCS(pBluecript SK~) ~ multicloning ~ite of plasmid pBluecript SX~
.
., . .
- ~ , Restrictlon enzymes : E = EcoRI
B = BamHI
H = HindIII
X = XbaI
Fig. 2 illustrates the SDS-PAGE patterns of mouse-human chimeric antibody MH162 in comparison with those of monoclonal antibody MRK16.
Fig. 3 is a graph showing the antibody-dependent cell-mediated cytotoxicity activity of MH162 or MRK16 against 2780AD cells. The bars in Fig. 3 represent standard deviations ~SD).
Fig. 4 is an explanatory view showing the amino acid sequence ~from a to b) of MRK16 derived H chain variable region, and the base sequence coding for it.
Fig. 5 is an explanatory view showins the amino acid sequence (from a to b) of MRK16-derived L chain variable region, and the base sequence coding for it. ~ -.
One approach to circumvent the antigenicity of a murine monoclonal antibody in humans is to COnstruGt murine V~human C
chimeric immunoglobulins. Since most immunoglobulin antigenicity :: :
,: . - . ,: ~ . - i.
resides in the C Jomain, creation of murine V/human C chimeric immunoglobulin should result in an antibody that has the specificity of a murine monoclonal antibody, but does not ellcit a human immune response (20, 46, 47). Furthermore, such chimeric proteins may interact more e~fectively with the human cellular i.~mune system by virtue of their human C domain, and thus provide more beneficiaL therapy ~han would the corresponding murine antibody (48)~ Mouse-human chimeric antibodies were shown to retain their ability ~o react with hapten antigens (49-51) as well as some carcinoma-assoclaeed antigens (52-55)~ Some ~rials in cancer immunotherapy using these chimeric antibodies are now in progress (56).
Recombinant chimeric antibodias in which th~ antigen-recognizing variabl~ (V) region~ of the monoclonal antibody MRK16 arQ ~oined to the con~tant (C) region~ of human antibodie~
(20,21) were con~tructed. When human effector cell~ were used~
the chimeric antibody was much more effective in killing drug-resistant tumor cell~ than the all-mous~ NRR16, as determined by antibody-dependent cell-medLated cytotoxicity. Ba~ed on thi~
~ind~ng, the pre~ent invention ~a~ accomplished.
In detail, the chimeric antibody against drug-resistant cancers in accordance with this inventio~ comprises varia~le regions having amino acid sequen~es sub tantially homologous with the variable regions of mouse monoclonal antibody agains~
drug-resistant cancers, and constant regions having amino acid sequ~nces substanti~lly homologous with the con~ nt regions of human immunoglobulin.
The process for preparing the chimeric antibody against drug-resistant c~ncers in accordance wi~h thi~ in~e~tion : . . :
~ 3 comprises the following steps (a) to (f):
(a) joining an upstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the constant region of the H chain of human immunoglobulin, to a downstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the variable region of the H chain of mouse monoclonal antibody against drug-resistant cancers, to prepare a DNA chain having a base sequence codin~ for a chimeric ~ chain, (b) joining an upstream site of transcription of a gene coding for an amino acid seguence substantially homologous with the constant region of the L chain of human immunoglobulin, to a downstream site of transcription of a gene coding for an amino acid sequence substantially homologous with ~he variable region o~ the L chain of mouse monoclonal antibody against drug-resistant cancers,..to prepare a DNA chain having a base sequence coding for a chimeric L chain, (c) incorporating each of the DNA chains obtained in the steps (a) and (b)~into the same or different expression vector(s) capable of expressing the relevant genetir info~mation, thereby to co~5truct r~combinant DNAs, (d) transforming a host cell with the recombinant DNAs obtained in the step (c~, to prepare a tr~nsformant, ~e) culturing the transformant obtained in the st~p (d), to produce a chimeric antibody a~ainst drug-resistant cancers in the cultured medium, and (f) collecting, if desir*d, th* chim*ric antibody produced C~7J~
in the cultured medium in the step (e).
(4) Effects of the invention:
The chimeric antibody in accordance with this in~ention is capable of selectively inhibiting the growth of cancer cells showing multidrug resistance, or has the ability to enhance their sen~itivity to drugs. The chimeric antibody is also characterized in that it has very low immunogenicity since its constant region is a C region of human origin, meaning that it minimally elicits a human immune response.
The chimeric antibody in accordance with this invention, therefore, can be an excellent means to attain the important goal of establishing a drug or method which produces few adverse reactions, has high selectivity, and is effective against cancer cells with multidrug resistance.
~5) Chimeric antibody:
The chimeric antibody in accordance with this invention, as mentioned previously, comprises variable (V) regions having amino acid sequences substantially homologous with the variable regions of mouse monoclonal antibody against drug~resistant cancers, and constant (C) regions ha~ing amino acid sequences ~ubstantially homologous with the constant regions o human immunoglobulin.
Basically, the chimeric antibody belongs to IgG, and has a structure in which two homologous H (hea~y) chains and two homologous L (lightl chains, each chain composed of a variable region and a constant region, are linked together by~disulfide bonds.
In the description herein, the monoclonal antibody agaInst 6 : ~ ;
:
~ . , , . . . , .. .... : .. . :: . . . ~
drug-resistant cancers, a source of the variable regio~ r~ ers specifically to that monoclonal antibody against the aforementioned P-glycoprotein which ful~ills ~he following requirements (a) to (d) and which is described in the specification of Japanese Patent Application No. 201445/19a5:
~a) To be produced by a hybridoma formed by ~using a mouse myeloma cell with a spleen cell from a mouse immunized with the adriamycin-resistant strain K562~ADM of humen myelogenous leukemia cell line ~552:
(b) To ha~e the ability t~ recognize an adriamycin-resistant strain specifically, (c) To be capable of inhibiting the cell growth of an adriamycin-resistant strain o~ enhancin~ i~s sensitivity eo vincristine or actinomyoin D; and (d) To belong to the IgG isotype.
Examples o~ such monoclonal antibody are MRX16 wi~h IgG2a as isotype, and MRK17 with IgGl as isotype.
Hybridoma MRK16 and MRK17 that produce monoclonal antibody MRK16 and MRX17 were deposited with the Ferment tion Research Institute, Agency of Industrial Science and Technology as FERM
BP-2200 and FERM BP-2201.
Monoclonal antibody MRK16 and MRX17 havo a seloctive aotion to inhibit the multiplication o~ drug-resistant hum~n c~ncer cells and a selective action to enhanc~ the drug ~ensitivlty of those cell~ (see tho aforementioned Japanese P~t~nt Applic~tion No. 201445/1985).
The amino acid sequences o~ the H- and ~-chain va~iable reg~ons o~ monoclonal antibody MRK16, and the b~g~ seguences - . . - - .
.:
2 ~
coding for them are shown in Figure 4 (from a to b) and Figure S
(from a to b), respectively.
The variable region referred to in ~his invention includes not only the above-mentioned variable region of mouse monoclonal antibody, but a variable region whose polypeptide is modified (subctituted, deleted or added) at some of the amino acid sequence, as far as the polypeptide has a specific antigen-binding capacity as mouse monoclonal antibody.
The human immunoglobulin, the source of the above constant region, arises upon a human immune response, and its fundamental units are polypeptide molecules comprising two homologous H
chains and two homologous L chains joined together by disulfide bonds. The gene sequence and amino acid sequence for the H chain constant region of human immunoglobulin have alraady been published (see IgM (62), IgD (63), IgGl (64), IgG2 (65), IgG3 (66), IgG4 (67), IgE (68) and IgA (69))~ The gene sequence and amino acid sequence of the constant region of the L chain (kappa type) are also of prior public knowledge (70).
The constant region referred to in this invention includes not only the abo~e-mentioned constant region of human immunoglobulin, but a constant region whose polypept~de is substituted, deleted or added at some of the amino acid sequence, as far as the poIypeptide has physiological ~unction (e.g.
complement-binding capacity) as the conistant region o~ human immunoglobulin.
Hence, examples of the chimeric antibody in accordance with this invention are an antibody prepared by joining constant regions having amino acid sequences substantially homologous with the constant regions of human IgGl, to variable regions having R
. ~ . . .. . . . .
~ ~ 2 ~
~mino acid sequences substantially homologous with the variable regionS o~ mouse monoclonal antibody MRK16; and an antibody prepared by joining constant regions having amino acid sequences substantially homologous with the constant r~gions of human IgGl, to variable regions having amino acid sequences substantially homologous with the variable regicns of mouse monoclonal antibody MRK17.
(6) Production of chimeric antibody:
The process for producing the chimeric antibody in accordance with this invention is characterized by including the following steps (a) through (f), as has been described hereinbe ore:
(a) joining an upstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the constan~ region of ~he H chain of human immunoglobulin, to a downstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the variable region of the H chain of mouse monoclonal antibody against drug-resistant cancers, to prepare a DNA chain ha~ing a ba e sequence coding ~or a chimeric H chain, (b) joining an upstream site of transcription of a gene coding for an amino acid sequence substantiall~
homologous with the constant region of tha L chain of human immunoglobulin, to a downstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the variable .~egion of the L chain of mouse monoclonal antibody :
..
- . :
.
, J''~ J''.~
against drug-resistant cancers, to prepare a DNA chain having a base sequence coding for a chimeric L chain, (c) incorporating each of the DNA chains obtained in the steps (a) and (b) into the same or different expression vector(s) capable of expressing the relevant genetic information, thereby to construct recombinant DNAs, td) transforming a host cell with the recombinant DNAs obtained in the step (c) to prepare a transformant, (e) culturing the transformant obtained in the step (d), to produce a chimeric antibody against drug-resistant cancers in the cultured medium, and ~f] collecting, if desired, the chimeric antibody produced in the cultured medium in the step (e).
ThP chimeric antibody of this invention can basically be produced by obtaining genes, which code ~or the variable regivns of the H and L chains of mouse monoclonal antibody, from a suitable gene source of murine origin by collection, cloning and the use of a suitable restriction enzyme; obtaining genes, which ~-code for the constant regions of the H and L chains of human immunoglobulin, from a suitable gene source of human origin in a similar manner; joining together both gene fragments for the variable regions and constant region~ by means of a ligase to :~
construct chimerlc H and L chain genes; introducing both~genes into a host cell, with the genes connected to the same or di.ferent suitable expression vector(s), to form a transformant;
and culturing the trans~ormant.
~` Such chimeric antibody, chimeric protein or recombinant protein can be produced by reference to literature on k~own recombination techniques i~ the releva~nt fieLds, such:as Maniatls 1 0: : ::
:
.
~ 3 t al., Molecular Cloning: A Laboratory manual, 2nd Ed. (1989) Co~d Spring Harbor Laboratory.
In this invention, the steps (a) and (c), or the steps (b) and (c), may be performed separately (namely, after a chimeric H
chain gene and a chimeric L chain gene are construc~ed, these genes may be inserted into expression vectors). A more preferred embodiment, however, would be ~o use in the steps (a) and (b) one of the two region genes, e~g. the gene coding for the constant region of human immunoglobulin, said gene having been connected with the expression vector of the ~tep (c). Since this procedure would result in the construction of recombinant DNAs in the steps ta) and (b), it would become unnecessary to carry out the step (c) in which the genes are inserted in different expression vectors to construct recombinant DNAs.
The process for production of the chimeric antibody in accordance with this invention will be described in greater detail below on the basis of preferred embodiments.
1) Construction of chimeric H chain gene and recombinant DNA
(i) Gene ~or variable region A gene coding for the variable region can be obtained by a customary method in gene engineering, e~g~, hybridization with a suitable probe, from the chromogene library of cells producing mouse monoclonal antibody against P glycoprotein, such as hybridoma MRK16 producing the aforementioned monoclonal antibody MRK16 or hybridoma MRK17 producing the monoclonal antibody MRK17 (both hybridomas deposited with the Fer~entation Research Institute as described horeinabove). A p~oferred probe is a ' , ,,, : ' , . .: - - : . . ~ , , mouse JH gene (a gene coding for the J region of the H chain variable region of mouse IgG) containing fragment (JH probe (26)).
(ii) Gene for constant region A gene coding for the constant règion can be obtained by preparing a gene library from human placental DNA, and performing a customary method in the field of gene engineering, such as hybridization with a suitable probe.
At least part of the chain length of the genes (i) and (ii) can be cnemically synthesized, if necessary, in accordance with the ordinary method of nucleic acid synthesis. These genes may be not only degenerate isomers different only in degenerative codons, but also genes having a bas~ sequence corresponding to the alteration (substitution, deletion ox addition) of the amino acid sequence of the polypeptide in each of the variable and constant regions; or their isomers.
When the gene (i) and the gene (ii) are joined together to construct a chimeric H chain gene, the use of the gene for the constant region connected with an expression vector is convenient for the preparation of a recombinant DNA as described previously.
The expression vector may be one capable of expressing the desired gen~s contained therein in a hos~ cel~, and gen~rally it is in the form of a plasmid. It should also hav~ a marker gene for the selection of a transformant (e.g. one concerned with drug resistance, auxotrophic properties, etc.).
A preferred example of the expression vector having the gene for the constant region of human origin joined thereto;~is ~ pSV2-HGl-gpt ~see the reference 24) whlch~has the Crl region of 12 ;
:
. .:, . - -, . - ~
~, ~ . , -: , : : . . , :
2 ~
uman origin.
~ A chimeric H chain gene and a recombinant DNA are constructed by joining an upstream site of transcription (5' site) of the expression vector-connected constant region gene (ii), to a downstream site of transcription (3' site) of the variable region gene (i) obtained from a gene library by use of a suitable restriction enzyme. It is possible to delete a suitable length from the connection end of the gene (i) or (ii), or add a suitable base sequence to that end, if this is necessary to secure the right base sequence. Fig. lA shows the preparation of the vector pSV2-VH16-HGlgpt for a chimeric antibody H chain by using pSV2-HGl-gpt having the Crl region of human origin as the above-mentioned expression vector, and joining a variabLe region gene of MR~-16 origin to the expression vec~or (see the Experimental Example ~2~
The expression vector should have a suitable promoter for expressing the genatic inormation of the chimeric H chain gene in host cells, namely, for transcribing its DNA to mRNA. In order to express a larger amount of antibody, the expression vector should also contain an enhancer.
When the gene coding for the H chain variable region of mouse monoclonal antibody against drug-resistant cancers is to be collected from genomic DNA, the gene may be cut out as a fragment containing a promoter residing upstream of the gene and an enhancer downstream of the gene. The use of such a ~ragment is convenient, since it eliminates the need to incorporate the promoter and the enhancer sepaFately.
, . ~
, ~
~)J ~ ~,J ,'~
2) Construction of a chimeric L chain gene and a recombinant DNA
(i) Gene for variable region Specifically, the gene coding for the variable region can be obtained, for example, by hybridization wlth a suitable probe from the chromogene library of hybridoma MRK16 or MRK17, as described earlier. A preferred example of ~he probe is a mouse JK gene (a gene coding for the J region of the kappa type L chain variable region of mouse IgG) containing fragment (JK probe (25)).
(ii) Gene for constant region The gene coding for the L chain constant region can be obtained from the chromogene library of human placenta, as in the case of the H chain constant region.
At least part of the chain length of the genes (i) and (ii) can be chemically synthesized, as can that of the H chain gene 1) as aforementioned. These gen~s may be not only degenerate isomers, but also genes having a base seguence corresponding to the alteration ~substitution, deletion or addition) of the amino acid sequence of the polypeptide in each of the variable and constant regions; or their isomers.
When the gene (i) and the gene (ii) are joined toge~her to construct, a chimeric L chain gene, the use of the gene for the constant region connected with an expression vector is convenisn~
as in the case of a chimeric H chain gene.
A preferred example of the expre~ion vector having the gene ~or the constant region of human ori~in joined th~reto is pSV2-HCk-neo (see ~he reference 24) which has the Ck region of .
':
~:
.
2 ~ J '~
uman origin.
A chimeric L chain gene and a recombinant DNA are constructed by joining an upstream site of transcription (5' site) of the expression vector-connected constant region gene (ii), ~o a downstream site of transcription (3' site) of the ~ariable region gene (i) obtained from a gene library by use of a suitable restriction enzyme. A suitable length may be deleted from, or a suitablP base sequence added to, the connection end of the gene ~i) or (ii), if this is necessary to secure the right base sequence. Fig. lB shows the preparation of the vector pSV2-Vkl6-HCkneo for a chimeric antibody L chain by using pSV2-HCk-neo having the Ck region of human origin as the above-mentioned expression vector, and joining a variable region gene of MRK-16 origin to the expression vector (see the Experimental Example ~2~).
The expression Yector should have a suitable promoter for expressing the genetic information of the chimeric L chain gene in host cells. In order to express a lar~er amount of antibody the expression ~ector should also contain an enhancer.
When the gene coding for the L chain variable region of mouse monoclonal antibody against drug-resistant cancers is to be collected ~rom genomic DNA, it is convenient to cut out the gene as a fragment containing a promoter and an enhancer and use it.
It is also possible to use the enhancer located upstream of the gene coding for the human L chain constant regio~l.
3) Transformation The so obtained chimeric H chain gene and L chain ~ene are introduced into suitable host cclls by a customary ~ , ~ . ' ' biotechnological technique to form a transformant, which can be made to produce the expression product of both chlmeric chain genes, i.e. a chimeric antibody in accordance with this invention.
Preferred as host cells for transformation are B
cell-derived tumor cells, myeloma cells, for the purpose of producing a large amount of antibody. ~he most effective examples of the myeloma cells are mutants of myeloma origin but not producing antibodies any more, such as NSl, P3Ul, and SP2/0.
The transformation of host cells by the expression vector havin~ the chimeric chain genes introduced therein, i.e.
recomb~nant DNAs, can be performed by any suitable methods in wide use in the gene engineering field. Preferred examples of such methods include transfection using calcium phosphate or calcium chloride, trans~ection by electroporation, or lipofection.
The transforman~ is the sam~ as the host cells used in terms of genotype, phenotype or microbiological properties, with the exception o~ new characters to be introduced by both chimeric chain genes (i.e. the ability to produce IgG chimeric chains), and the characters derived rom the vector used, as well as the possible omission of partial genetic information ~rom the vector at the time of gene recombination.
Preferred as host cells for transformation are B
cell-derived tumor cells, myeloma cells, for the purpose of producing a large amount of antibody. ~he most effective examples of the myeloma cells are mutants of myeloma origin but not producing antibodies any more, such as NSl, P3Ul, and SP2/0.
The transformation of host cells by the expression vector havin~ the chimeric chain genes introduced therein, i.e.
recomb~nant DNAs, can be performed by any suitable methods in wide use in the gene engineering field. Preferred examples of such methods include transfection using calcium phosphate or calcium chloride, trans~ection by electroporation, or lipofection.
The transforman~ is the sam~ as the host cells used in terms of genotype, phenotype or microbiological properties, with the exception o~ new characters to be introduced by both chimeric chain genes (i.e. the ability to produce IgG chimeric chains), and the characters derived rom the vector used, as well as the possible omission of partial genetic information ~rom the vector at the time of gene recombination.
4) Expr~ssion of chLmeric chain g0nes/production of chimeric antibody A chimeric antibody (IgG) is produced in the culture system (within cells and/or in medium) by culturing a clone of the transformant obtained in the above step.
. 16 `` : :
- :-. : - ~
The culture conditions for the transformant are essen2Qa~y~
thè same as for the host cells ~sed.
. 16 `` : :
- :-. : - ~
The culture conditions for the transformant are essen2Qa~y~
thè same as for the host cells ~sed.
5) Isolation of chimeric antibody The isolation of a chimeric antibody from the above culture can be performed in accordance with a customary method concerned with the isolation of protein or antibody. A preferred example of such a method is affinity chromatography o~er a protein A
Sepharose column (see the reference 33).
From the transformant ~hat has expressed the so produced chimeric antibody, it is possible to separate and purify m-RNA
coding for the chimeric antibody by a customary method. cDNA is prepared from the mRNA by a customary method, and a suitable promoter or enhancer region is incorporated into a site upstream of the cDNA codin~ for the chimeric antibody, whereby the chimaric antibody can be expressed and produced even in host cells other than myeloma cells, such as yeast, silkworm or plants.
As shown in the Experimental Examples l~ter, an expression vector including a chimeric mouse V/human C immunoglobulin gene was transfected into myeloma cells, with the result that a functional chimeric IgG with the sam~ affinity and binding specificity as those of the original hy~ridoma antibody was produced. This ChimeriG antibody was much lower ln immunogenicity than the all-mouse antibody. Furthermore, the human C region of the chimeric antibody permits the human effector function to work more effecti~ely.
. - ~ ~. .: :
.. . . .
- , . : ' : . ' ~ ~:.: ` ' .
? ~
Experimental Examples ~1~ Materials and MethOds (a) vectors, clones, probes and cells:
Hybridoma MRK16 (FERM-BP-2200) producing MRK was used as a source of a gene coding for the variable region of mouse monoclonal antibody against drug-sensitive cancers. The ~phage ~gtlO (22) was used as a subcloning vec~or including the mouse monoclonal antibody H chain variable region that had been cut out by EcoRI. ~EMBL3 (23) was used as a subcloning vector including the mouse monoclonal antibody L chain variable region cut out by 3amHI.
Mouse Jk gene-containing fragment (Jk probe) (25) isolated from clone Igl46 was used as a probe for the mouse monoclonal antibody L chain variable region. Mouse ~H probe isola~ed from MEP203 (26) was used as a probe for the mouse monoclonal antibody H chain variable region.
pSV2HGlgpt was used as an expression vector having a gene coding for the human IgG H chain constant region joined theretoO
pSV2HCkneo was used as an expression vector having a gene coding fo~ the human IgG L chain constant region joinsd thereto (24).
` Mouse myeloma Sp2/0 (Sp2/0-Agl4) obtained from ATCC
~Rockville, MD) was used as a host cell for the expression vector.
Human drug-resist~nt cell lines (~562/ADM a~d 27~0AD) and their parent drug-sensitlve cell lines (K562 and A2780~) for use in antibody-dependent cell-mediated cytotoxicity test were maintained as described previously (27).
la ~
,, ..
c~ J
(b) Cloning of size-fractionated DNA:
A fragment containing a gene coding fo~ the mouse monoclonal antibody H chain variable region was id~ntified in Southern hybridization of EcoRI-digested genomic DNA with JH probe. A
fragment containing a gene coding for th~ mouse monoclonal antibody L chain variable region was iden~ified in Southern hybridization of BamHI-diges~ed genomic DNA with J~ probe. These genes were confirmed to be rearranged.
The respective fragments were eluted from the relevant regions separated on agarose gel, ligated with ~gtlO and ~EMBL3 arms, and packaged into ~phage.
Plaque hybridization was carried out according to the Banton-Davis method (29).
(c) DNA transfection of mouse Sp2/0 myeloma cells:
200 ~g each of plasmids pSV2-VH16-HGlgpt and pSV2-VK16-HC~neo (see Fig. 1) were cotransfected into 107 mouse Sp2/0 cells ~CRL1581, ATCC) by electroporation (30,31). Transformants were selected in RPMI 1~40 medium supplemented with 10% fetal bovine serum and 0~8 mg/ml G418 (GI8CO, Grand Island, NY). A chimeric antibody in the growth medium ~as detected by enzyme-linked immunosorbent assaf (ELISA) to collect antibody-producing cells ~16,27).
(d) Isolation of chimeric antibody:
Antibody-producing cells were ~rown in RPMI 1640 medium supplemented with 1. 0% fetal bovine serum, which had been precleared of protein A-binding bovine immunoglobulin by af~inity chromatography using protein A-Sepharose CL-4 (Pharmacia, Uppsala, Sweden) ~32). Purification o~ the antibody was carried out as described previously ~33), using~protein A-Sepharose CL-4B
., . .- : . : .
~:., '. : '', : -, : . - . : .
)J ~ J ~J
arfinity chromatography.
(e) SDS-PAGE:
Denaturins gel electrophoresis was performed according to Laemmli ~34) with a 4-20% polyacrylamide linear gradient gel, and gels were stained with 0.05~ Coomassie brilliant blue.
(f) Antibody~dependent cell-mediated cytotoxicity:
Mononuclear cells from the peripheral blood of normal volunteers were used as the effector cell source. Target cells were labeled with 51Cr, as describ2d previously (17). A cell suspension (100,~1) containing 104 labeled target 2780A3 cells was incubated at 37C for 30 min with various concentrations of monoclonal antibody in a 96-well microculture plate. Then, 100 ,ul of a cell suspension containing effector mononucl~ar cells was added to each well. The pla~e was incubated at 37C for 6 h in a humidified 5% C02 atmosphere. After the insoluble 2780AD was removed by centrifugation, the radioactivity in 100 ~1 of supernatant was counted in a gamma counter. Determination was carried out in triplicate. The percent specific cytolysis was calculated from the 51Cr release of test samples and control samples, as follows:
~ specific release = (E-S~(M-S) x 100, where E = experimental release (cpm in supernatant from target cells incubated with effector cells and exp~rimental antibody), S - spontaneous release Icpm in supernatant from target cells incuba~ted with medium only), and M - maxium release (cpm released from target cells lysed with 1~ Triton X-100~.
~2) Experiments The materials and methods of [1~ were u3ed to prepare a ~ ~ :
:
'~23~
himeric antibody and confirm its properties below.
1) Preparation of chimeric antibody (a) Construction of the chimeric H chain gene:
The variable region gene of mouse heavy chain was cut out of MRK16-producing hybridoma cells as a 3.0-kb fragment by EcoRI, and subcloned into ~gtlO. It was identified by hybridization with mouse JH probe. The variable region gene had rearranged to the J3 segment (V-D-J). The so obtained mouse variable region gene was used as an EcoRI fragment for constructing a chimeric heavy-chain gene (Fig. lA). The mouse variable region gene was joined to the 5' site of the human constant region in the same transcriptional direction to construct pSV2-VH16-HGlgpt (Fig.
lA).
(b) Construction of the chimeric L chain gene:
The L chain variable region gene was cut out of genomic DNA
of ~RK16-producing hybridoma as a ll-kb BamHI fragment, and subcloned into ~EMBL3. It was identified by hybridization with mouse JK probe. The variable region gene had rearranged t~ the Jl segment. Multicloning site from pBluescript SK M13 (Stratagene, La Jolla, CA) was induced into the HindIII site of pSV2HCkneo (Fig. 1~). Then, the mouse variable region gene was trimmed to a 7-Xb BamHI-Xbal fragment and was subcloned into the BamHI-XbaI site of pBluescript SK M13 ~ The resulting 7-kb fragment of mouse variable region was cut out by NotI/SalI
digestion and was cloned into the multicloning site of the pSV2HCX neo. Thus, the mouse L chain variable region gen~ was joined to a S' site of the Xuman constant region in the same transcriptional direction, to construct pSV2-Vk 16-HCk neo (FigO
lB).
~` ' ' '' ''" - ' ~
~ ''' ''` ` ' ~
(c) Transformation of ~ouse myeloma cells:
SP2/0, a nonproducer mouse myeloma cell line, was cotransfected by the chimeric H and L chain genes using electroporation methods (30,31). ~he transformed cells were selected with G418. The res~ltin~ stable transformants, which were obtained about 2 weeks after the electroporation, were screened to select clones that produced ~he antibody specific to the multidrug-resistant cell line K562/ADM. Screening was done by enzyme-linked immunosorbent assay using parent K562 cells (adriamycin-sensitive strain) as negative control (16). Two positive clones producing chimeric antibody were established from 1 x 107 cells. The product of a clone with a larger output among the recloned transformants was designated as MH162, and used for further analysis.
The SP2/0 transformants yielded sufficient amounts of chimeric antibody (5-lO~ug/ml in the medium supplemented with 1%
serum, which had been deprived of Protein A-binding immunoglobulins). The apparent affinity of the chimeric antibody (MH162) to the K562/ADM cell antigen was similar to that of the mouse antibody tMRK16), as determined by enzyme-linked immunosorbent assay.
2) Properties of chimeric antibody (a) Test for antibo~y specificity:
The antibody (MH162) specificity was tested by enzyme-linked immunosorbent assays (data not shown). Multidrug-resistant cell lines K562/ADM and 2780 D bound the chimeri~ antlbody to approximately the same extent as the original mouse MRK16~
Parent drug-sensitive lines K562 and A2780, on the other hand, ,~
:
~a~
did not bind it ei~her. Th~s, this MH162 has the same binding specificities as MRK16 (16,35).
(b) SDS-PAGE analysis of the chimeric antibody:
The chime~ic antibody M~162 was purified to apparent homogeneity by single-step affini~y chromatography using Protein A-Sepharose (Fig. 2). 0.5 ~g each of monoclonal antibody MRK16 (lane 1) and chimeric antibody MH162 ~lane 2) were subjected to SDS-PAGE analysis (A) after treatment with mercaptoe~hanol or (B) without mercaptoethanol treatmentO A molecular weight marker was obtained from Amersham, Japan.
(c) Antibody-dependent cell-mediated cytotoxicity by tbe chimeric antibody:
The chimeric MH162 was used in antibody-dependent cell-mediated cytotoxicit,y a~says with human mononuclear cells as effectors. Fig. 3 shows the findings of experiments in which ~780AD cells were exposed to 1 ~g/mL of antibody and varying doses of human e~fector cells. The ADCC tests were performed as described in the Materials and Methods using l,ug/ml of MH162 (O), or l,ug/ml of MR~16 (~J, or without the monoclonal antibody (~). The determination was performed in triplicate. The chimeric MH162 produced significant ~ytotoxicity even at an effector/target cell ratio of 10:1, while the mouse MRK16 monoclonal antibody showed only an insignificant level of ADCC
activity.` Cells from the parent A2780 line were not lysed by the chimeric MH162 or by mouse MRK16 5data not shown).
References !
.: .`,~ ' ' `, 'i,~.,~ : . `
': ` . ~ , ,' . ; `::
HOE 90/5 01~2;~9 REF EREP-ICES
l) Riordar., J.R., and Llng. V. Gene~lc and blochemical characterizatlon of multldrug resist~nce. Pharmacol. Ther., 28:51-75, 1985.
2) Tsuruo. T. Mechanlsms of ~ul~ldru~ resls~ance and implica~lons for therapy. Jpn. J. Canc~r Res., 79:2B5-296, 1987.
3) Ronlnson, I.B. (ed.). ~Molecular and Cellular B~ology o~
M~ltidrug Resistance in Tu~or Cells." New York, Pleu~um P~ublishing ~orp.. in press, 1989.
4~ Sa~a, A. X.. Glo~er. C. J., MeYers~ M.B., Bledler, J. L., and Felsted, R. L. Vinblastine photoa~finl~y lab~lln~ of a hlgh molecular weight surface membrane ~lycoproteln speclflc ~or multidrug-resistant cells. J. ~lol. Chem., 261:6137-6140, 1986.
5) Cornwell, M.M.. Sa~a. A.R., Fels~ed, R.L., Go~t~sman, M~ M., and Pastan, I. Membrane ~eslcle~ ~rom ~ultldrug resl~tant human cancer cells contain a speclflc 150- to l~0-kDa proteln detected by photoa~initY labelin~. Proc. Natl. Acad. Scl.
USA, 83:3847-3850, 1986.
Sepharose column (see the reference 33).
From the transformant ~hat has expressed the so produced chimeric antibody, it is possible to separate and purify m-RNA
coding for the chimeric antibody by a customary method. cDNA is prepared from the mRNA by a customary method, and a suitable promoter or enhancer region is incorporated into a site upstream of the cDNA codin~ for the chimeric antibody, whereby the chimaric antibody can be expressed and produced even in host cells other than myeloma cells, such as yeast, silkworm or plants.
As shown in the Experimental Examples l~ter, an expression vector including a chimeric mouse V/human C immunoglobulin gene was transfected into myeloma cells, with the result that a functional chimeric IgG with the sam~ affinity and binding specificity as those of the original hy~ridoma antibody was produced. This ChimeriG antibody was much lower ln immunogenicity than the all-mouse antibody. Furthermore, the human C region of the chimeric antibody permits the human effector function to work more effecti~ely.
. - ~ ~. .: :
.. . . .
- , . : ' : . ' ~ ~:.: ` ' .
? ~
Experimental Examples ~1~ Materials and MethOds (a) vectors, clones, probes and cells:
Hybridoma MRK16 (FERM-BP-2200) producing MRK was used as a source of a gene coding for the variable region of mouse monoclonal antibody against drug-sensitive cancers. The ~phage ~gtlO (22) was used as a subcloning vec~or including the mouse monoclonal antibody H chain variable region that had been cut out by EcoRI. ~EMBL3 (23) was used as a subcloning vector including the mouse monoclonal antibody L chain variable region cut out by 3amHI.
Mouse Jk gene-containing fragment (Jk probe) (25) isolated from clone Igl46 was used as a probe for the mouse monoclonal antibody L chain variable region. Mouse ~H probe isola~ed from MEP203 (26) was used as a probe for the mouse monoclonal antibody H chain variable region.
pSV2HGlgpt was used as an expression vector having a gene coding for the human IgG H chain constant region joined theretoO
pSV2HCkneo was used as an expression vector having a gene coding fo~ the human IgG L chain constant region joinsd thereto (24).
` Mouse myeloma Sp2/0 (Sp2/0-Agl4) obtained from ATCC
~Rockville, MD) was used as a host cell for the expression vector.
Human drug-resist~nt cell lines (~562/ADM a~d 27~0AD) and their parent drug-sensitlve cell lines (K562 and A2780~) for use in antibody-dependent cell-mediated cytotoxicity test were maintained as described previously (27).
la ~
,, ..
c~ J
(b) Cloning of size-fractionated DNA:
A fragment containing a gene coding fo~ the mouse monoclonal antibody H chain variable region was id~ntified in Southern hybridization of EcoRI-digested genomic DNA with JH probe. A
fragment containing a gene coding for th~ mouse monoclonal antibody L chain variable region was iden~ified in Southern hybridization of BamHI-diges~ed genomic DNA with J~ probe. These genes were confirmed to be rearranged.
The respective fragments were eluted from the relevant regions separated on agarose gel, ligated with ~gtlO and ~EMBL3 arms, and packaged into ~phage.
Plaque hybridization was carried out according to the Banton-Davis method (29).
(c) DNA transfection of mouse Sp2/0 myeloma cells:
200 ~g each of plasmids pSV2-VH16-HGlgpt and pSV2-VK16-HC~neo (see Fig. 1) were cotransfected into 107 mouse Sp2/0 cells ~CRL1581, ATCC) by electroporation (30,31). Transformants were selected in RPMI 1~40 medium supplemented with 10% fetal bovine serum and 0~8 mg/ml G418 (GI8CO, Grand Island, NY). A chimeric antibody in the growth medium ~as detected by enzyme-linked immunosorbent assaf (ELISA) to collect antibody-producing cells ~16,27).
(d) Isolation of chimeric antibody:
Antibody-producing cells were ~rown in RPMI 1640 medium supplemented with 1. 0% fetal bovine serum, which had been precleared of protein A-binding bovine immunoglobulin by af~inity chromatography using protein A-Sepharose CL-4 (Pharmacia, Uppsala, Sweden) ~32). Purification o~ the antibody was carried out as described previously ~33), using~protein A-Sepharose CL-4B
., . .- : . : .
~:., '. : '', : -, : . - . : .
)J ~ J ~J
arfinity chromatography.
(e) SDS-PAGE:
Denaturins gel electrophoresis was performed according to Laemmli ~34) with a 4-20% polyacrylamide linear gradient gel, and gels were stained with 0.05~ Coomassie brilliant blue.
(f) Antibody~dependent cell-mediated cytotoxicity:
Mononuclear cells from the peripheral blood of normal volunteers were used as the effector cell source. Target cells were labeled with 51Cr, as describ2d previously (17). A cell suspension (100,~1) containing 104 labeled target 2780A3 cells was incubated at 37C for 30 min with various concentrations of monoclonal antibody in a 96-well microculture plate. Then, 100 ,ul of a cell suspension containing effector mononucl~ar cells was added to each well. The pla~e was incubated at 37C for 6 h in a humidified 5% C02 atmosphere. After the insoluble 2780AD was removed by centrifugation, the radioactivity in 100 ~1 of supernatant was counted in a gamma counter. Determination was carried out in triplicate. The percent specific cytolysis was calculated from the 51Cr release of test samples and control samples, as follows:
~ specific release = (E-S~(M-S) x 100, where E = experimental release (cpm in supernatant from target cells incubated with effector cells and exp~rimental antibody), S - spontaneous release Icpm in supernatant from target cells incuba~ted with medium only), and M - maxium release (cpm released from target cells lysed with 1~ Triton X-100~.
~2) Experiments The materials and methods of [1~ were u3ed to prepare a ~ ~ :
:
'~23~
himeric antibody and confirm its properties below.
1) Preparation of chimeric antibody (a) Construction of the chimeric H chain gene:
The variable region gene of mouse heavy chain was cut out of MRK16-producing hybridoma cells as a 3.0-kb fragment by EcoRI, and subcloned into ~gtlO. It was identified by hybridization with mouse JH probe. The variable region gene had rearranged to the J3 segment (V-D-J). The so obtained mouse variable region gene was used as an EcoRI fragment for constructing a chimeric heavy-chain gene (Fig. lA). The mouse variable region gene was joined to the 5' site of the human constant region in the same transcriptional direction to construct pSV2-VH16-HGlgpt (Fig.
lA).
(b) Construction of the chimeric L chain gene:
The L chain variable region gene was cut out of genomic DNA
of ~RK16-producing hybridoma as a ll-kb BamHI fragment, and subcloned into ~EMBL3. It was identified by hybridization with mouse JK probe. The variable region gene had rearranged t~ the Jl segment. Multicloning site from pBluescript SK M13 (Stratagene, La Jolla, CA) was induced into the HindIII site of pSV2HCkneo (Fig. 1~). Then, the mouse variable region gene was trimmed to a 7-Xb BamHI-Xbal fragment and was subcloned into the BamHI-XbaI site of pBluescript SK M13 ~ The resulting 7-kb fragment of mouse variable region was cut out by NotI/SalI
digestion and was cloned into the multicloning site of the pSV2HCX neo. Thus, the mouse L chain variable region gen~ was joined to a S' site of the Xuman constant region in the same transcriptional direction, to construct pSV2-Vk 16-HCk neo (FigO
lB).
~` ' ' '' ''" - ' ~
~ ''' ''` ` ' ~
(c) Transformation of ~ouse myeloma cells:
SP2/0, a nonproducer mouse myeloma cell line, was cotransfected by the chimeric H and L chain genes using electroporation methods (30,31). ~he transformed cells were selected with G418. The res~ltin~ stable transformants, which were obtained about 2 weeks after the electroporation, were screened to select clones that produced ~he antibody specific to the multidrug-resistant cell line K562/ADM. Screening was done by enzyme-linked immunosorbent assay using parent K562 cells (adriamycin-sensitive strain) as negative control (16). Two positive clones producing chimeric antibody were established from 1 x 107 cells. The product of a clone with a larger output among the recloned transformants was designated as MH162, and used for further analysis.
The SP2/0 transformants yielded sufficient amounts of chimeric antibody (5-lO~ug/ml in the medium supplemented with 1%
serum, which had been deprived of Protein A-binding immunoglobulins). The apparent affinity of the chimeric antibody (MH162) to the K562/ADM cell antigen was similar to that of the mouse antibody tMRK16), as determined by enzyme-linked immunosorbent assay.
2) Properties of chimeric antibody (a) Test for antibo~y specificity:
The antibody (MH162) specificity was tested by enzyme-linked immunosorbent assays (data not shown). Multidrug-resistant cell lines K562/ADM and 2780 D bound the chimeri~ antlbody to approximately the same extent as the original mouse MRK16~
Parent drug-sensitive lines K562 and A2780, on the other hand, ,~
:
~a~
did not bind it ei~her. Th~s, this MH162 has the same binding specificities as MRK16 (16,35).
(b) SDS-PAGE analysis of the chimeric antibody:
The chime~ic antibody M~162 was purified to apparent homogeneity by single-step affini~y chromatography using Protein A-Sepharose (Fig. 2). 0.5 ~g each of monoclonal antibody MRK16 (lane 1) and chimeric antibody MH162 ~lane 2) were subjected to SDS-PAGE analysis (A) after treatment with mercaptoe~hanol or (B) without mercaptoethanol treatmentO A molecular weight marker was obtained from Amersham, Japan.
(c) Antibody-dependent cell-mediated cytotoxicity by tbe chimeric antibody:
The chimeric MH162 was used in antibody-dependent cell-mediated cytotoxicit,y a~says with human mononuclear cells as effectors. Fig. 3 shows the findings of experiments in which ~780AD cells were exposed to 1 ~g/mL of antibody and varying doses of human e~fector cells. The ADCC tests were performed as described in the Materials and Methods using l,ug/ml of MH162 (O), or l,ug/ml of MR~16 (~J, or without the monoclonal antibody (~). The determination was performed in triplicate. The chimeric MH162 produced significant ~ytotoxicity even at an effector/target cell ratio of 10:1, while the mouse MRK16 monoclonal antibody showed only an insignificant level of ADCC
activity.` Cells from the parent A2780 line were not lysed by the chimeric MH162 or by mouse MRK16 5data not shown).
References !
.: .`,~ ' ' `, 'i,~.,~ : . `
': ` . ~ , ,' . ; `::
HOE 90/5 01~2;~9 REF EREP-ICES
l) Riordar., J.R., and Llng. V. Gene~lc and blochemical characterizatlon of multldrug resist~nce. Pharmacol. Ther., 28:51-75, 1985.
2) Tsuruo. T. Mechanlsms of ~ul~ldru~ resls~ance and implica~lons for therapy. Jpn. J. Canc~r Res., 79:2B5-296, 1987.
3) Ronlnson, I.B. (ed.). ~Molecular and Cellular B~ology o~
M~ltidrug Resistance in Tu~or Cells." New York, Pleu~um P~ublishing ~orp.. in press, 1989.
4~ Sa~a, A. X.. Glo~er. C. J., MeYers~ M.B., Bledler, J. L., and Felsted, R. L. Vinblastine photoa~finl~y lab~lln~ of a hlgh molecular weight surface membrane ~lycoproteln speclflc ~or multidrug-resistant cells. J. ~lol. Chem., 261:6137-6140, 1986.
5) Cornwell, M.M.. Sa~a. A.R., Fels~ed, R.L., Go~t~sman, M~ M., and Pastan, I. Membrane ~eslcle~ ~rom ~ultldrug resl~tant human cancer cells contain a speclflc 150- to l~0-kDa proteln detected by photoa~initY labelin~. Proc. Natl. Acad. Scl.
USA, 83:3847-3850, 1986.
6) Hamada, H., ~d Tsuruo. T. Purl~lc tlon o~ the 170- to 180-kilodalton me~brane ~lycoprotein assoclat~d wlth multldrus resistance: The 170- to 180-kilodalton ~e~brane ~lycoprot~ln is an ATPase. J. Blol. Che~., 263:1454~1458, 1988.
7) Hamada, H., and Tsuruo. T~ Chara~terizat~on o~ ~h~ ATPase Actlvlt~ of the 170- to 180-klladalto~ me~br~ne ~lycoproteln lP-~lycoproteln~ asso~iated ~lth ~ultldru~ ~eslsta~ce.
, :, 2~2~
Cancer Res., 48:4926-4932. 1988.
, :, 2~2~
Cancer Res., 48:4926-4932. 1988.
8) WillinghaQ, M. C., Richert, N. D., Cornwell, M. M., Tsuruo, T., Hamada, H.. Go~tesman, M. M., and Pastan, I.
Immunocytochemial localization of P170 at the plasma membrane of multidrug-reslstant human cells. J. Histochem. Cytochem., 35:1451-1456, 1987.
Immunocytochemial localization of P170 at the plasma membrane of multidrug-reslstant human cells. J. Histochem. Cytochem., 35:1451-1456, 1987.
9) Sugawara, I., Kataoka, I., Morishita, Y., Hamada, H., Tsuruo, T., ItoYama~ S., and Mori, S. Tissue distribut~on o~ P-glycoproteln encoded by a mutlidrug-resistant gene as revealed by a monoclonal antibody, MRK16. Cancer Res., 48:
1926-1929, 1988.
1926-1929, 1988.
10) Shen. D-W.. Fo~o, A., Roninson, I.B., Chln, J.E., Soffier, R., Pastan, I., and Gottesman, M. M. Multidrug resistance in DNA-medlated transformants is l~nked to transfer of the human mdrl gene. Mol. Cell. Biol., 6:4039-4045, 1986.
11) Gros, P., Neriah, U. B., Croop, J. M., and ~ousman, D. E.
Isolation and expression of a complementary DNA ~mdr) that confers multldrug resistance. Nature, 32~:728-731, 1986.
Isolation and expression of a complementary DNA ~mdr) that confers multldrug resistance. Nature, 32~:728-731, 1986.
12) Ueda, K., Cardarelli, C., Gottesman, M. M., and Pastan, I.
Expresslon of a full-length cDNA ~or the human mdrl (P-glYcoprotein) gene confers multldrug reslstance. Proc. Natl.
Acad. Sci, USA, 84:3004-3008, 1987.
Expresslon of a full-length cDNA ~or the human mdrl (P-glYcoprotein) gene confers multldrug reslstance. Proc. Natl.
Acad. Sci, USA, 84:3004-3008, 1987.
13) Bell, D.R., Gerlach, J.H., Kartner, N., Bulch, R.N., and Llng, V. Detection of P-glycoproteln in ovarlan cancer: a molecular marker associated with multidrug resistance.
J, Clln. Oncol.,3:311-315, 1~85.
.
.. . . . . .
J, Clln. Oncol.,3:311-315, 1~85.
.
.. . . . . .
14) Fojo. A. T., Ueda. K., Slamon. D.J., Poplack, D.G., Gottesman, ~1. M., and Pastan, I. Expresslon of a multidru~-resistance gene in human tumors and tissues. Proc. Natl.
Acad. Sci. USA. 84:265-269. 1987.
Acad. Sci. USA. 84:265-269. 1987.
15) Tsuruo, T., Suglmoto, Y., Hamada, H., Roninson. I., Okumura, ., Adachl, K., Morishima. Y., and Ohno, R. Detection of multldrùg reslstance markers, P-glycoproteln and mdrl mRNA.
ln human leukemia cells. Jpn. J. Cancer Res.. 7~:1415-1419.
__ 1~87.
ln human leukemia cells. Jpn. J. Cancer Res.. 7~:1415-1419.
__ 1~87.
16) Hamada, H., and Tsuruo, T. Functlonal role for the 170- to 180-kDa glycoprotein speciflc to drug-resistant tumor cells as revealed by monoclonal antibodies. Proc. Natl. Acad. Sci.
US~, 83:7785-7789, 1~86.
US~, 83:7785-7789, 1~86.
17) Tsuruo, T., Hamada, H., Sato, S., and Helke, Y. Inhibition of multldrug-reslstant human tumor growth ln athymlc mlce by anti-P-glycoprotein monoclonal antibodies. Jpn. J. Cancer Res., 80:627-631, 1989.
18) Meeker, T.C., Lowder, J. Maloney, D.G., Miller, R. A., Thielemans, K., Warnke, R., and Levy, R. ~ cllnical ~rlal of anti-id~otype therapy ~or ~ cell mallgnancy. Blood, 65:1349-1363, 1985.
19) Miller, R. A., Lowder, J., Meeker, T. C., Bro~n, S.. and Levy, R. Anti-idiotYpes ln B-cell tumor therapy. Natl. Cancer Inst. Monogr., 3:}31-134, 1987.
20) Marx, J. L. Antlbodies made to order: Chlmeric antibodies -whlch are part mouse and part human - may help sol~e the problems hlnderlng the therapeutic use o~ monoclonal : ,, -. .
~ . . . : ~.
antibodies. Science, 229:4s5-4s6, 1987.
~ . . . : ~.
antibodies. Science, 229:4s5-4s6, 1987.
21) Morrison, S.L., and 01, V.T. GeneticallY en~ineered antibody mlecules. Adv. Immunol. 44:6~-92, 1989.
22) Huynh, T.V., Young, R.A., and Davis, R.W. Constructlng and screening cDNA libraries in AgtlO and Agtll. In: ~lover, D.M.
(Ed.) DNA Cloning, Vol.l, pp.49-78, Oxford, IRL Press, 1985.
(Ed.) DNA Cloning, Vol.l, pp.49-78, Oxford, IRL Press, 1985.
23) Frlschauf, A., Lehrach, H., Poustka, A., and Murray, N.
Lambda replacement vectors carrying Polyllnker sequences. J.
Mol. Biol., 170:827-842, 1983.
Lambda replacement vectors carrying Polyllnker sequences. J.
Mol. Biol., 170:827-842, 1983.
24) ~ameyama, K., Imai, K., I~oh, T., Taniguchi, M., Miura, X., and Kurosawa, Y. Convenient plasmid vectors for construction of chimeric mouse/human antibodies. FEBS Lett.,244(2), 301-306,1989 25) Sakano, H., H~ppi, K., Heirich, G., and Tonegawa, S.
Sequences at the somatic recomblnatlon sltes o~
immunoglobulln light-chain genes. Nature, 280:288-294, 1979.
Sequences at the somatic recomblnatlon sltes o~
immunoglobulln light-chain genes. Nature, 280:288-294, 1979.
26) Roeder, W., Makl, R., Traunecker, A., and Tonegawa, S.
Linkage of the four r subclass heavy chain genes. Proc.
Natl. Acad. Scl. USA, 78:474-478, 1981.
Linkage of the four r subclass heavy chain genes. Proc.
Natl. Acad. Scl. USA, 78:474-478, 1981.
27) Hamada, H., Okochi, E., Oh-hara, T., and Tsuruo, T.
Purification of the Mr2~,000 calicum-binding protein (sorcln) associated with multidrug resis~ance and lts detectlon with monoclonal antibodies. Cancer Res., 48:3173-3178, 1988.
Purification of the Mr2~,000 calicum-binding protein (sorcln) associated with multidrug resis~ance and lts detectlon with monoclonal antibodies. Cancer Res., 48:3173-3178, 1988.
28) Southern, E. Detectlon of specific sequences amon~ DNA
fragments separat~d by gel electrophoresis. J. Mol. Biol., 98:503-517, 1975.
fragments separat~d by gel electrophoresis. J. Mol. Biol., 98:503-517, 1975.
29) Benton, W.D., and Davis, R.W. Screening Agt recombinant clones by hybridization to slngle plaques in sltu.
.
~2~
Sclence, 196:180-182. 1977.
.
~2~
Sclence, 196:180-182. 1977.
30) Potter, H., Weir, L., and Leder, P. Enhancer-dependent expression of human immunoglobulin genes introduced into mouse pre-3 lYmphocytes by electroporation. Proc. Natl. Acad.
- Sci . USA . 81: 7161-7165, 1984 .
- Sci . USA . 81: 7161-7165, 1984 .
31) Toneguzzo, F., Hayday, A.C., and Keatlng, A. Electric fleld-mediated DNA transfer: transient and stable gene expresslon in human and mouse lymphoid cells. Mol. Cell. Blol., 6:703-706, 1986.
32) Underwood, P.A. Removal of bovine immunoglobulin from serum in hybridoma culture media using protein A. Methods EnzYm 121:301-306, 1986.
33) Goding, J.W. Monoclonal Antibodies: Principles and Practlce.
New York: Academic Press, 1983.
New York: Academic Press, 1983.
34) Laemmli, U.K. Clea~age of s~ructural proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685, 1970.
35) ~amada, H., and Tsuruo. T. Growth lnhibi~ion of multidrug-resistant cells by monoclonal antlbodies against P-glycoprotein. In: I.B.~Ronlnson (ed.) Molecular and Cellular 3iology of Multldrug Resistance ln Tumor Cells. New Yor~:
Plenum Publishlng Corp., in Press> 1989.
Plenum Publishlng Corp., in Press> 1989.
36) Levy, R.L., and Miller, R.A. Biolo~ical and cli~cal implications of lYmphocyte hybrldomas: tumor therapy with monoclonal antibodies. Annu. Re~. Med., 34:107-116i 1983.
37) Mi}ler, R.A., Maloney, D.G., Warnke, R., and Levy, R.
Treatment of B cell lymphoma w1th monoclonal anti-idiotype antibody. N. Engl. J. Med., 306:5l7-522, 19~2.
- .
'' ` ~ ,. '',: ' . ' ~ , ' ` ~ ' ' ;
- ' '' ' : ' . "' .' . ` ' ' ' ': `
~2~
Treatment of B cell lymphoma w1th monoclonal anti-idiotype antibody. N. Engl. J. Med., 306:5l7-522, 19~2.
- .
'' ` ~ ,. '',: ' . ' ~ , ' ` ~ ' ' ;
- ' '' ' : ' . "' .' . ` ' ' ' ': `
~2~
38) HerlYn, D. and Koprowski, H. IgG2~ monoclonal antlbodles inhibit human tumor grow~h through interaction w~th ef~ector cells. Proc. Natl. Acad. Sci. USA, 79:4761-4765, 1982.
39) ,~asui, H., Kawamoto, T., Sata, J.D., Wolf, B., Sato. G. and Mendelsohn, J. Growth lnhibltlon of human tumor cells in athymic mice by antl-epidermal growth factor receptor monoclonal antibodies. Cancer Res.. 44:1002-1007, 1984.
40) Thorpe, P.E., Brown, A.N., Bremmer. J.A.. Foxwell, B.M., and Stirpe, F. An immunotoxin composed o~ monoclonal antl-Thyl.l antibody and a ribosome-inactivating pro~ein from Sa~anaria officinalis: potent antitumor effects in vitro and in vivo.
J~CI, 75:1~1-159, 1985. ..
J~CI, 75:1~1-159, 1985. ..
41) Pastan, I , Willingham, M.C., and FitzCe~rald, D.J.P.
Immunotoxins. Cell, 47:641-6~8, 1986.
Immunotoxins. Cell, 47:641-6~8, 1986.
42) FitzGerald, D.J., Willingham, M.C., Cardarelli, C.O., Hamada.
H., Tsuruo, T., Gottesman, M.M. and Pastan, I. A monoclonal antibody-Pseudomonas toxin conjugate that specifically kills multidrug-resistant cells. Proc. Natl. ~cad. Sci. USA, 84:4~88-7292, 1987.
H., Tsuruo, T., Gottesman, M.M. and Pastan, I. A monoclonal antibody-Pseudomonas toxin conjugate that specifically kills multidrug-resistant cells. Proc. Natl. ~cad. Sci. USA, 84:4~88-7292, 1987.
43) Kaizer, H., Levy, R. and Santos, G. Autologous bone marrow transplantation in T cell malignancies: Use o~ in vitro monoclonal antibody treatment of remission marrow to elimi~
nate tumor cells~ J. Cell. Biochem., Suppl. 6, Abstrt . ~0114, P . 41, 1982.
nate tumor cells~ J. Cell. Biochem., Suppl. 6, Abstrt . ~0114, P . 41, 1982.
44) Houghton, A.N., Mintzer, D.. Cordon-Cardo, C., Welt, SO.
Fliegel, B., Vadhan, S., Carswell, E., Melamed, M.R., Oettgen, H.F.. and Old, L.J. Mouse monoclonal IgG3 antibodY
detecting GD3 ~anglloslde: A phase I trlal in patients with ~:
, . . , ., , ~ i ~
~2~
malignant melanoma. Proc. Natl. Acad. Sci. USA, ~2:1242-1246, 1985.
Fliegel, B., Vadhan, S., Carswell, E., Melamed, M.R., Oettgen, H.F.. and Old, L.J. Mouse monoclonal IgG3 antibodY
detecting GD3 ~anglloslde: A phase I trlal in patients with ~:
, . . , ., , ~ i ~
~2~
malignant melanoma. Proc. Natl. Acad. Sci. USA, ~2:1242-1246, 1985.
45) Larrlck, J.W., and Buck, D.W. Practlcal aspects of human monoclonal antibody production. Biotechniques, ~:6, 1984.
46) Morrison, S.L. Transfectomas provide no~el chlmerlc antibodies. Science, 229:1202, 1985.
47) Steplewski, Z., Sun, L.K., Shearman, C.W., Ghrayeb, J.
Daddona, P., and Koprowski, H. Biologlcal acti~lty of human-mouse IgGl, I~G2, IgG3 and IgG4 chimeric monoclonal antibodies with antitumor specificity. Proc. ~atl. Acd. Sci.
USA, 85:4852-4856, 1988.
Daddona, P., and Koprowski, H. Biologlcal acti~lty of human-mouse IgGl, I~G2, IgG3 and IgG4 chimeric monoclonal antibodies with antitumor specificity. Proc. ~atl. Acd. Sci.
USA, 85:4852-4856, 1988.
48) Morrison, S.L., Johnson, M.J., Herzenberg, L.A., and Oi.
V.T. Chimeric human antlbody molecules: mouse antlgen-blndlng domains wlth human constant region domains. Proc, Natl. Acad.
Scl. USA, 81:6851-6855, 1984.
V.T. Chimeric human antlbody molecules: mouse antlgen-blndlng domains wlth human constant region domains. Proc, Natl. Acad.
Scl. USA, 81:6851-6855, 1984.
49) Boulianne, G.L., Hozumi, N., and Shulman, M.J. Production of functional chimaeric mouse/human antibody. Nature, 312:643-~46, 1984.
50) Neuberger, M.S., Williams, G.T., Mitchell, E.B., Jouhal, S.S., Flanagan, J.G., and Rabbitts, T.H. A hapten-speclfic chimaerlc IgE antibody with human physiological ef~ector functlon. Nature, 314:268-270 ,1985.
51) Sahagan, B.G., Doral, H., Saltzgaber-Muller, J.~ Toneguzzo.
F., Guindon, C.A.. LillY~ S.P., McDonald, ~.W., MorrisseY.
D.V., S~one, B.A., Davis, G.L., McIntosh, P.K., and Moore, G.P. A genetlcally engineered murlne/human chlmeric antlbodY
retains specificity ~or human tumor-assoclated antlgen. J.
.
, ~
, ~ . : . :
. . . . . .
2 ~
Immunol., 137:1066-1074, 1986.
5~) Sun, L.K., Curtis, P., Rakowicz-SzulczYnska, E.. GhraYeb, J., Chang, N., ,~orrlson, S.L., and Koprowski, H. Chlmeric anti~ody wlth human constant region and mouse varlable regions directed agains~ carcinoma-associated antigen 17-lA.
Proc. Natl. Acad. Sci. USA, 8 :214-218, 1987.
53) Nishimura, Y., Yokoyama, M., Araki, ~., Ueda, R., Kudo. A., Watanabe, T. Recom~lnant human-mouse chimerlc monoclonal antibody speclfic for common acu~e lymphocytic leukemia antigen. Cancer Res., 47:999-1005, 1987.
54) Liu, A.Y., Ro~inson. R.R., Murray, E.~., Ledbetter, J.A., ~el~str~m, I., and Hellstr~m, K.E. Production o~ a mouse-human chimeric monoclonal antlbody to-CD20 with potent Fc-dependènt biologic activity. J. Immunol., 139:3521-3526, 19~7.
Sa) Liu. A.Y., Roblnson, R.R., Hellstr~m. K.E., Murray. E.D..
Chang, C.P., and Hellstr~m. I. Chimeric mouse-human IgGl antibody that can medlate lysis o~ cancer cells. Proc. Natl.
Acad. Sci. USA., 84: 34~9-3443, 1987.
SB) LoBuglio, A.F., Wheeler, R.H., Trang, J., Haynes, A., - Rogers, K., HarYey, E.B., Sun, L., Ghrayeb, J., and Khazaeli, M.B. MouseJhuman chimerlc monoclonal sntlbody in man: ~inetlcs and immune respons~. Proc. Natl. Acad. Sci.
USA, 86: 4220-4224, 1989.
57) Thiebaut, F., Tsurus. T., Hamada, H.. Gottesman, M.M., Pastan, I and ~illingham, M.C. Cellular localizat~on oi ~he multidrug-reslstance gene product P-glycoprotein ln normal human tissues. Proc. Natl. Acad. Scl. USA, 84:7735-7738!, , :
: . : ,-, . . -: . , . , :
~.
- . . .
2~2~
1987.
58) Sugawara, I.. .~akahama. M., Hamada. H., Tsuruo, T.. and Morl, S. Apparent stronger expresslon in the human adrenal cortex than in the human adrenal medulla o~ M~ 170,00-180.O P-glycoprotein. Cancer Res., 48:4611-4614, 1988.
59~ Cordon-Cardo, C., O'Brien, J.P., Casals, D., Rittman-Grauer, L., Biedler, J.~., Melamed, M.R., and ~ertlno, J.R.
Multidrug-resistant gene (P-glycoprotein) ls expressed by endothelial cells at blood-braln barrier sites. Proc. Natl.
Acad. Sci. USA, 86:695-698, 1989.
~0) Thiebau~, F., Tsuruo, T., Hamada, H., Gottesman, M.M., Pastan, I., and Willingham, M.C. Immun~histochem~cal localizatlon in normal tlss~es o~ dl~erent epltopes ln the multidru~ transport proteln P170: E~ldence ~or locallzation in braln capillaries and crossreactlvlty of one antibody with a muscle protein. J. Hlstochem. Cytochem., 37:159-164, 1989.
61) Y~ng. C-P. H., DePinho, S.G., Greenber~er, L.M., Arceci, R.J~, and Horwltz, S.B. Progesterone lnteracts with P-glycoprotein in multidrug-resistant cells and in the endometrIum of gravid uter~s. J. Biol~ Chem., ?64:782-788, 1989.
..
- , , - ~
- :
F., Guindon, C.A.. LillY~ S.P., McDonald, ~.W., MorrisseY.
D.V., S~one, B.A., Davis, G.L., McIntosh, P.K., and Moore, G.P. A genetlcally engineered murlne/human chlmeric antlbodY
retains specificity ~or human tumor-assoclated antlgen. J.
.
, ~
, ~ . : . :
. . . . . .
2 ~
Immunol., 137:1066-1074, 1986.
5~) Sun, L.K., Curtis, P., Rakowicz-SzulczYnska, E.. GhraYeb, J., Chang, N., ,~orrlson, S.L., and Koprowski, H. Chlmeric anti~ody wlth human constant region and mouse varlable regions directed agains~ carcinoma-associated antigen 17-lA.
Proc. Natl. Acad. Sci. USA, 8 :214-218, 1987.
53) Nishimura, Y., Yokoyama, M., Araki, ~., Ueda, R., Kudo. A., Watanabe, T. Recom~lnant human-mouse chimerlc monoclonal antibody speclfic for common acu~e lymphocytic leukemia antigen. Cancer Res., 47:999-1005, 1987.
54) Liu, A.Y., Ro~inson. R.R., Murray, E.~., Ledbetter, J.A., ~el~str~m, I., and Hellstr~m, K.E. Production o~ a mouse-human chimeric monoclonal antlbody to-CD20 with potent Fc-dependènt biologic activity. J. Immunol., 139:3521-3526, 19~7.
Sa) Liu. A.Y., Roblnson, R.R., Hellstr~m. K.E., Murray. E.D..
Chang, C.P., and Hellstr~m. I. Chimeric mouse-human IgGl antibody that can medlate lysis o~ cancer cells. Proc. Natl.
Acad. Sci. USA., 84: 34~9-3443, 1987.
SB) LoBuglio, A.F., Wheeler, R.H., Trang, J., Haynes, A., - Rogers, K., HarYey, E.B., Sun, L., Ghrayeb, J., and Khazaeli, M.B. MouseJhuman chimerlc monoclonal sntlbody in man: ~inetlcs and immune respons~. Proc. Natl. Acad. Sci.
USA, 86: 4220-4224, 1989.
57) Thiebaut, F., Tsurus. T., Hamada, H.. Gottesman, M.M., Pastan, I and ~illingham, M.C. Cellular localizat~on oi ~he multidrug-reslstance gene product P-glycoprotein ln normal human tissues. Proc. Natl. Acad. Scl. USA, 84:7735-7738!, , :
: . : ,-, . . -: . , . , :
~.
- . . .
2~2~
1987.
58) Sugawara, I.. .~akahama. M., Hamada. H., Tsuruo, T.. and Morl, S. Apparent stronger expresslon in the human adrenal cortex than in the human adrenal medulla o~ M~ 170,00-180.O P-glycoprotein. Cancer Res., 48:4611-4614, 1988.
59~ Cordon-Cardo, C., O'Brien, J.P., Casals, D., Rittman-Grauer, L., Biedler, J.~., Melamed, M.R., and ~ertlno, J.R.
Multidrug-resistant gene (P-glycoprotein) ls expressed by endothelial cells at blood-braln barrier sites. Proc. Natl.
Acad. Sci. USA, 86:695-698, 1989.
~0) Thiebau~, F., Tsuruo, T., Hamada, H., Gottesman, M.M., Pastan, I., and Willingham, M.C. Immun~histochem~cal localizatlon in normal tlss~es o~ dl~erent epltopes ln the multidru~ transport proteln P170: E~ldence ~or locallzation in braln capillaries and crossreactlvlty of one antibody with a muscle protein. J. Hlstochem. Cytochem., 37:159-164, 1989.
61) Y~ng. C-P. H., DePinho, S.G., Greenber~er, L.M., Arceci, R.J~, and Horwltz, S.B. Progesterone lnteracts with P-glycoprotein in multidrug-resistant cells and in the endometrIum of gravid uter~s. J. Biol~ Chem., ?64:782-788, 1989.
..
- , , - ~
- :
Claims (7)
1. A chimeric antibody against drug-resistant cancers comprising variable regions having amino acid sequences substantially homologous with the variable regions of mouse monoclonal antibody against drug-resistant cancers, and constant regions having amino acid sequences substantially homologous with the constant regions of human immunoglobulin.
2. A chimeric antibody as claimed in Claim 1, wherein the antigen of the drug-resistant cancers is P-glycoprotein.
3. A chimeric antibody as claimed in Claim 1 or 2, wherein the mouse monoclonal antibody is produced by hybridoma MRK16 or MRK17 formed by fusing a mouse myeloma cell with a spleen cell from a mouse immunized with the adriamycin-resistant strain K562/ADM of human myelogenous leukemia cell line K562.
4. A process for producing a chimeric antibody as claimed is Claim 1, which comprises the following steps (a) to (f):
(a) joining an upstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the constant region of the H chain of human immunoglobulin, to a downstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the variable region of the H chain of mouse monoclonal antibody against drug-resistant cancers, to prepare a DNA chain having a base sequence coding for a chimeric H chain, (b) joining an upstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the constant region of the L chain of human immunoglobulin, to a downstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the variable region of the L chain of mouse monoclonal antibody against drug-resistant cancers, to prepare a DNA chain having a base sequence coding for a chimeric L chain, (c) incorporating each of the DNA chains obtained in the steps (a) and (b) into the same or different expression vector(s) capable of expressing the relevant generic information, thereby to construct recombinant DNAs, (d) transforming a host cell with the recombinant DNAs obtained in the step (c), to prepare a transformant, (e) culturing the transformant obtained in the step (d), to produce a chimeric antibody against drug-resistant cancers in the cultured medium, and (f) collecting, if desired, the chimeric antibody produced in the cultured medium in the step (e).
(a) joining an upstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the constant region of the H chain of human immunoglobulin, to a downstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the variable region of the H chain of mouse monoclonal antibody against drug-resistant cancers, to prepare a DNA chain having a base sequence coding for a chimeric H chain, (b) joining an upstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the constant region of the L chain of human immunoglobulin, to a downstream site of transcription of a gene coding for an amino acid sequence substantially homologous with the variable region of the L chain of mouse monoclonal antibody against drug-resistant cancers, to prepare a DNA chain having a base sequence coding for a chimeric L chain, (c) incorporating each of the DNA chains obtained in the steps (a) and (b) into the same or different expression vector(s) capable of expressing the relevant generic information, thereby to construct recombinant DNAs, (d) transforming a host cell with the recombinant DNAs obtained in the step (c), to prepare a transformant, (e) culturing the transformant obtained in the step (d), to produce a chimeric antibody against drug-resistant cancers in the cultured medium, and (f) collecting, if desired, the chimeric antibody produced in the cultured medium in the step (e).
5. A process for producing a chimeric antibody as claimed is Claim 4, wherein the antigen of the drug-resistant cancers is P-glycoprotein.
6. A process for producing a chimeric antibody as claimed in Claim 4 or 5, wherein the mouse monoclonal antibody is produced by hybridoma MRK16 or MRK17 formed by fusing a mouse myeloma cell with a spleen cell from a mouse immunized with the adriamycin-resistant strain K562/ADM of human myelogenous leukemia cell line K562.
7. The chimeric antibody as claimed in claim 1 and substantially as described herein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2-51563 | 1990-03-02 | ||
| JP2051563A JP2564412B2 (en) | 1990-03-02 | 1990-03-02 | Chimeric antibody against drug-resistant cancer and production thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2025695A1 true CA2025695A1 (en) | 1991-09-03 |
Family
ID=12890444
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002025695A Abandoned CA2025695A1 (en) | 1990-03-02 | 1990-09-19 | Chimeric antibody against drug-resistant cancers and process for production thereof |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP2564412B2 (en) |
| CA (1) | CA2025695A1 (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6147500A (en) * | 1984-08-15 | 1986-03-07 | Res Dev Corp Of Japan | Chimera monoclonal antibody and its preparation |
| JPS6261596A (en) * | 1985-09-11 | 1987-03-18 | Japan Found Cancer Res | Monoclonal antibody related to chemical resistance and production thereof |
-
1990
- 1990-03-02 JP JP2051563A patent/JP2564412B2/en not_active Expired - Fee Related
- 1990-09-19 CA CA002025695A patent/CA2025695A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| JP2564412B2 (en) | 1996-12-18 |
| JPH03254691A (en) | 1991-11-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fell et al. | Homologous recombination in hybridoma cells: heavy chain chimeric antibody produced by gene targeting. | |
| Oren et al. | TAPA-1, the target of an antiproliferative antibody, defines a new family of transmembrane proteins | |
| EP1124568B1 (en) | Polyspecific binding molecules and uses thereof | |
| AU737910B2 (en) | Chimeric antibody fusion proteins for the recruitment and stimulation of an antitumor immune response | |
| Sahagan et al. | A genetically engineered murine/human chimeric antibody retains specificity for human tumor-associated antigen. | |
| JP4439808B2 (en) | T cell receptor fusions and conjugates and methods for their use | |
| US5644033A (en) | Monoclonal antibodies that define a unique antigen of human B cell antigen receptor complex and methods of using same for diagnosis and treatment | |
| EP0369566A2 (en) | Bifunctional chimeric antibodies | |
| AU728911B2 (en) | Mutants of the LAG-3 proteins, products for the expression of these mutants and use | |
| Hamada et al. | Mouse-human chimeric antibody against the multidrug transporter P-glycoprotein | |
| US5281699A (en) | Treating B cell lymphoma or leukemia by targeting specific epitopes on B cell bound immunoglobulins | |
| Maecker et al. | Prevalence of antigen receptor variants in human T cell lines and tumors. | |
| CA2128151A1 (en) | Receptors of interleukin-12 and antibodies | |
| AU2002238537B2 (en) | Hybridoma cell line G250 and its use for producing monoclonal antibodies | |
| Seon et al. | Monoclonal antibody SN2 defining a human T cell leukemia-associated cell surface glycoprotein. | |
| EP0511308B1 (en) | Chimeric immunoglobulin for cd4 receptors | |
| AU2002238537A1 (en) | Hybridoma cell line G250 and its use for producing monoclonal antibodies | |
| CA2025695A1 (en) | Chimeric antibody against drug-resistant cancers and process for production thereof | |
| Lugasi et al. | Murine spontaneous T‐cell leukemia constitutively expressing IL‐2 receptor—a model for human T‐cell malignancies expressing IL‐2 receptor | |
| CA2019323A1 (en) | Chimeric mouse-human km10 antibody with specificity to a human tumor cell antigen | |
| Parsons et al. | A novel CEA vaccine stimulates T cell proliferation, γIFN secretion and CEA specific CTL responses | |
| WO2012015662A1 (en) | Il-18 receptor as a novel target of regulatory t cells in cancer | |
| Ariyoshi et al. | Mouse‐human chimeric antibody MH171 against the multidrug transporter P‐glycoprotein | |
| Ragnarsson et al. | Multiple myeloma cells are killed by syndecan-1-directed superantigen-activated T cells | |
| WO1994014847A1 (en) | Genetically engineered immunoglobulins |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |