Note: Descriptions are shown in the official language in which they were submitted.
~ 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 ~ ~
'
,
.
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~
3A
.
., . .
- ~ ,
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
:: :
3B
,: . - . ,: ~ . - 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
7 4
: . . :
~ 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
11
'
,
,,, : ' ,
. .: - - : . . ~ , ,
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.
13
,
. ~
, ~
~)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
14
.
':
~:
.
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.
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.
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
1 9
., . .- : . : .
~:., '. : '', : -,
: . - . : .
)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,
22
,~
:
~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
!
23
.: .`,~ ' ' `, '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.
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.
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.
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.
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.
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.
.
.. . . . . .
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.
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.
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.
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
26
: ,, -. .
~ . . . : ~.
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.
23) Frlschauf, A., Lehrach, H., Poustka, A., and Murray, N.
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.
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.
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.
28) Southern, E. Detectlon of specific sequences amon~ DNA
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.
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 .
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.
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.
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.
28
- .
'' ` ~ ,. '',: ' . ' ~ , ' ` ~ ' ' ;
- ' '' ' : '
. "' .' . ` ' ' ' ': `
~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. ..
41) Pastan, I , Willingham, M.C., and FitzCe~rald, D.J.P.
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.
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.
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
29
~:
, . . , ., , ~ 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.
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.
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.
..
- , ,
- ~
- :
