Note: Descriptions are shown in the official language in which they were submitted.
WO 91/09134 P~/JP9OtO1631
DESCRIPIION
EIIOSPECIFIC ANTIBODY TO CANCER CEL~ AND ENZYM~ WIT~
PRODnUG-ACTIVATING CEII~RACTERISTICS.
The present invenl;ion relates to a bi~pecific ~ntibody en~ne comple~
that serves well as a~ anticancer therapeutic drug. More specifically, the
present inve~tion relates to a hybrid monoclonal antibody thereinafter also
10 referred to a9 hybrid MoAb) wherein o~e of the two specificities is to human
cancer cells and the ot;her is to a prodrug-activating enzyme, ~nd a polydoma
that produces s~id an~body.
The present inve~tion also relates to an anti-human-cancer-protei~
complex obtained by immunologically binding the above-mentioned en~yme
1~ to the above-mentioned hybrid MoAb.
Many investigations have been made of what are called "antibody
missile therapy drugs", sntitumor immunocomplexe~ prepared by binding an
antitllmor antibody to a chemotherapeutic agent or a bioto~cin which aims at
20 selective destructiorl of cancer cells; some successes have been achieved in
blood-related cancers such as leukemia and lymphoma. However, no
sati3factory results have been obtained in actual clinical application.
Particularly with respect to solid cancers, much remains unsolved, including
the problerrl of serious side effects. l~is is mainly because 1) it is impossible
25 to introduce a sufficient amount of antica~cer agent into cancer cells due tothe limited number of tumor-related antigens present on the cancer cell
surfaces, 2) ~evere side effects hamper clinical application of highly cytoto2icbiotoxins (e.g., ricin, P~eudomon~ aeru~inosa e~otoxin) to obtain a lethal
effect on cancer cells in the prese~ce of 80 few tumor-related antigens, and 3)
30 cancer cells without target a~tigens are capable of prolif~ra~on uninfluencedby the cytoto~c action of mi~sile therapeutic drugs, since human cancer cells
are generally highly diverse and there is almost no possibility that all have
the same kind of tumor-relatet antigen. It i~ particularly diff~cult to develop
a therapeutic method that overcome~ the tiversity of cancer cells. Proposed
3B m~thod8 include therapy using numerous kinds of anticancer antibody
.~ , . . ... ., . , : ~ . .. ......
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.- ... . ~. . ..
. ~ .. .. .. . . .. ..
,
wo gl/09134 2 ~ 6 9 ~ 3 ~ 2 - pcr/Jpso/ol~3l
(antibody cocktail therapy), but this therapy is unrealistic, since it is very
difficult to prepare numerous kinds of ca~cer-specific antibody.
SUMMARY OF 'f ~; ~}3NIION
With the aim of solving the above mentioned problems with
conventional anticancer missile therapeutic drugs, the present inventors
investigated a rece~tly developed bispeci~lc antibody a~d developed the
present inveIltion. Accordingly, the inventors prepared a bispecific a~tibody
capable of binding to both an enzyme that converts an irlactive an~cancer
prodrug into active type and ~o a human cancer cell, and administered an
immunocomplex comprising said antibody and said enzyme, as well as an
inactive prodrug, to cancer patients, thus developing an anti-h~ cancer-
protein comple2 e:~ibiting a cytotoxic effect selectively on cancer cells,
regardless oftheir diversi~.
The prodrug itself is present in blood and other organs and tissues in an
inactive form; only in the vicinity of the target cancer tissue is the prodrug
decomposed and activated by the anti-human-cancer-protein comples of the
present invention, as bound to human cancer cells, to eshibit its anticancer
activity. Thus, the prodrug has almost no side effects in the administration
method using the anti-human-cancer-protein comple2 of the present
invention. Administration using the anti-human-cancer-protein ~omplex cf
the present invention is also characterized in that anticancer activities are
e~hibited against cancer cells which are present in the vicinit3r of the target
cancer tissue but are free of target antigens, cytotoxic effect being e~hibited
regardless of the diversity of cancer cells. Accordingly, one object of the
present invention is to provide a bispecific hybrid monoclonal antibody
wh~rein one of the two specificities is to human cancer cells and the other is to
a prodrug-activating enzSnne, and a polydoma that produces said antibody.
Another object of t~e present invention i8 to provide an anti-human-
cancer-protein complex compri~ing the bispecific hybrid MoAb described
above and a prodrug-activating enzyme ~mmunologically coupled thereto.
BRIEF DESCRIPIION OF l ~; DRAW~GS
. ,
2~9~39
WV 91~09134 ~ 3 ~ PCr/JP90/01631
Fig. 1 show.s the cytoto~icity of a tripeptidated drug (Boc-Gly-Gly-Arg-
ADR; - ~ -) and its activated body (ADR; - o -) on gastric car~cer cell line
NUGC4 (see Example 4).
Fig. 2 shows the cytoto~icity of a tripeptidatsd drug (Boc-Pro-Gly-Arg- -
TAN-1120; ~ d its activated body (TAN-1120; - o -~ on rensl caIlcer cell
line AM-RC-6 (see Example 4).
Fig. 3 shows the cytoto~icity of a tr;peptidated drug (Boc-Gly-Gly-Arg-
PM; - 1~ -) and its activated body ¢M; - o -) o:n re:tlal cancer cell li:lle AM-RC-6
(see E~ample 4). ~ ~ .
Fig. 4 shows a chromatographic patl;ern of the trypsin-hydrolyzed
product of Boc-Gly-Gly-Arg-ADR ~see Example 6).
Fig. 5 shows a chromatographic pattern of the UK-hydrolyzed product
of Boc-Gly-Gly-Arg-PM ~see E~ample 6).
Fig. 6 shows the antibody dilution curve of the culture supernat~nt of
~nti hl~ anti UK bispecific antibody producing mou~e tetraoma IJTF 20-7.
(see Esample ~)
DETAILED DESCRIPIlON OF l~IE I~IENIION
The above-mentioned polydoma that produces a bispecific hybrid MoAb
is prepared, for example, by fusing a hybridoma that produces an anti-
human-cancer antibody with another hybridoma that produces an antibody
against a prodrug-activating en~nne. Any anti-h~ an-cancer-antibody-
producing hybridoma can serve for this purpose, as long as it produces an
an~body capable of specifically binding to human cancer cells. Examples of
2~ such hybridomas include mouse hybridoma 22C6 [IFO ~0172, P~13RM BP-
20~4] [cf. Jap~ese Unes~m;ned Patent Publication No. 79970/19901, which
produces aIl MoAb against human transferrin receptor (hereinafter also
referred to as h~YR), and mouse hybridoma RCS-l [IF() ~0184, FBRM BP-
2333] [cf. Japanese Patent Application No. 62939/1989], which produces an
MoAb against human rena} ca~cer. Representative e:cample~ of target
antigens for the anti-human-cancer antibodies produced by these
hybridomas~ i.e., target alltigens for cancer cell~ to which the bispecific hybrid
MoAb of the pre~ent invention binds specifically, include cancer cell
membrane surface antigens such as tumor-related antigens,
~5 immunocompetent cell surface receptors and virus-infected cell surface
a~tigens. Of thes0, hTfR, a tumor-related anligen, is often used. Other
wo gl/09134 2 0;6 9:4 3 9 4 PCT~/JP90/01631
e~amples of target antigens include carcinoembryonic antigen (CEA), a-
fetoprotein, some cancer-related sugar chain antigens such as CA19-9 ~S.
Hakomori: Cancer Research, 45, 2405 (1985)], the B-cell lymphoma
membrane immunoglobulin idio-type [R. A. Miller et al.: New England
Journal of Medicine, 306, 517 (1982)] and the T-cell lymphoma membrane
immunoglobulin idio-type [L. L. Lanier et al.: Journal of Immunology, 137,
2286 (1986)].
In preparing a hybndoma that produces sln antibody against a prodrug-
activating enzyme, an ordinary hybridoma preparation method is used EG.
Kohler et al.: Nature, 256, 495 (197~)]. For e~cample, a~imals are immunized
with the enzyme in accordance with a standard method, and the resulting
antibody-producing cells are fused with myeloma cells etc.
Examples OI immune animals include rabbits, rats, mice and guinea
pigs, with preference given to mice in the case of MoAb preparation.
Inoculation can be achieved by an ordinary method. For e2~ample, the mouse
receives subcut~neous or intrapelitoneal inoculation of the en~me at the
back or abdomen at a dose of 1 to 100 lIg, preferably 10 to 25 llg, in emulsion
in an equal volume (0.1 m~) of saline, in the presence of Freund's complet,e
adjuvant, 3 to 6 times once every 2 to 3 weeks.
Of these immune aI~imals, for example, mice, individuals with high
antibody titer are selected. Three to five days after final immunization,
spleens and/or lymph nodes are collected, and antibody-producing cells
contained therein are fused with myeloma cells. Fusion can be achieved in
accordance with a known method. E~camples offusogens include polyethylene
2~ glycol (hereinafter al~o referred to as PEG) and Sendai ViI us, with preference
given to PE~. Esample myeloma cell lines include N~1, P3U1 and SP210,
with preference given to P3U1. A preferred ratio of, for e~cample, splenocytes
and myeloma cells, is 1:1 to 10:1. It is recommended that this cell mi2ture be
incubated at 20 to 37C, preferably 30 to 37C, in the presence of a PEG with a
molecular weight of about 1,000 to 9,000 at a concenh ation OI 10 to 80% for 3
to 10 minutes.
Various methods are available for screeni~g the antibody-producing
hybridomas described above. For e~ample, human cancer cells or enzyme ~ `
proteins are adsorbed to a micI~oplate to prepare an an'dgen-sensit;i~ed plate,
to which i8 added the culture supernatant of the hybridomas obtained by cell
~usion. This is followed by determination of antibody titer in the culture
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. ::
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2069~39
WO 91/09134 -- 5 - PCr/JP90/01631
supernatant by enzyme immunoassay (hereinafter also referred to as EIA) for
detection of plate-bound specific antibody. Hybridomas positive for antibody
activity are selected, cultured in HAT (hypoxanthine-aminopterin-
thymidine) medium etc. and immediately subjected to cloning, which can be
done easily by the limiting dilution method. The antibody titer of the culture
supernatant of the cloned hybridomas is also determined by the EIA
procedure described above; monoclonal hybridomas that stably produce a
potent ant ibody can thus be se}ected and cultured. Irl this case, a hybridoma
that produces a neutralizing antibody against a prodrug-activating enzyme,
10 su~h as urokinase, can also be used as a parent cell in polydoma preparation.There are several methods of preparing a polydoma that produces the
bispecific hybrid MoAb of the present inven~ion [e.g., Yoji Aramoto et al.:
Protein~, Nucleic Acids and Enzymes, 33, 217 (19883]. All of them are
suitable; examples are as follows: 1) The above-mentioned HAT-resistant
,~ 15 hybridoma, th~t produces an antibody against a prodrug-activating enzg~ne,
is acclimated step-by-step to a culture medium containing 6-bromode-
o:cyuridine (hereinafl;er also referred to as 6-BrdU), and a thymidine kinase-
deficient strain is clolled to make it HAT-sensitive. Similarly, an HAT-
resistant hybridoma that produces an anti-human-cancer specific antibody is
20 made resistant to ~a~aguanine (hereinafter also referred to as 8-AZO, and a
hypoxanthine-guanine-phosphoribosyl transferase-deffcierlt strain is cloned
to make it HAT-sensitive. Next, these two cloned strains are fused by a
standard method to yield tetraomas, from which a tetraoma that secretes a
hybrid MoAb capable of binding to both human cancer cells and a prodrug-
25 activating enzyme i9 selected on HAT medium and cloned. 2) A hybridoma'chat produces an anti-human-cancer-cell specific antibody is labeled with
~luorescein isothiocyanate (hereinafter also referred to as FITC), and another
hybridoma that produees an antibody against a prodrug-activating enzyme is
labeled with tetramethyl rhoda~nine isothiocyanate (hereinafter also referred
30 to as TEUTC), followed by fusion of these t~vo in accordance ~vith a standardmethod. The resulting cell suspension is applied to a fluorescence-activated
cell sorter (hereinafter also referred to as FACS), and a tetraoma that shows
both the green fluorescence of ~llC and the red fluorescence of TR~C is
selected and cloned. Also, it is possible to use the markers for the two parents3~ in totally revers~ combination to select and clone the desired tetraoms.
.
.
. ~ ,, , ;;, . . . . . .
wo 91~09134 2 0 ~i 9 ~ ~ 9 6 - PCI/JP90/01631
These procedures of cell fusion employ a fusogen such as Sendai virus
or PEG, or a means such as electric stimulation. It is preferable to use PEG.
An example mode of PEG use is described below, but this is not to be
construed as limitative. A PEG with a molecular weight of about 1,000 to
9,000 is used at a concen~ation of about 10 to 80%; treatrnent time is about
0.5 to 30 minutes. As a preferred mode of use, about 35 to 5~% PEG 6,000 is
kept in contact with cells at 37C for about 4 to 10 minutes to achieve efficient
fusion.
Polydoma selection can be carried out using the EIAT medium
described above and other means. For this purpose, 8-AZG, 6-thioguanine (6-
TG) or 5-BrdU i~ used for drug acclimation to obiain corresponding drug-
resistant strains. Also, various election media are used to introduce a new
marker into fused cells. ~ ample~ of such selection media include media
supplemented with neomyci~ or hygromycin B [B. Sugden et al.: Molecular
and Cellular Biology, 5, 410 (1985)].
A~ stated above, it is also possible to use a method in which hybridomas
labeled with dif~erent fluorescent pigments are fused, followed by sorting of a
double-labeled hybrid hybridoma by means of FACS [L. Karawajew et al.:
Journal of Immunological Methods, 96, 266 (1987)~.
Various methods are available ~or screening hybrid antibody-
producing polydomas, including combinations of the following methods and
their modifications: (1) the method employing two kinds of EIA techniques
using the above-mentioned antigen-~ensitized plate to which human cancer
cells or en~ne have been adsorbed, (2) the EIA method in which the subject
culture supernstant is added to a micropl~te to which human cancer cells are
adhered, followed by addilion of a prodrug-activating enzyme labeled with
horseradish peroxidase (hereinafter also referred to as ~IRE') and detection of
bispecific antibody, and, when using an antibody against a prodrug-
activating enzyme belonging to a ~ubclass different from that of the a~ti-
human-cancer specific antibody, (3) the EIA method, in which the subject
culture supernat~nt is added to a microplate to which human cancer cells are
adhered, followed by addition of an ~IRP-labeled specific antibody against the
mouse IgG subclass and detecl~on of bispecific an~body.
The polydoma positive for bispeci~lc antibody activity is immediately
3~ subjected to cloning, easily be achieved by the limiting dilution method etc.
The antibody titer of the culture supernatant of the cloned polydoma is
.
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;~69~39
WO gl/0913~ - 7 - P~r/JP9û~Olfi31
determined by the method described above, and a polydoma that stably
produces a potent antibody i~ selected, whereby the desired polydoma ~e.g.,
mouse tetraoma UTF20-7(IF0 ~0260, ~ERM BP-3156) obtained in following
Example, or other methods) that produces the monoclonal bi~pecific antibody
5 can be obtained.
The polydoma of the prese~t invention described above can be
cultivated normally in liquid medium, or ill the abdominal cavity of animals
(e.g., mammalians such as mice) by a known method. Pu~ificat;ion of the
antibody in the culture broth or a~cites can be achieved using a combination
10 of known biochemical techni~ues. For ex~nple, the cell culture broth or
ascites fluid is centrifuged, and ~he resulting supernatant separated and
æubjected to salting-out (nonnally ~ith ammonium sulfate or sodium sulfate).
The re~ulting protein precipitate is dissolved in an appropriate solution,
followed by dialysis. The ~olution is then subjected to column
15 chromatography (u~ing an ion e~change column, gel filtration column,
protein A column, hydro~cyapatite column etc.) to separate and purify the
desired antibody. From each liter of culture supernatant, the separation and
purification procedures described above yield about 1 to 10 mg of a bispecific
MoAb of a purity not less than 90% by protein weight. Also, from 20 m~ of
20 ascites fluid, the same MoAb i9 obtained in an amount of about 2 to 20 mg.
The bispecific MoAb thus obtained is uniform as a protein and, for
e2ample, F(ab')2 fragments etc. capable of binding to both human cancer cells
and a prodrug-activa~ng e~zyme can be obtained, for e~ample, by l~eatment
wi1~h protease (e.g., pepsin). These fragments can be used for the same
25 purpose as the bispecific MoAb of the present invention.
A tetraoma formed between a hybridoma that produces an anti-human-
cancer-cell MoAb alld another hybridoma that produces an antibody against a
prodrug-activating enzyme i8 included i~ the polydoma that produces the
hybrid ~oAb of the present invention, but any trioma formed b0tween a
30 hybridoma that produces one MoAb and a cell that produces t~e other MoAb,
or a~y hybridoma obtained by immortalizing two kinds of cells that produce
respective Mo~bs using Epstei~-Barr virus or other means. and then fusing
them, can be used for the same purpose as 1 he above-mentioned tetraoma, as
long as it produces the bispecific MoAb of the present invenl;ion.
3~ When these polydomas produce mouse IgG MoAb, it is possible to
prepare a mou~e-human chimeric antibody by derivi~g a DNA that encodes a
' '' " ', ' " : : . ' "' ' ' ' ' ~' '
~69~39
wo gl/09~ 8 - PCr/JPsO/01637
variable or hypervariable region containing the ~ntigen recog~ition site for
said bispecific hybrid MoAb and binding thereto a gene that encodes the
constant region of human IgG, using a gene manipulation technique [Z.
Steplewski et al.: Proceedings of National Academy of Science, 85, 4852
5 (1988)]. Such a chimeric antibody serves well in administration to humans,
due to its low antigenicity.
In anticancer therapy using the bispecific MoAb of the present
invention or a selective anti-human-cancer-protein complex prepared from a
prodrug-activating en~yme and said bispecific MoAb, se~veral methods are
lO available, including (1) the method in which the bispecific MoAb of the
present invention is administered to the cancer patient, and after suf~lcient
time has elapsed for it to bind to cancer tissues and cells, the enzyme and thenthe prodrug are administered, (2) the method in which the bispecific MoAb
and the enzyme are administered simultaneously, followed by prodrug
1~ administration, and (3) the method in which the hybrid MoAb is reacted with
the enzyme, a~d after separation of the unreacted portion of the enzyme, the
resulting anti-human-cancer-protein complex is administered to the cancer
patient, followed by prodrug administration.
The bispecific MoAb or prodrug-activating enzyme of the present
20 invention, or an anti-human-cancer-protein complex prepared therefrom, can
be used for the treatment of various cancerous diseases in the form of a
preparation such as an injection, with or without being formulated with an
appropriate pharmacologically acceptable carrier, ewipient, diluent or other
additive, after gelm removal by filtration using, for example, a membraile
25 filter, as desired. Dose volume varies depending upon the type of target
cancer, symptoms, route of administration and other aspects; but, for
example, in intravenous admini~tration to an adult human patient, it is
normally about 0.02 to 1.0 mg/l{g, preferably about 0.04 to 0.4 mg/kg, daily, asthe bispecific antibody, or about O.Ol to 0.6 mg/kg duly, as the prodrug-
30 activating enzyme.
Any prodrug-activating enzyme can serve for the present invention, as
long as it shows prodrug-activating action, but it is preferable to use a
protease (e.g., urokinase (UE), trypsin), which cleaves peptide bonds, or a
glycosidase (e.g., glucuronida~e), which cleaves sugar chain bonds. Of these
35 enzymes, a protease, particularly urokinase, i8 preferred. Also, it is desirable
that the en~;yme be a human-derived en~nne whose blood level is low or
.
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wo 91~09134 - 9 - PCr/JP90~01631
which is not present in blood, and that the enzyme be produced by c~ncer cells
[J. C. Kirchheimer et al.: Proceedings of National Academy of Science, U~3A,
B6, 5424 (1989)]. For exarnple, in the case of urokinase, prodrugs comprising
a drug active body and an appropriate peptide (e.g., Gly-(~ly-Arg, Pro-Gly-
Arg, Pyr-Gly-Arg) bound thereto can be used. In the case of glucuronidase,
glucuronidated drugs can be used as prodrugs. VVhether the prodrug is a
peptidated drug or a glucuronidated drug, its toxicity is expected to be
extremely lower than that of the original drug active body, or even nontoxic;
therefGre, it is capable of being activated in the vicinity of the cancer tissue10 a~d selectively destroying the cancer tissue when used in combination with
an anti-human-cancer-protein complex comprising the bispecific antibody of
the present invention acnd a prodrug-activating enzyme immunologically
bound thereto.
Any anticancer agent can be used as the original drug for the above-
15 mentioned prodrug, but preference is given to those in clinical application
such as adriamycin, ci~platin, melphalan, methotrexate, mitomycin C,
vincristine, puro~mycin and phenylenediamine mustard. A~lso, highly
cytotoxic ansamitocins, TAN-1120 (represented by the following formula (II)
wherein X represen~s OH) and related compounds may be used as anticancer
20 agents. Examples of such compounds include compounds represented by the
following formula (I) ~cf. Japanese Patent Application No. 18B60/1989,
European Patent Publication No. 376176], their 4,~-deoa~y bodies, and
compounds represented by the following fo~nula (II) [c Japanese Patent
Application No. 178634/1989, Europea~ Patent Publication No. 376177] aDd
25 their salts. :~n this case, any ansa nitocin or TA~-1120 related compound canbe used, as long as it possesses anticancer activity. In any case, as stated
above, the drug is administered to the patient in the fo~m of a nonto~cic or
weakly toxic prodrug, and is decomposed by the anti-human-cancer-protein
complex of the pre~ent invention in the vicinity of t~e cancer tissue to exhibit30 its pharmacological activities.
... .. . ... , , . .. ;. - .-.. . ; .
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wo 91/0913q 2 Q 6 9 4 ~ 9 - 1~ - PCr/JPg0/01631
Angarrlitocins include compounds repre~e~ted by the following formula: :
OR
C~3
~o~ c~3
0 ~f~ N ,l o
~,
C~3 3 ~ `
: ~ .
15 whereiIl R represents a hydrogen atom or a carbo~ylic acid-derived acyl
group; Q represents a hydrosyl group (OH) or a mercapto group (SH); X
represents a chlorine atom or a hydrogen atom; Y repre~ents a hydrogen
atom, a lower alkylsulfonyl group, an alkyl group or an aralkyl group which
may have a substituent.
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WO 91/09134 ., - ?1-. pCI`/JP90/01631
TAN-1120 related compound~
o o~
5 ~X33
oc~3 0 08
1~ l~EI :~,'
cl3
~\a~, ' . ,,
ca3 ~3
wherein X represent~ a hydro2~rl group or a hydrogen atom. ~ :
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.:
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W~ 91/091~ 2 0 ~ 9 ~ 3 9 PCl/JP90/0163~ ~
EX~MPLES
The present invention is hereinafter described in more detail by means ::
of the following reference a~d working examples, but these are not to be
5 cons~ued as limitations on the scope of the invention.
The animal cell lines used in the reference and working e~amples are -:
in deposition as listed in the table below.
_ _ . _ _ '
. . (IFO) (FRI)
Ammal cell lme :CFO No. P 13RM No.
_ _ _
Mouse hybridoma UK 1-3 60176 BP-2083 ; - ~
. . _ . . ~
Mouse hybridoma UK 1-87 50177 BP-2084
Mou~e hybridoma UK 1-6 ~0208 BP-2~48
Mouse hybridoma RCS-1 50184 BP-2333 : ~:
. _
Mouse hybridoma BG1-~ ~0219 BP-2688
Mouse hybridoma 2ac6 ~0172 BP-2064
Mouse tetraoma U~20-7 60260 BP-3166
. :
I:FO: InstituteforFermentation,Osaka
FRI: Fe~mentation Research Ins~tute, Agency of Industrial Science
and Technology, ~ulinistry of International Trade and Industry,
Japan
- .
~ - . .. .. ., . - - , . , . ., .. .. , . - - - ,: :. , i
~uv~
WO 91/09134 - 13 - , P~r/JP90/01631
In the present specification, amino acids and peptides are represented
by the abbreviation system adopted by the IUPAC-IUB Commission on
Biochemical Nomenclature (CBN). For e~ample, the symbols given below are
used. When there is a possibility that an optical isomer is present in a give~
amino acid, the symbol represents the ~body unless otherw~se stated.
Gly: Glycine residue
Pro: Proline residue
Arg: Arginine residue
Pyr or PyroGlu: Pyroglutamic acid residue
ReferenceE~amplel Mi~edhçma~lutination(MHA) -
Of the subject cells, adherent cells were dispensed to a 60-well
microplate (produced by Nunc Intermed) at 500 cells per well and cultured for
24 to 48 hours. Non-adherent cells were suspended in a ser~m-free culture
16 medium and dispen~ed to a plate at the same ratio 8S above, followed by
centrifugation at 400 X g for ~ minut~s to adsorb them to the plate, on the day
of examination.
For preparing indicator blood cells, sheep red blood cells were wa~hed
three times with phosphate buffered sali~e (20mM bisodiumphosphate,
0.15M NaCl; pE7.5) (hereinafter also referred to as PBS) and suspended in
PBS to obtain a 2% suspension. This suspension was mised with an equal
amount of mouse anti-shee~red-blood-cell antibody (produced by Ortho Co.),
diluted with PBS to a concentration 2.5 times the ma~cimum agglutination
value, followed by reaction at 37C for 30 minutes. These blood cells were
wa~hed ~hree 'dmes with PBS and resuspended at a concentration of 2%.
Then, an equal amount of rabbit anti-mou~e-IgG antibody (produced by
Cappel Laboratories), diluted 25-fold ~rith PBS, was added, followed by
reaction at 37C for 80 minutes. The reac~on mixture was then washed three
times with PBS and stored as a 2% suspension.
The cell-adsorbed plate was washed by sequential additions of a
veronal-buffered saline (pH 7.4; hereinafter also referred to as VBS)
containing 0.1 M MgC~2-0.03 M CaC~2-0.1% glucose and a solution
containing 6% fetal calf serum (6% FC~VBS). Subsequently, sample solu~on
cont~ning a mouse an~i-human-cancer antibody was dispensed to each well,
and the plate was kept standing at room temperature for 1 hour. After plate
washing with VBS, indicator blood cells, in 0.2% dilution in 6% FCS-VBS, -~
:' ~
wo gl/09134 2 ~ 6 9 '1 3 9 PCI`/JP90~01631
~'
were dispensed to each well, and the plate was kept standing at room
temperature for 40 minutes. Negt, the unreacted blood cells were washed
away with VBS, and the plate was observed microscopically. In the con~rol
test, in which no antibody was added, a rosette was found in not greater than
5 1% of the cells. A "positive" test was definded as a rosette formed by not less
than 25% of the target cells.
ReferenceE~ample2 ~nmunofluorescence(IF)
After cultivation, the subject cells were suspended in a 0.02% EDTA~
10 PBS solution. This suspension was washed with a serum-free culture
medium, and a solution containing a mouse anti-human-cancer antibody was
added, ~ollowed by reaction at 4C for 1 hour. After washing with culture
medium, fluorescein-labeled anti-mouse-Ig~ antibody was added, ~ollowed by
reaction at 4C for 1 hour. After washing with PBS, the reaetion product was
15 observed using a fluorescent microscope.
Reference Esample 3 Cell EIA usin~ tumor cells
Target tumor cells were seeded to a Nunc :[~termed 96-well microplate
at 10,000 to 40,000 cells per well, followed by incubation in a carbon dio~ide
20 incubator at 37C for 1 day. Af~er culture supernatant removal, a solution
containing a mouse anti-human-cancer antibody was added, followed by
reaction at room temperature for 2 hours. The plate was then washed with a
medium ~upplemented wil~ 0.2% bovine ser~m albumin (hereinafter also
referred to as BSA); a rabbit anti-mouse-IgG antibody labeled with
25 horseradish perosidase (hereina~cer also referred to as HRP) was then added,
followed by reaction at room temperature for 2 hours.
After plate washing, a 0.1 M citrate buf~er containing ortho-
phenylenediamine a the enzyme substrate and H22 ~ras added to each well,
followed by en~ne reaction at room temperature. ~tier ~a reaction was
30 te~minated by the addition of 1 N sulfur~c acid, the developed color was
measured at a wavele~gth of 492 nm using Multiscan (produced by Flow Co.).
Reference E~ample 4 Pre~paration of hybridoma that Produces anti-human-
renal-cell-cancer monoclonal antibodY
(1) Euman renal cell cancertransplalltation and serumimmunization
., - ,. . . . . .
~ J ~
Wo 91/09134 - 15 - PCr/JP~01631
A 2 x 2 mm tissue section was collected from a renal csncer patient's
tumor tissue and ~ransplanted ~ubcutaneously to a nu/nu-BALB/c mouse.
Upon subculture (nonnally 3 to 4 weeks after transplantatiorl) of stable cell
line AM-RC-3, serum wa~ collected from the mouse recipient. A 0.5 m~
portion of the serum was mixed and suspended in an equal amount of
Freund's complete adjuvant. The resulting suspension wa~ intraperitoneally
administered to the same line of BALB/c mou~e. Therea~er, the mouse was
immunized with 0.~ m~ serum from the above-mentioned recipient nude
mouse at intervals of absut 7 to 10 days. After a total of 7 immuni~ations,
a~tibody titer was dete~mined.
By the MHA method described ill Reference E~ample 1, mice showing a
high antibody titer again~t renal cancer cell line AM^RC-7 were subjected to
the following e~perixnent.
(2) eparationofhs~bri oma
The immune mou~e splenocyte~ obtained in (1) were fused with mouse
myeloma cell line N~l by a standard method, ~ollowed by selection culture
using ~AT medium. The hybridomas grown were subjected to ~creening by
the MHA method described in Reference Example 1, and the group of
hybridomas showing high antibody titer were further cloned to yield the
desired mouse hybridoma RCS-1 (FERM BP-2333, IFO 50184), which
produces an anti-human-renal-cell-cancer MoAb. The RCS-1 antibody
produced by the mouse hybridoma RCS-1 proved to belong to the IgG
subclass.
(3) Production of mouse MoAb
5 X 106 cells of mouse hybridoma RCS-1 were intraperitoneally
administered to MCEt(AF)-nu mice. About 4 weeks later, ~ to 10 m~ of ascites
fluid was collected. After salting-out with ammonium sulfate, the ascites
~uid wns puri~led using a DEAE-cellulose columm About 200 mg of pulified
mouse anti-human renal-cell-ca~cer MoAb RC~1 was obtained from 50 m~ of
ascites fluid.
(4) ProPerties of mouse MoAb
Reacti~i1y of RC~1 sntibody against various tumor cell lines was
determined by the methods described in Reference E2amples 1, 2 and 3. RC~
,, . ~. .
wo 91/09134 2 ~ b' 9 ~ 3 9 - PCI/JP90/01631
1 antibody was found to be positive for renal cancer cell lines AM-RC-3, AM-
RC-6 and AM-RC-7, bladder cancer cell line T24 and lung cancer cell lines
Luci-10 and PC-10, and negative for other cancers, namely gastric cancer,
intestinal cancer, breast cancer and leukemia cpncer cell lines; it was also
negative for normal renal tissues.
:
Reference Example 6 EIA for anti-UX aIItibod~assa~r
A 5 ll~/m~ UK solution was di3pensed to a 96-well microplate at 100 p~
per well. After the microplate was kept ctanding at 4C overnight, 150 1l~ of
10 PBS containirlg 2% casein and 0.01% thimerosal was added, to prepare a
sensiti~ed plate. After remoYing tlle added solutio~, the plate was washed
with PBS containing 0.06% Tween 20 (hereinaf~er also referred to as PBS-
l~v), and 100 }1~ ofthe subjectmouse antibody solution was added, followed by `~
reaction at room temperature for 2 hours. Similarly, after the plate was
15 thoroughly washed with PB~I~r, an HRP-labeled rabbit anti-mouse-IgG
antibody was added, followed by reaction for a hours.
The procedure described in Reference E~cample 3 was then followed to
determine the HRP activity bound to the solid phase.
20 Reference Example 6 EIA for anti-low-molecular-UK antibodY assaY
Using a low molecular UR (two chain-low molecular UK, supplied by
JCR Co.) in place of the UK described in Reference Example 5, a plate
sensitized with a low molecular UK was prepared, and anti-low-molecular-
IJE antibody titer was deteImined by the same method.
Reference Example 7 FibrinolYsis neutralization e~cPeriment
To a UE solution (final concentration 2B ng/m~), the subject anti-UK
antibody solution was added, followed by reaction at 37C for 1 hour. The
reaction mi~ture was then i~ected to a fibrin agarose plate at 6 }1~ per well.
30 After incubation at 37C for 2 to 6 hours, ~lbri~olysis plaques (diameter) were
measured to calculate the neutralizing capability of the anti-UK MoAb on
UK enzyme activi1 y.
.
Reference ~xample 8 Preparation of h,ybridoma that Produces mouse anti-
3~ URmonoclonalantibody
,~
; .
''` ' ' .' '` ,'''` i' '`', ` ' ` , . . ' . ." ' . ' ' '
2069~39
WO 91/09~ 17 - PCr/JPgO/01631
(1) Immuni~ahon
To a 200~ug/me solution of a commercially available UK (produced by
Nippon Seiyaku) in saline, an equal amount of Freund's complete adjllvant
was added; this mixture was thoroughly emulsified. The resulting emulsion
5 was intraperitoneally and subcutaneously (at the back) administered to
BALB/c mice (female, 20 ~g/0.2 mUmouse), ~ollowed by booster immunization
at intervals of 2 to 3 weeks. The animals sho~nng ma~imum ser Lm antibody
titer at 10 days after the third booster immuni~ation received intravenous
administration of a UE antfgen solution (50 ~g/0.1 m~ saline/mouse).
(2) Cell fusion
Spleens were excised 3 days after final immunization, alld a splenocyte
suspension was prepared by a standard method (about 108 cells). Then, 2
x 107 mouse myeloma cells (P3U1) were added, and cell fusion was carried
out using PEG 6000 by the method of Kohler a~d Milstein [Nature, 256, 495
(197~1].
After completion of fusion, the cell mi~ture was suspended in what is
called H~T medium, containing hypoxanthine, aminopterin and thymidine?
followed by cultivation for 10 days. l~nrnediately after parent cell selection,
the HAT medium was replaced ~rith IIT medium of the same composition a~
EAT medium but lacking aminopte~in, and cultivation was continued.
(3) HYb~doma selectio~l and cloning
The aIltibody titer of the hybridoma culture supernatant was
2~ determined by the EIA metllod described in Reference E~ample ~, u~ing a
UK-coupled microplate. At lV to 20 days following fusion, hybridomas began
to appear, along with an aIltibody that specifically binds to UE. The
hybridomas showing particularly high af~mity were ~ubjected to cloning by
the limiting dilution method.
The culture supernatant of the cloned hybridoma was subjected to
screening in the same man~er; those having high UK af~mity were selected.
As a result, UK1-3 [~ERM BP-2Q83, IFO 50176] and UE1-87 ~FERM BP-
2084, D~O 50177] were obtained, both mouse hybridomas that produce an
MoAb that specifically binds to UR:. The immunoglobulin classes and
subclasses of the antibodies produced by these hybridomas were identified by
the Ouchterlonymethod as IgGl and IgGzb, respectively.
.
WQ 9 1 /09 134 -18 - PCr/JP90/01631
~6'gg~9
Reference Example 9 Preparation of hYbridoma that produces mouse anti-
low-molecular-UK monoclonal antibody ~:
6 (1) Tmn~unization
Mice were immunized in the same manner as in Reference E~ample 8-
(1) e2ccept that a commercially a~railable two chain-low molecular UK
~supplied by JCR Co.) was used in place of the UE described therein. ;~
(2) Cell fusion
Cell fusion was camed out in accordance with the method desc~ibed in
ReferenceEsample 8-(2).
(3) Hsrbridoma selection and clonin~
;
Hybridoma screening was carried out by the ~IA method, described in
~; Reference Example 6, using a microplate to which low molecular UE was
adsorbed; hybridomas that produced an anti-low-moleculsr-UK MoAb were
obtained in the same manner as in Reference Example 8-(3) . Out of them,
mouse hybr~doma UK1-6 ~O 50208, FERM BP-2548], a hybridoma that
produces an anti-low-molecular-U~ MoAb capable of specifically binding to
UK without degrading the ffbrinolytic capability thereof, was obtained. The
immunoglobulin class and subclass of the antibody UK 1-6, produced by the
hybridoma thus obtained, was identi~led as IgGl (~ chain) by the Ouchterlony
; method.
; 25
Reference Example 10 EIA for anti-~lucuronidase antibodY assaY
A~ antigen-sensitized plate was prepared in the same manner as in
Reference Example ~ e~cept that @-glucuronida~e (produced by Sigma Co.)
was used in place of the UE described therein. EIA was then carried out in
the saIne manner as in Reference E~ample 5, to determine the anti-
glucuronidase antibody titer.
Reference 13~cample 11 Glucuronidase enzYme reaction neutralization
e~Periment
36 An antibody-sensitized plate was prepared in the same manner as in
Reference Example 5 except that an anti-mouse-IgG antibody was used in
'' :.
"
~6g~39
WO 91/09134 - 19- PCI/JP90/~1631
place of the UK described therein. The subje~t mouse anti-glucuronidase
antibody solution was then added, followed by reaction at room temperature
for 2 hours. A~er plate washing with PB~I~v, a 25 ~ -glucuronidase
solution was added, followed by reaction at room temperature for 2 hours.
After the plate was thoroughly washed, 1.0 mM synthetic substrate p-
nitrophenyl-~-D-glucuronide in solution in 0.14 M acetate buf~er (pE 4.6)
containing 0.14% l~iton X-100 was added, followed by enzyme reaction at
37C for 40 minutes. After te~ninating the reaction by the addition of 2.6 M
2-amino-2-methyl-1,3-propanediol, the amount of pigment formed was
10 measured at 415 nm using Multiscan.
:`
Reference Example 12 Pre~aration of anti-hTfR-antibodY-Producin~
hYbridoma
(1) Puriflcation of hTfR
1~ 1.5 kg of human placenta tissue was cut into small pieces and blended
in PBS (p~I7.5), followed by centrifilgation. The resulting sediment was
homogenized in PB~3 containing 4% Iriton X-100. This homogenate was
ultrasonicated and then centrifuged. To th~ resulting supernatantwas added
ammonium sulfate at about 32 g per 100 m~ supernatant. After salting-out,
20 thi9 mi2ture was applied to a colurnn coupled with anti-hTf antibody, followed
by thorough washing with 20mM disodium phosphate buffer (hereinafter also
referred to as PB), pE[ 7.5, containing 0.5 M NaCl. The hl~ fraction eluted
with a 0.02 M glycine buf~er solution (pH 10.0) containing 0.5 M NaCl and
0.5% Triton X-100 was applied to an hTf-coupled column. After the column
25 was washed with PB containing 1 M NaCl, elution was conducted using a
0.05 M glycine buffer solution (pH 10.0) containing 1 M NaCl and 1% Triton
X-100 to yield about 1.5 mg of a purified sample of hTfR.
(2) Immunization
To a 200~g/m~ solution of the above purified sample of hTfE~ in
30 physiological saline was added nn equal volume of Freund's complete
adjuvant, followed by thorough emulsi~lcation. I~e resulting emulsion was
then administered intraperitoneally and subcutaneously at the back to
BALB/c mice ~female, n = 10, 20 ~g/melmouse). Additional immunization was
conducted at intervals of 3 weeks. The animal that showed the ma~imum
3~ serum antibody titer 2 weeks after 4 additional immunizations ~vas
. .
wo gl/ngl34 2 0 6 9 ~ ~ 9 20- pcr/Jp9o~ol63r
intravenousl~ given the same hTfR antigerl solution as speci~led above
~30 ~lg/0.1 m~ physiological saline/mouse).
(3) Cell fusion
3 days after the final immunization, the spleen was excised and a
5 splenocyte suspension was prepared by a conventional method
(approximately 108 cells). To this suspension was added 2 X 107 mouse
myeloma cells (P3U1)9 followed by cell fusion in accordance wit~ t~e method
described in Reference Example 8-(2). After selection of parent cells in HAT
medium, cultivation was continued using ET medium which had the same
10 composition as that of HAT medium, but not including aminopterin.
(4) Selection and clonin~ of hYbridomas
A commercially available anti^mouse IgG rabbit antibody solution
(20 llg/mO was dispensed to a 96-well microplab at 100 p~ per well. After
this microplate was allowed to stand at 4C overnight, PBS (plI 7.3)
1~ containi~ng 2% BSA was added to prepare a sensitized plate. The purified
sample of hl~R obtained in (1), aflier being labeled with HR~? in accordance
with a conventional method, was used for EL9 [T. Eitagawa: Yuki Gosei
Kagaku, 42, 283 (1984)]. Accordingly, the culture supernatant of hybridomas
was added to the above second antibody-sensitized plate, and reaction was
20 carried out at room temperature for 2 hours. After the plate was washed with
PBS, HRE'-labeled hlYR ~vas added, followed by reaction at room temperature
for 2 hours. En~nne reaction was then carried out by the method described in
Reference Example 3, to determine the antibody titer.
The hybridoma showing especially high binding actiuity was subjected
25 to cloning by limil~ng dilution method to yield ænti-hlYR-antibody-producing
hybridoma 22C6. The present antibody was identified as the IgGl (K chain)
subclass, e~chibiting high affinity to human leul~emia cell strain K562 aIld
human epidermoid carcinoma cell line A431.
. '` ', . . ' . ' ._ ' , ," ' ~ . , ' ' ,~ ' '
~ u u ~
WO 91/0913'~ - 21 - . PCT'/JP90/01631
Example 1 Preparation of hybridoma that_produces anti-~lucuronidase
monoclonal antibodY
(1) Immunization
To a 500 ~g/m~ solution of a commercially available ~-glucuronidase in
saline, an equal amount of Freund's complete adjuvant was added; this
mi2ture was thoroughly emulsified. The resulting emulsion was
intraperitoneally and subcutaneously (at the back) administered ~ BAI.B/c
mice (female, ~0 ~g/0.2 ~/mounse), followed by booster immu~ization at
intervals of 2 to 3 weeks. The animals showing ma~imum serum antibody
ti~er at 10 days after 'che third booster immunization receiYed i~t~avenous
administration of a ,~glucurollidase solution ~100 1lg/0.2 m~ salineJmounse).
(2) Cell fusion
Spleens were e~cised from mice that showed high serum antibody titer,
as determined by the EL~ mei;hod described in Reference Example 10; cell
~usion was carried out in accordance with the method described in Reference
Example 8-(a).
,
(3) H~rbridoma selection and clonin~ ' '
.
Fused cells that appeared at 10 to 20 days following fusion were ~''
screened by the ELA method described in Re~erence E~ample 10; the
hyb~idomas showing particularly high af~nity were subjected to cloning by
th~ limited dilution method.
The cloned hybridomas were selected in the same EIA method,to yield
BG1-5 [~ q BP-2688, ~FO 60219], a mouse hybridoma that produces an
MoAb that specifically binds to glucuronidase. The iantibody produced by this
hybridoma was identified as IgGl. The neutraliza~on e~periment described
in Reference E~ample 11 revealed that this antibody does not neutralize
glucuronida~e enzyme activity.
E2ample 2 Preparatio,n of hybrid hybridoma that produces anti-human-
cancer-cell-anti-UE bisPecific antibod
3i6
(1) Cell fusion ' -
wo gl~ogl~ ~ O ~ ~ ~ 3 9 22 - PCr/JP90/Q1631 ~
Hybridoma RC~1, which produces an ~ti-hum~n-renal-cell-cancer MoAb,
obtained in Reference E~gample 4, and hybridoma UK1-6, which produces an
anti-UK MoAb, obtained in Reference Example 9, were each incubated in
Iskove-Ham F-12 mixed medium containing 0.~ Ilg/m~ ~TrC and 1.5 llg/m~
5 TRITC at 37C ~or 30 minutes for fluorescent staining. A~ LSM ~olution
(commercially available from Wako Pure Chemical Irldustries Ltd.) was then
added, and the dead cells were remoYed; the two hybridomas were then mixed
at a ratio of 1 to 1 for cell fusion using P~G 6000 by the method described in
Reference E~ample 8-(2).
After incuba~ion at 37C for 2 hours, the cell ~xture was applied to
FACS, and 2~00Q lluorescein-rhodamine double stained cells were separated
and seeded, at 10 cells per well, $o a 36-well microplate seeded with 5 X 105
cells/well mou~e thymocytes as feeders, and c~l'dvated.
1~ (2~ Hybrid hybridoma selection and clonin~
The culture supernatant from each well in which cell gro~vth occurred 1
to 2 weeks after fusio~ was subjected to Cell-EIA to determine the bispecific
antibody titer. Specifically, to the microplate coupled with renal cancer cell
AM-RC-7, prepared in Reference Example 3, the subject hybrid hybridoma
20 culture supernatant was added, followed by reaction at room temperature for
a hours. After plate washing with 0.2% BSA medium, biotin-labeled UK was
added, followed by reaction at room temperature for 2 hours. After FlRP_
labeled avidin reaction at room temperature for 1 hour, the plate was washed
a~d the enzyme activity bo~ded to the solid phase was dete~mined by the
25 method described in Refere~ce Example 3.
The cells in wells showing high bispecific antibody titer were subjected
to cloning by the limiting dilution method, yielding the desired bispecific-
antibody-producing tetraoma.
30 (3) Purification of bis~ecific antibod.Y
To BALB/c mice pretreated by intraperitoneal administration of 0.5 m~
mineral oil, mouse hybrid hybridomas (tetraomas) were inoculated
intraperitoneally at 5 X 106/mouse. Ascites fluid, whose rete~tion occurred
about 10 to 20 days after inoculation, was collected and subjected to salting-
35 out with 50% saturated ammonium sulfate to yield an IgG fraction. Afterdialysis with 20 mM PBS (pH 7.5), the IgG fraction was applied to a U~-
;
......... . . . .. . . . . .................. . .
. ~ , . ,., . ., . . .: .. . . . . . . , : ~
2~69~9
Wo 91J0913 - 23 - PCl/JP90/01631
coupled Cellulofine colnmn, followed by elution wi'ch 0.2 M glycine-HCl buffer
at pH 2.9. After dialysis with PBS, the acid-eluted fraction was applied to a
hydro2yapatite column to purify the de~ired bispecific anti-human-cancer-
cell-anti-UK antibody.
E~ample 3 Svnthesis of triPePtidated drUF 1 :
(1) Svnthesis of G1Y Ar~F OMe
To a solution of 4.6 g of carbobenzylo~yglycine (ZGly) in 20 m~ of
dimethylform~mide (DMlF), 4.34 g of N-hydroxy-~-norbornene-2,3-
dicarbo~yimide (~IONB) and 4.9 g of dicyclohesylcarbodiimide (DCC~ were
added with ice coolirlg, follo~ed by s~irring for 3.~ hours. Then, 0.7 g more ofHONB and 0.8 g more of DCC were added, followed by stirring for 2 hours.
Af~er the reaction mi~ture was filtered, the resulting filtrate, along with 5.22g of arginine methyl ester (Arg-OMe) hydrochloride, was added to a solution
of 2.8 m~ of triethylamine in 30 me D~!CF with ice cooling. After stirring at
room temperature for 2 hours, the misture was kept standing overnight.
After precipitate removal by filtration, the filtrate was concentrated under
reduced pres~ure. The resulting residue was diluted with water and washed
2a wit~ ethyl acetate. The water layer was concentrated under reduced pressure
to yield a colorless oily substance (9.65 g). This oily substance was dissolved
in 170 m~ of methanol (MeO~I) containing 20 me of 1 N ~IC~, followed by
catalytic reduction in a hydrogen stream in the presence of palladium black
(500 mg). After sti~ring in the hydrogen streiun for 4.6 hours, the catalyst
2~ was removed by filtration, and 1 he filtrate was concentrated under reduced
pressure. The resulting re~idue was stored in DMF (30 me) solution for use
for the nest reaction.
(2) ~nthesis of Boc-Gly GlY-Ar~-OEI
To a solution of 176 mg of t-butylo~carbonylglycine (Boc-Gly) in 1 m~
of D~F, 197 mg of ElONB and 2~L8 mg of DCC were added, followed by stirring
with ice cooling. After precipitate removal by filtration, the filtrate was
added to a mixture of a solution of the Gly-Arg-OMe obtained in (1) in DMF (2
mO and triethylamine (187 p~) ~hile stirring. Aflcer the resulting mixture
was kept standing in a re~rigerator overnight, the precipitate was r~noved by
filtration. The residue obtained by concentrating the filtrate under reduced
; . . , ~ .. - , ,, . , . ; .. ,. ., ~ .... . ~ , . ...
WO 91/09134 ~ o ~ 9 PCl'/JP90/0163
pressure ~as subjected to colu~ chromatogr~phy using silica gel (10 g),
followed by elution with ethyl acetate-pyridine-acetic acid-water (60:20:6:10)
to yield 210 mg of Boc-Gly-Gly-Arg-OMe. Then5 170 mg of the Boc-Gly-Gly-
Arg-OMe was dissolved in 1 m~ of a 1 N NaOH solution with ice cooling, and
5 the reaction mixture was applied to cationic exchange resin (Biorex 70). The
effluent fraction wa3 collected and converted to HC~ salt by the addition of 1
N HC~. The solvent was distilled of ~under reduced pressure to yield Boc-Gly-
Gly-Arg-OE (148 mg).
NMR ~D20) o: 1.42 (9H, s, CH3), 1.~7-1.80 (4H, m, CH2), 3.19 (2H, t,
CH2N), 3.81 (2E, s, CH2CO~, 3.95 (2H, s, CH2CO), 4.13-4.30 ~1H, m, N-
CHCV)
MS mlz: 389 [M+E]+, 289 ~M-Boc+ 2H]+
(3) S~rnthesis of Boc-Pr~GlY-ArF-OH
To a solution of 410 mg of t~ut;yloxycarbonylglycîne (Boc-Pro) in 2 m~
of I)MF, 430 mg of HONB and 494 mg of DCC were added, followed by stirring
with ice cooling. Afl;er precipitate removal by filtration, the fllltrate was
added to a mi~ture of a solution of the Gly-Arg-OMe obtained in (1) in DMF (4
mO and triethylamine (374 u~) while stirring. After this mixture was kept
standing over~ight, the precipitate was removed by filtration. The residue
obtained by concentrating the filtrate under reduced pressure was subjected
to column chromatography using silica gel (20 g), followed by elution with
ethyl acetate-pyridine-acetic acid-water (60:20;6:10) to yield 718 mg OI Boc-
Pro-Gly-Arg-OMe. Then, 398 mg of the Boc-Pro-Gly-Arg-OMe was dissolved
in 2 m~ of a 1 N NaOH solution with ice cooling, and the reaction mi2~ture was
applied to anionic e~change resin (AG-1 X 8). The effluent fraction was
collected and applied to cationic exchange resin tBiorex 70). The effluent
fraction was collected and converted to HC~ salt by the addition of 1 N ~IC~.
The solvent was distilled off under reduced pressure to yield Boc-Pro-Gly Arg-
OlI(280mg).
MS m/z: 429 [M~lI] +, 829 [M-Boc~ 21I]+
(4)SYnt~esisofPvroGlu-GlY-Ar~-OH
To a solutiun of 129 mg of pyroglutamic acid in 1 m~ of D~?, 197 mg of
:EIONB and 248 mg of DCC were addedt followed by stirring with ice cooling.
Afl;er precipitate removal by filtration, the filtrate was added to a mixture of a
. ~ . - . . . ,., - .,, ~ , . . - . . . ... .. . . . . .
. ~ - .. . .. . . ~ . . . - . .
. . . , . . ~. - . . ................................... .
-: - . ; . . . . . . : .
2~9~39
WO 9 1 /09 1 34 - 25 - PCI /JP90/01 631
Gly-Arg-OMe solution in DMF t2 me) and triethylam~ne (187 11~), followed by
the same treatment as (2) to yield 244 mg of PyroGlu-Gly-Arg-OMe Then,
244 mg of the PyroGlu-Gly-Arg-OMe was dissolved in 1 m~ of a 1 N NaOH
solution with ice cooling. After stirring, the mixture was applied to cationic
e:cchange resin (Biorex 70), and the effluent fraction wa collected and
converted to HC~ salt by the addition of 1 N HC~. The solvent was distilled off
under reduced pressure to yield PyroGlu-Gly-Arg-OH ~200 mg).
MS m/z: 343 [M+H~+
(5) Synthesis of Boc-Glsr-~ -adriamycin
A solution of 28 mg of Boc-Gly-Gly-Arg-OH and 11 mg of 1-
hydro~ybenzotriazole (HOBT) in 0.3 m~ of DMF wa~ added to a solution of 6.4
mg of adriamycin (ADR) hydrochloride and 3 lle of N-ethylmorpholine in 0.1
m~ of DMF, followed by stirring. After 3.8 mg of an aqueous ~olution of
carbodiimide (WSC) was added to the reaction mi~ture described above, the
solvent was distilled off under reduced pressure. To the residue, û.2 m~ of
DMF, and then 8 mg of EIONB, 9 ~u~ of N-ethylmorpholine and 24 mg of WSC
were added, followed by stirring at room temperature. After the reaction
mi2cture was concentrated under reduced pressure, water was added to the
residue. This residue dilution was added to a suspensioll of reversed phase
silica gel ~ 8) in 5% CH3CN-H20, followed by puriffcation with an
increasing density gradient of CH3CN. Finally, elution was camed out using
CE3CN-H20-2M ammonium acetate (2:2:1). Af~er the C~I3CN was distilled
o~, the eluted frac~on was estracted with n-butanol. The resul~ng n-butanol
est~act was dried over sodium sulfate, and the solvent was distilled off under
reduced pressure to yield Boc-Gly-Gly-Arg-ADR (3.55 mg).
MS m/z: 914 [M~:~I]+
~6) S~rnthesis of Boc-Pro-GlY-Ar~-ADR
To a solution of 5 mg of ADR and 3 lI~ of N-ethylmorpholine in 0.5 m~ of
DMF, a solution of 18.1 mg of Boc-Pro-Gly-Arg-OE, lO mg of ~IONB and 10
mg of WSC in Dl!lCF was added, followed by stirring. The misture was then
treated in ~he same manner as (6) to yield Boc-Pro Gly-Arg-ADR.
MS m/z: 9~4 ~M~l~]+
36
(7) S~nthesis of P~rroGlu-Glv-Ar~-ADR
. . - .. . . . , . . . ~ i . . . ~ ~ . .. .
- . - - ,, .. - .,.. ,, - ........ . ,. , ........ . ,, ,. , , , ~, . - . . ..
..... .-.. . , ~ ; ~ ; . . . - , , . .. ; . , .
WO 91/09134 - 26 - PCr/JP9~)/û1631
2~9d39
To a solution of 5 mg of ADR and 3 ~ of N-ethylmorpholine in 0 5 m~ of
D~?, a solution of 20.1 mg of PyroGlu-Gly-Arg-OH, 10 rng of ~IONB and 10
mg of WSC in DMF was added, followed by stirring. The mi~ture was then
treated in the same manner as (5) to y~eld PyroGlu-Gly-Arg-ADR (0.14 g).
5 MS mlz: 867 [M+ H]+
(8) SYnthesis of Boc-Pro GlY-ArF-T~-~:120
To a solution of 1.7 mg of TAN-1120, 4.2 mg of Boc-Pro-Gly-Arg-OH
and 1.3 mg of HONB in 0.5 me of DMF, 3.0 mg of WSC was added, followed by
10 stirring at room temperature. The same treatment as (~) was followed to
yield Boc-Pro-Gly-Arg-TAN-1120 from the fraction eluted with 40% CH3CN-
H20.
MS ml~: 1082 [M~E]+
(9) S~ hesis of Boc-Gly-My-Ar~-purom~cin
To a solution of 6.2 mg of puromycin (PM)-2~IC~ and 61l~ of N-
ethylmorpholine in û.1 m~ of DMF, 17.7 mg of Boc-Gly-Gly-Arg-OH, 7 mg of
l-hydro~ybellzotriazole and 4 mg of water-soluble carbodiimide were added,
followed by stirring at room temperature overnight. After the solvent waæ
distilled off under reduced pressure, the residue was diluted with water and
subjected to colum~ chromatography using reversed phase silica gel (RP-~)
for purification with an increased CH3CN density gradient from 5% CH3CN-
H20. The fraction eluted with CH3CN-H20-2M ammonium acetate (2:2~
was collected. After the CH3CN was distilled of ~ under reduced pressure, n-
butanol estraction was carried out. Af~er the n-butanol extraet was dried
over anhydrous sodium sulfate, the solvent was distilled off under reduced
pressure to yield Boc-Gly-Gly-Arg-PM (3.0 mg).
MSm/z: 842 [M+H]+, 742 EM-Boc+21~13~
Example 4 C~rtotoxici~r oftripePtidated dru~s
'l~he cytotoxicities of the three kinds of tripeptid~ted drugs obtained in
Esample 3 ~Boc Gly-Gly-Arg-ADR, Boc-Pro-Gly-Arg-'rAN-1120 and Boc-Gly-
Gly-Arg-PM, respectively) [Figs, 1 through 3; - ~ -] on gasl~ic cancer cell lineNUGC4 or re~al cancer cell line AM-RC-6 were compared wil~h those of their
activated bodies (ADR, TAN-1120 and PM, re~pect;~vely) [Figs, 1 through 3; -
o -]. Specifically, each drug was added at various concentrations to a
microplate ~eeded with cultured human cancer cells at 5 X 103 cells/well,
" .
. . , ~, .. .
h~ ~J u ~ ~ ~J a
wo 9~/09134 - 27 - Pcr/Jp9~/01631
followed by cultivation for 4 days. Then, in accordance with a known method,
viable cells were counted using 3-(4,5-dimethylthiazol-2-yl)-2,6-
~phenylte~azolium bron~de (~rrr) ~H. Tada et al.: Jounalof~mununolo~cal
Methods, 93,157 (1986)].
The results are shown in F~gs. 1 through 3. ~JDR prodrugbody~Fig.1;
- o -] ~howed an about 1% activity relative to that ofthe activated body rFig.
l; - o -] on NUGC4 or AM-RC-6 cancer cells. TAN-1120 pradrug body ~Fig. 2;
- -] showed a 1/10 to 11100 or les~ cytoto3icity relative to that of the
activated body [Fig. 2; - o -]. PM prodrug body [Fig. 3; - ~ -] showed an about
1/4 activity relative to that of the activated body [Fig. 3; - o -].
E~ample ~ Tripeptidated dru~ activatin~ reac~on (1)
To a microplate æeeded ~;vith 5 X 103/well renal cancer cell line AM-RC-
6, the Boc-Gly-Gly-Arg-ADR or Boc-Gly-Gly-Arg-PM obtained in E~cample 3
1~ was added, followed by the addition of trypsill to the Boc-Gly-Gly-Arg-ADRcell ~cture, or UR to the Boc-Gly-Gly-Arg-PM cell mi~re, and cultivation
at 37C. Three day~ later, viable cells were counted by the method described
in Example 4, hnd prodrug activating reaction of trypsin or UK was
measured.
The results are shown in Tables 1 and 2. Boc-Gly-Gly-Arg-ADR was
decomposed by trypsin and showed significant increase in cytoto~icity. As
well, Boc-Gly-Gly-Arg-PM was decomposed by UK and showed significant
increase in cytotoxicil y.
Table 1
: , r .
% increase in cell count in the
Boc-Gly-Gly ~rg ADR1) prese: lce of ~ ~in (1: ~/me)
. . ` O 1.0 10 100 ~`
302.0 ~glm~ 100 67 4B 32 ~, .
_ _
4.0 llg/m~ 100 64 ~4 28
1) Tr~peptidsted adriamycin
wo gl~oglW 2 ~ 6 9 ~ 3 ~ 28 - pcl~/Jpso/~163r
Table 2
_ _ _
% increase in cell cou~t in the
Boc-Gly-Gly Ar~ PM1) pro~encc ( f urokina ie(llg/me)
o a.o ~.o
. .
1.0 ~glme 100 90 B5
_. _
200 llg/m~ 100 33 12
1) Tripeptidated puromycin
Example6 Tripeptidatedd~ugactivatin~rea~ion(2)
The trgpsin decomposition product of Boc-Gly-Gly-Arg-ADR and the :-UE decomposition product of Boc-(~ly-Gly-Arg-PM obtained in :~ample 5
were subjected to reversed phase high perfo~nance liquid chromatography
using an ODS column (YMC A-302 ODS 120A columD, 4.6 X 1~0 mm,
1~ commercially available from Y~IC EE), and their chromatographic patterns
were compared with tho9e of their ac~vated bodies ADR and PM. Elution was
carried out with an eluent of 30% acetonitrile/0.01 M phosphate buffer ~pH
3.0) at a flow rate of 1.0 me/mill; the ultraviolet absorbance of the column
ef~luent was dete~ined at 254 ~n.
The results are shown in ~igs. 4 and 5. Fig. 4 reveals that Boc-Gly-Gly-
Arg-ADR (peak B; eluted at 10.2 minutes) was decomposed and activated into
ADR (peak A; eluted at 3.6 minutes) by trypsin. Fig. ~ ~eveals t hat Boc-Gly-
Gly-Arg-PM (peak B; eluted at 3.2 minutes) was decomposed and activated
25 into PM (peak A; eluted at 1.8 minutes) by UR.
Example 7 SYnthesis of prodrugs 2
(1) S~rnthesis of Boc-Gl~Gly-Ar~phenYlenediamine mustard
Phenylenediamine mustard (PDM) was synthesized i~ accordance with
a known method [W. C. J. Ro~s: Journal of Chemical Socisty, 183 (1949) and
J. L. Everett et al.: Journal of Chemical Society, 1972 (1949)].
To a solution of 6.1 mg of PDM a~d ~ of N-ethylmorpholine in 0.3 m~
of D~F was added7 a D~!CF solution of 9.0 mg of Boc-Gly-Gly-Arg-OlI, 7.7 mg
of HOBT and 6 mg of WSC, followed by s~mng. Tne misture was then
` ~ :
4 3 9
wo 91/09134 - 2g- PCr/JP90/01631
b:eated in the same manner as in Example 3-(5) to yield Boc-Gly-Gly-Arg-
PDM.
MS m/z: 603 [M + H] + ~ 503 ~M-Boc + 2~I] +
(2) SYnthesis of Boe-Gly-Gly Ar~-Val-ADR
A solution of 20 mg of ADR, 4 1l~ of N-ethylmorpholine, 28 mg of 3-
nitro-2-pyridinesulfonyl-I~valine (Npys-Val), 12 mg of HOBT and 24 mg of
WSC in 1 m~ of DMF was s~irred at room temperature. Du~ing 1;he reaction, 4
~ of N-ethylmorpholine was added twice. After the solvent was distilled off
under reduced pressure, extraction was carried out with ethyl acetate. The
e~tract was washed with ~ater a~d dried, a~d the solvent wa~ distilled off
u~lder reduced pressure. The resulting residue was purified by column
chromatography using silica gel (3 g). The 2% methanol-chlorofo~m eluted
fraction was concentrated under reduced pres~ure to yield Npys-Val-ADR as
1~ an orange-red Cl'y9tal (a7 mg). To a solution of 13.1 mg of the Npys-Val-ADR
in 1 m~ of dio~ e, 0.1 m~ of a 1 N aqueous hydroshloric acid was added,
followed by stim~g at room temperature for 40 minutes. Aqueous sodium
bicarbo~ate was then added to neutralize the solution, followed by n-butanol
extraction. After the e~tract was washed with water and dried~ the solvent
wa~ distilled off under reduced pressure. The resulting residue was purified
by column chromatography using silica gel (3 g) to yield Val-ADR (7 mg) as
an orange-red oily substance from the 10% methanol-chloroform eluted
fraction.
MSm/z: 643 [M+H]+
To a D~F ~olution of 2.0 mg of Val-ADR, 7.~ mg of Boc-Gly-Gly-Arg-
OH and 6.9 mg of ElOBT, 7.0 mg of WSC was added, followed by stirring at
room temperature. The ~olvent was di~tilled off under reduced pressure and
the resulting re~idue was treated in the same m~nner as in E~ample 3-(~) to
yield Boc-Gly-Gly-Arg-Val-ADR.
MSm/z: 1035~M+Na]+
(3) S~rnthesis of QS4-GlY-Gl~r-Ar~-PM
To a suspension of 628 mg of glycine ethyl ester (Gly-OEt) in 5 m~ of
DMF, 630 1l~ of N-ethyImorpholine was added, followed by stirring at room
temperature. A solution of 1.178 g of 6-(3-~arboa~ypropyl-2,3-dimethosy-5-
methyl-1,4-ben~oquinone (QS-4) in 3 mr of DMF was then added, followed by
~ o ~ 9 ~ 3 ~ PCI tJP90/0163
the addition of 95~ mg of WSC and stirring at room temperature overnight.
A~ter the solvent was distilled of~ under reduced pressure, the residue was
diluted with water and extracted with ethyl acetate. Af~er the e~t~act was
washed with water and dried, the solvent was distilled of~ under reduced
pressure. The resulting residue was purified by column chromatography
using silica gel (4~ g~. The 1% methanol-chloroform eluted fraction was
concentrated under reduced pressure and the residue was crystallized from
ethyl acetate-n-hesane to yield ~2,3-dime~oxy-5-methyl-1,4-benzoquinone-
6-yl)butanolglycine ethyl ester (Q~Gly-O~t, ~61 mg) i~ the form o$ an
orange-yellow needle. An ethyl acetate solution of 94 mg of the Q~4 Gly-OEt
was converted to a hydroquinone by mi~cing in an aqueous solution of
hydrosulfite while shaking; the ethyl ace~te was then distil}ed of~ under
reduced pressure. Afl;er the residue was dissolved in MeO~I, 1 N NaOEI ~as
added and hydrolysis was carried out in an N2 gas stream. The hydrolyzate
1~ was acidated with 1 N ~lC~ and e~ctracted with ethyl acetate. After the
est;ract wa~ washed with water and dried, the solvent was distilled off under
reduced pressure. The resulting residue was dissolved in DMF (0.2 mO,
followed by addition of Gly-Arg-OMe (141 mg), EIOBT (39 mg) a~d WSC ~110
mg) and stimng at room temperature overnight. EOBT (39 mg) and WSC (5~
mg) were further added, followed by stirringO After the solvent was distilled
off under reduced pressure, water was added, and the unreacted substance
was extracted with ether. The residue obtained by concentrating the aqueous
solution under reduced pressure was subjected to column chromatography
using silica gel (6 g), followed by elution with ethyl acetate-pyridine-acetic
acid-water (60:20:6:10) to yield QS-4-Gly-Gly-Arg-OMe. To an aqueous
solution of 1~e obtained QS-4-Gly-Gly-Arg-OMe, an aqueous solutio~ of
hydrosulfite wa~ added, followed by stirring. Then, 1 N NaOE was added,
and hydrolysis ~as carried out. Af~er neutraL~zation with 1 N ~IC~, the
hydrolyzate was applied to cationic exchange resin (Biorex 70). The ef~uent
fraction and the ~ N pyridine eluted fraction were combitned, and the solvent
was distilled off under reduced pressure to yield Q~4-Gly-Gly-Arg (80 mg).
To a DMF solution of 10 mg of puromycin hydrochloride (PM-2HC~) and 1011~
of N-ethylmorpholiIIe, 20 mg of the afiorementioned Q~Gly-Gly-Arg, 7 mg
of HOBT and 4.6 mg of WSC were added, followed by ~tirring at room
temperature overnight. After the ~olvent was distilled o~ under reduced
pressure, the re ulting residue was dissolved in MeOH and oxidized to a
~ , . - . .
WO 91/09134 - 31- PCI~/JP90/01631
quinone body with an aqueous solutioll of ferric chloride. The solve~t was
then distilled of~ under reduced pressure. The residue was treated in the
same manner as in Example 3-(~) to yield Q~4-Gly-Gly-Arg-PM in the form
of a yellow oily sub~tance.
MS m/z: 994 [M + 3H] +
(4) Synthesis of Q~3-10-Gl~-Gly-Ar~-PM
The star1;ing mate~ial 6-(9-carboxynonyl-2,3-dimethoxy-5-methyl-1,~
benzoqui~one (Q~10) was treated in the same manner a~ in (3) above and
crlrstallized from ethyl acetate-n-he~ane to yield 10-~2,3-dimethogy-5-methyl-
1,4-methyl-benzoquinone-6-yl~decanoylglyci~e ethyl ester (Q~10-Gly-OEt)
in the fo~m of an orange-yellow needle (mp. 77 to 77.6C).
~ (CDCe3) 8: 1.23-1.40 (l~H, m, CE3, CH2), 1.63 (2~I, m, CH2), 2.01 (3H,
s, nuclear CH3), 2.24 (~I, t, CH2CON), 2.44 (2EI, t, nuclear C~I~), 3.99 (6H, s,OCEt3), 4.03 (2~3E, d, NCH2COO), 4.22 (2E, q, OC~12)
Af~Ger hydrolysi~, Q~10-Gly-OEt was condensed with the dipeptide
Gly-Arg-OMe to yield Q~10-Gly-Gly-Arg-OMe in the form of a yellow oily
sllbstance.
~IS m/z: 639 ~d[+3H]+
After hydrolysis, Q~10-Gly-Gly-Arg-OMe was condensed with PM in
the presence of WSC to yield Q~10-Gly-Gly-Arg-PM in the form of a yellow
oily substance.
MSm/z: 1076[M+H]+
26 ~5) ~rnthesis of B~GlY-Ala-Pro-GlY-Arz-PM
To a solution of 7û mg of B~Gly-Ala-Pro (Peptide Eenkyukai~ in 1 m~
of D~F, 6~ mg of HONB and 68 mg of DCC were added, followed by stir~ng
at room temperath~re for 2 hours. A~ter l~ t ~ ls reaction misture was filtered and
the precipitate was removed, the filtl~a~ was added to a solution of 30 ~u~ of
triethylamine a~d 112 mg of Gly-Arg-OMe hydrochloride i~ 1 m~ of DMF,
followed by stirring at room temperature overnight. After the precipitate was
removed by filtration, the filtrate was concentrated u~der reduced pressure.
The resulting re~idue was subjected to column chromatography using silica
gel (6 g) for elution with ethyl acetate-pyridine-acetic acid-water (60:20:6:10)3~ to yield E~z~Gly-Ala-Pro-Gly-Arg-OMe. To an aqueous solu~on of the obtainedB~Gly-Ala-Pr~Gly-Arg-OMe, 1 N NaOH was added, followed by stirring at
':
:~
wo 9~/09l34 2 ~ g 9 4 3 9 32- PCI/JP90/0163
room temperature. The react;on mia:ture was applied to cationic e~change
resin (Biorex 70). The ef~luent fraction and the 1 N pyridine eluted fraction
were combined, and the solvent was distilled of~ under reduced pressure to
yield Bz-Gly-Ala-Pro-Gly-Arg (70 mg) i~ the form of a colorless oily
substance. To a solutiorl of 10.3 mg of PM and 10 1l~ of N-ethylmorpholine in
300 ~e of DMF, a solution of 20 mg of the above-mentioned B~Gly-Ala-Pro-
Gly-Arg in 300 1l~ of D~? alld 7 mg of HOBT was added, followed by the
additio~ of 9.0 mg of WSC and stirring at room temperature for 4 hours. After
the mi~ture was kept standi~g in a cold room overnight, the solvent W8S
distilled offunder reduced pre sure, a~d the residue was treated in the same
manner as i~ E~ample 3-(~) to yield B~Gly-Ala-Pro-Gly-ArgPM in ~he form
of a colorle~s oily substa~ce.
MSm/z: 1014[M+H+]
(6) S~thesis of ~-Gly-Pro-Leu-GlY-GlY-Ar~-PM
The starting material commercial Z-Gly-Pro-Leu-Gly was treated in
the same manner as in (5) above to yield Z-Gly-Pro-Leu-Gly-Gly-Arg-PM in
the form of a colorless oily substance.
MSm/z: 1143~M~lI+]
Example 8 Prodru~ activa~n~ reaction (3)
To a microplate seeded ~1vith human epide~oid carcinoma cell line
A431 at 7 X 103 celVwell, the Boc-Gly-Gly-Arg-PDM, Boc-Gly-Gly-Arg-Val-
ADR, Q~Gly-Gly-Arg-PM, QS-10--Gly-Gly-Arg-PM and B~Gly-Ala-Pro-
26 Gly-Arg-PM obtained in Example 7 ~vere added, followed by addition of U~
and cultivation at 37C. Ihe prodrug aclivating reaction of UK was then
dete~mined by the method described in Ea:ample 5.
The re8ult8 are ~hown in Table 3. All prodrug bodies were activated by
UE and ~howed 8t~0ng cytotoxici~.
... r............ . . . ~ . . .. . . . . . . .
20~33
wo 91/09134 - 33 - PCI/JPgO/01631
Table 3
_ _ _ .
IJK concentration
Prodrug body (llg/m~) % cell growt~
_ .
Boc-Gly-Gly-Arg-PDM 0 100
1.0 llg/m~ 1 87
4 66
. . _ _ _ _
Boc-Gly-~ly-~rg-Val-ADR O 100
B.0 ~lg/m~ 1 92 : ~
50 ::
_ . . _ .
Q~Gly-Gly-Arg-PM 0 100
1.0 llg/m~ 1 84
4 64
. _ _ _ . ~. . .
Q~10-Gly-Ç~ly-Arg-PM O 100
2.0 llg/m~ 2 8~
~5 8 ~6
. _
B~Gly-Ala-Pr~Gly-Arg-PM O 100
l.o llgln,e 2 73
20 Example 9 eparation of hybrid hYbridoma that produces anti-hlY~-anti-
UK bisPecific antibodY
(1) Cell filsion
:EIybridoma 22C6, whi~h produces an anti-hlYR MoAb, obtained in Reference
25 Example 12, and hybridoma UEl-6~ ~hich produces an anti-Ug MoAb,
obtained in Reference Esample g, were each incubated in Iskove-Ham F-12
mixed medium containing 0.6 llg/m~ ElTC and 1.5 ~g/m~ TRlTC at 37C for
80 minutes for 1uorescent staining. An LSM solution (commercially
available from Wako Pure Chemical :lndustries Ltd.) ~as l;hen added, aIld t~he
30 dead cellg were removed; the two hy~ridomas were then mised at a ratio of 1
to 1 for cell fusion u~ing PEG 6000 by the method des~bed in RefereIlce
Example ~(2). :
After incubation at 37C for 2 hours, t~e cell misture wa~ applied to
FACS, and 2~000 fluorescein-rhodamine double stained c211s were separated
3~
.
wo g I /091 3-~ 2 ~ ~ 9 ~ 3 9 34 PCT/JP9o/0163~
and seeded, at lO cells per well, to a 96-well microplate seeded with 5 x 105
cells/well mouse thymocytes as feeders, and cultivated.
(2) Hybrid hYbridoma selection and clo~in~
The culture supernat~nt from each well in which sell gro~wth occurred 1
to 2 weeks after fusion was subjected to Cell-ElA to determine the bispecific
antibody titer. Specifically, to the microplate coupled with human cancer cell
A431, prepared in Reference E~ample 3, the subject hybrid hybridoma
culture supernatant was added, followed by reaction at room temperature for
2 hours. After plate washing with 0.2% BSA medium, biotin-labeled UK was
added, followed by reaction at room temperature for 2 hours. After HRP-
labeled a~idin reaction at room temperature for 1 hour, the plate was washed
and the enzyme activity bound to the solid phase was determined by the
method described in Reference E~nmple 3.
1~ The cells in wells showing high bispecific antibody titer were subjected
to cloning by the limiting dilution method, the desired bispecific-antibody-
producing mouse tetraoma Ul~ 20-7 was obtained.
The result is shown in Figure 6.
(3) Purification of bispecific antibod~,r
To BALB/c mice prel;reated by intraperitoneal administration of 0.6 m~
mineral oil, mouse hybrid hybridomas (tetraomas) were inoculated
intraperitoneally at 5 X 106/mouse. Ascites fluid, whose retention occurred
about 10 to 20 days after inoculation, was collected and subjected to salting-
out with 60% saturated ammonium sulfate to yield an Ig~: ~raction. Afl;er
dialysis with 20 mM PBS (pE 7.5), the IgG~ f~action was applied to a UK-
coupled Cellulofine column, followed by elution with 0.2 M glycine-l~lCl buffer
at pE 2.9. After dialysis with Pl~S, the acid-eluted fraction was applied to a
hydroxyapatite column, the desired bispecific anti~hl~-anti-UK antibody
was puri~led.
By the present method, about 8.2 mg of the desired bispeci~lc antibody
UTF 20-7 was obtained.
Egample 10 Prodru~ activatin~ reaction by bispecific arltibody
3~ To a microplate see~led with 1.0 X 104 cell/well of human epide~moid
carcinoma cell line A431 and mouse leukemia cell line P388, the
~U~33
wo 91~09134 - 35 ~ PCI/JP90/01631
immunocomple~ comprisi~g the purified bispecific antibody obtained in
Example 9 and UK tl:1~ was added, followed by reaction at 5C for 30
minutes. After cells were w~shed at a low temperature, the prodrug Boc-Gly-
Gly-Arg-Val-ADR, described in Example 7-(2) or the prodrug Q~10-Gly-Gly-
5 Arg-PM described in Example 7-(4) was added at a ffnal concentration of ~.0
g/ml and 0.2 ~g/ml, respectively. I~e prodrug activating reaction of the
immunocomplex comprising UK and the bispecific antibody, bound to cell
sur~ace, was then determined by the method described in Esample ~. The
results are shown in Table 4. All prodrugs were activated by the :~
10 immunocomple:~: comprising U:E~ and the bispecific antibody and showed
strong cytoto~icity agsinst t}~e target cell line A431. On the other hand, they
showed no cytoto~iu~y against t;he ~on-target eell line P388. ~ .
Table 4
. _ immnocomple2~ % cell growth
prodrug body concenl;ration _
(Il.g/ml as UE) A431 P388
. _
Boc-Gly-Gly-Arg-Val-AD~ O 100 100
(0.5 llglm~) 3 75 103 :. `
58 95 :`:
_
Q~10-Gly-Gly-Arg-PM 0 100 100 :
(2.0 llg/me) 3 84 98
_ _ 62 95
2~
''` ~ ~ '
', `
.'. ` ' ` ' ` '` ' ' ` ' " ` ~ ~ ' ` " `' '` ' ', ',` ' ` ` " ` '
. ' . . . ` .:, '`" ' . . . ` ' , ' ' . . . , ` '"' . ~ . . 1 . ' . ' , ' ' ` `, ' ' '`'
