U.S. patent application number 17/352652 was filed with the patent office on 2021-12-16 for cell injury inducing therapeutic drug for use in cancer therapy.
The applicant listed for this patent is Chugai Seiyaku Kabushiki Kaisha. Invention is credited to Mika ENDO, Takahiro ISHIGURO, Yumiko KAWAI, Yasuko KINOSHITA, Yuji SANO, Toshiaki TSUNENARI.
Application Number | 20210388110 17/352652 |
Document ID | / |
Family ID | 1000005798521 |
Filed Date | 2021-12-16 |
United States Patent
Application |
20210388110 |
Kind Code |
A1 |
KINOSHITA; Yasuko ; et
al. |
December 16, 2021 |
CELL INJURY INDUCING THERAPEUTIC DRUG FOR USE IN CANCER THERAPY
Abstract
The present invention provides anticancer agents comprising as
an active ingredient a bispecific antibody that comprises a domain
comprising a glypican 3-binding antibody variable region, a domain
comprising a T cell receptor complex-binding antibody variable
region, and common L chains that can enhance the affinity for the
two antigens, as well as pharmaceutical compositions comprising the
bispecific antibody as an active ingredient, the compositions being
for use in combination with other anticancer agents. The bispecific
antibodies are novel molecules which are produced with high
efficiency and have strong anti-tumor activity as well as safety
and excellent pharmacodynamics. The bispecific antibodies can be
expected to be applied to various cancers.
Inventors: |
KINOSHITA; Yasuko;
(Kanagawa, JP) ; KAWAI; Yumiko; (Kanagawa, JP)
; TSUNENARI; Toshiaki; (Kanagawa, JP) ; ISHIGURO;
Takahiro; (Tokyo, JP) ; ENDO; Mika; (Kanagawa,
JP) ; SANO; Yuji; (Kanagawa, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chugai Seiyaku Kabushiki Kaisha |
Tokyo |
|
JP |
|
|
Family ID: |
1000005798521 |
Appl. No.: |
17/352652 |
Filed: |
June 21, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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16083975 |
Sep 11, 2018 |
11072666 |
|
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PCT/JP2017/007033 |
Feb 24, 2017 |
|
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17352652 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2809 20130101;
A61P 35/00 20180101; C07K 16/303 20130101; A61K 39/3955 20130101;
C07K 2317/73 20130101; A61K 45/06 20130101; A61K 39/39558 20130101;
C07K 2317/31 20130101; C07K 16/22 20130101; A61K 2039/505 20130101;
C07K 2317/92 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61P 35/00 20060101 A61P035/00; A61K 39/395 20060101
A61K039/395; A61K 45/06 20060101 A61K045/06; C07K 16/22 20060101
C07K016/22; C07K 16/28 20060101 C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 14, 2016 |
JP |
2016-050095 |
Claims
1-15. (canceled)
16. A method for treating cancer, comprising administering an
effective amount of a bispecific antibody that comprises an
antibody variable region having glypican 3-binding activity and an
antibody variable region having CD3-binding activity, wherein the
cancer is liver cancer, gastric cancer or head & neck cancer,
wherein the antibody variable region having glypican 3-binding
activity and the antibody variable region having CD3-binding
activity comprise a common light chain variable region, and wherein
(a) the antibody variable region having glypican 3-binding activity
comprises heavy chain variable region complementarity-determining
region (CDR) 1, CDR2, and CDR3 comprising the amino acid sequences
of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 206,
respectively; (b) the antibody variable region having CD3-binding
activity comprises heavy chain variable region CDR1, CDR2, and CDR3
comprising the amino acid sequences of the CDR1, CDR2, and CDR3
regions comprised in SEQ ID NO: 168, respectively; and (c) the
common light chain variable region comprises CDR1, CDR2, and CDR3
comprising the amino acid sequences of the CDR1, CDR2, and CDR3
regions comprised in SEQ ID NO: 223, respectively.
17. The method of claim 16, wherein (a) the antibody variable
region having glypican 3-binding activity comprises a heavy chain
variable region comprising a sequence having at least 80% identity
to the amino acid sequence of SEQ ID NO: 206; (b) the antibody
variable region having CD3-binding activity comprises a heavy chain
variable region comprising a sequence having at least 80% identity
to the amino acid sequence of SEQ ID NO: 168; and (c) the common
light chain variable region comprises a sequence having at least
80% identity to the amino acid sequence of SEQ ID NO: 223.
18. The method of claim 16, wherein the bispecific antibody
comprises (a) an antibody heavy chain comprising a sequence having
at least 80% identity to the amino acid sequence of SEQ ID NO: 385;
(b) an antibody heavy chain comprising a sequence having at least
80% identity to the amino acid sequence of SEQ ID NO: 402; and (c)
a common light chain comprising a sequence having at least 80%
identity to the amino acid sequence of SEQ ID NO: 410.
19. The method of claim 16, wherein the bispecific antibody
comprises (a) an antibody heavy chain comprising the amino acid
sequence of SEQ ID NO: 385; (b) an antibody heavy chain comprising
the amino acid sequence of SEQ ID NO: 402; and (c) a common light
chain comprising the amino acid sequence of SEQ ID NO: 410.
20. The method of claim 16 further comprising administering an
effective amount of another anticancer agent.
21. The method of claim 20, wherein the bispecific antibody is
administered simultaneously with said another anticancer agent.
22. The method of claim 20, wherein the bispecific antibody is
administered before or after administration of said another
anticancer agent.
23. The method of claim 20, wherein said another anticancer agent
is a chemotherapeutic agent, a T cell-activating agonist agent, an
immune checkpoint inhibitor, or an angiogenic inhibitor.
24. The method of claim 23, wherein said another anticancer agent
is a chemotherapeutic agent, wherein the chemotherapeutic agent is
an antimetabolite, a plant alkaloid, or a platinum compound.
25. The method of claim 24, wherein the antimetabolite is
capecitabine, the plant alkaloid is paclitaxel, and the platinum
compound is cisplatin.
26. The method of claim 23, wherein said another anticancer agent
is an immune checkpoint inhibitor, wherein the immune checkpoint
inhibitor is an anti-PD1 antibody, an anti-PDL1 antibody, an
anti-CTLA-4 antibody, an anti-TIM3 antibody, or an anti-LAG3
antibody.
27. The method of claim 23, wherein said another anticancer agent
is bevacizumab.
28. The method of claim 16, wherein the cancer is liver cancer.
29. The method of claim 17, wherein the cancer is liver cancer.
30. The method of claim 19, wherein the cancer is liver cancer.
31. The method of claim 20, wherein the cancer is liver cancer.
32. The method of claim 16, wherein the cancer is gastric
cancer.
33. The method of claim 16, wherein the cancer is head & neck
cancer.
34. A method for treating cancer, comprising administering an
effective amount of an antibody that binds CD3.
35. A method for treating cancer, comprising administering an
effective amount of an antibody that binds glypican 3.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/083,975, .sctn. 371 date Sep. 11, 2018,
which is a U.S. National Phase of PCT Application No.
PCT/JP2017/007033, filed Feb. 24, 2017, which claims priority to
Japanese Patent Application No. 2016-050095, filed Mar. 14, 2016,
each of which is incorporated herein by reference.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The content of the electronically submitted sequence listing
(Name: 6663_0178_Sequence_Listing.txt; Size: 616 kilobytes; and
Date of Creation: Jun. 15, 2021) filed with the application is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0003] The present invention relates to multispecific
antigen-binding molecules, uses thereof, and such.
BACKGROUND ART
[0004] Antibodies are drawing attention as pharmaceuticals because
of their high stability in plasma and few adverse reactions
(Non-patent Documents 1 and 2). Antibodies are known to induce not
only an antigen-binding action, an agonistic action, and an
antagonistic action, but also effector-mediated cytotoxic
activities (also called effector functions) such as
antibody-dependent cellular cytotoxicity (ADCC), antibody dependent
cell phagocytosis (ADCP), and complement-dependent cytotoxicity
(CDC), and exhibit antitumor effects against cancer cells
(Non-patent Document 3). ADCC is a cytotoxicity exhibited by
effector cells against antibody-bound target cancer cells via
binding of the antibody Fc region to an Fc receptor present on
effector cells such as NK cells and macrophages. A complement
complex binds to the complement-binding site present in an antibody
structure. CDC is a cytotoxicity that results from cell destruction
where an influx of water and ions into cells is promoted by pore
formation on the cell membrane of the antibody-bound cells by
complement components present in the complex. A number of
therapeutic antibodies showing excellent anti-tumor effects have
been developed as pharmaceuticals for cancer treatment (Non-patent
Document 4); and while existing therapeutic antibodies have shown
excellent actions, the therapeutic outcome achieved by
administration of these antibodies is still not satisfactory.
[0005] For an antibody to show ADCC, ADCP, and CDC, it is necessary
for the antibody Fc region, the antibody receptor (Fc.gamma.R)
present on effector cells such as NK cells and macrophages, and
various complement components to bind. In humans, isoforms of
Fc.gamma.RIa, Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIIa, and
Fc.gamma.RIIIb have been reported as the Fc.gamma.R protein family,
and the respective allotypes have been reported as well (Non-patent
Document 5). Among these isoforms, Fc.gamma.RIa, Fc.gamma.RIIa, and
Fc.gamma.RIIIa carry a domain called the Immunoreceptor
Tyrosine-based Activation Motif (ITAM) in the intracellular domain,
and transmit activation signals. On the other hand, only
Fc.gamma.RIIb carries a domain called the Immunoreceptor
Tyrosine-based Inhibitory Motif (ITIM) in the intracellular domain,
and transmits inhibitory signals. Every one of the Fc.gamma.Rs is
known to transmit signals via crosslinking by immune complexes and
such (Non-patent Document 6). When antibodies actually exert an
effector function on cancer cells, Fc.gamma.Rs on the effector cell
membrane form clusters at the Fc regions of several antibodies
bound on the cancer cell membrane, and activation signals are
transmitted by effector cells. A cytocidal effect is exerted as a
result, but since Fc.gamma.Rs are crosslinked only in effector
cells present near cancer cells this time, activation of immunity
is shown to occur locally in cancer cells (Non-patent Document
7).
[0006] Naturally-occurring immunoglobulins bind to antigens at
their variable regions, and bind to receptors such as Fc.gamma.R,
FcRn, Fc.alpha.R, and FcsR, and complements at their constant
regions. FcRn is one of the binding molecules that interact at the
IgG Fc region, and since each of the antibody heavy chains binds
one molecule of FcRn, two molecules of FcRn have been reported to
bind one IgG-type antibody molecule. However, unlike FcRn and such,
Fc.gamma.R interacts at the antibody hinge region and CH2 domain,
and only one molecule of Fc.gamma.R binds to one molecule of
IgG-type antibody (Non-patent Document 8). Furthermore, a common
naturally-occurring IgG-type antibody recognizes and binds a single
epitope via its variable region (Fab); therefore, it can bind to
only one antigen. On the other hand, many types of proteins are
known to be involved in cancer and inflammation, and there may be
crosstalk among the proteins. For example, several inflammatory
cytokines (TNF, IL1, and IL6) are known to be involved in
immunological diseases (Non-patent Document 9). Furthermore,
activation of other receptors is known as one of the mechanisms of
cancer in acquiring drug resistance (Non-patent Document 10). In
such cases, common antibodies that recognize a single epitope would
be unable to inhibit multiple proteins.
[0007] Antibodies (bispecific antibodies) that bind to two or more
types of antigens with one molecule are being studied as molecules
that inhibit multiple targets. It is possible to confer binding
activities to two different antigens (a first antigen and a second
antigen) by modifying naturally-occurring IgG-type antibodies
(Non-patent Document 11). Accordingly, there will not only be
neutralization of two or more types of antigens by a single
molecule, but also enhancement of antitumor activity due to
crosslinks between cells having cytotoxic activity and cancer
cells. As molecular forms of a bispecific antibody, a molecule
comprising an antigen-binding site added to the N or C terminus of
an antibody (DVD-Ig and scFv-IgG), a molecule having different
sequences for the two Fab regions of an antibody (common L-chain
bispecific antibody and hybrid hybridoma), a molecule in which one
Fab region recognizes two antigens (two-in-one IgG), and a molecule
having a CH3 region loop site as a new antigen-binding site (Fcab)
have been reported so far (Non-patent Documents 12 and 13). Since
all bispecific antibodies interact at their Fc regions with
Fc.gamma.R, antibody effector functions are preserved. Thus, the
bispecific antibody binds to any antigen that it recognizes and at
the same time binds to Fc.gamma.R, and exhibits ADCC activity
against cells expressing the antigen.
[0008] If all the antigens recognized by the bispecific antibody
are antigens specifically expressed in cancer, the bispecific
antibody exhibits cytotoxic activity to cancer cells when it binds
to any of the antigens. Therefore, in comparison to a conventional
antibody pharmaceutical that recognizes one antigen, a more
efficient antitumor effect can be expected from such an antibody.
However, in the case where any one of the antigens recognized by
the bispecific antibody is expressed in normal tissues or cells
expressed on immunocytes, damage on normal tissues or release of
cytokines occurs due to crosslinking with Fc.gamma.R (Non-patent
Document 14). As a result, strong adverse reactions are
induced.
[0009] A T-cell redirecting antibody that employs cytotoxicity
mobilizing T cells as effector cells as the mechanism for its
antitumor effect has been known from the 1980s as a bispecific
antibody (Non-patent Documents 15, 16, and 17). Unlike antibodies
that employ ADCC mobilizing NK cells or macrophages as effector
cells as the mechanism for their antitumor effects, a T-cell
redirecting antibody is an antibody against any one of the subunits
constituting the T-cell receptor (TCR) complex on T cells, and is
specifically a bi-specific antibody comprising an antibody that
binds to the CD3 epsilon chain and an antibody that binds to an
antigen on the target cancer cell. T cells come close to cancer
cells via simultaneous binding of the CD3 epsilon chain and a
cancer antigen by a T-cell redirecting antibody. As a result,
antitumor effects against cancer cells are considered to be exerted
through the cytotoxic activity possessed by T cells.
[0010] Catumaxomab, which is known as a T-cell redirecting
antibody, binds at two Fabs each to a cancer antigen (EpCAM) and to
a CD3.epsilon. (CD3 epsilon) chain expressed on T cells.
Catumaxomab induces T cell-mediated cytotoxic activity by binding
to the cancer antigen and the CD3.epsilon. at the same time, and
induces cytotoxic activity mediated by antigen-presenting cells
such as NK cells and macrophages, by binding to the cancer antigen
and Fc.gamma.R at the same time. By use of these two cytotoxic
activities, catumaxomab exhibits a high therapeutic effect on
malignant ascites by intraperitoneal administration and has thus
been approved in Europe (Non-patent Document 18). In addition,
there are cases where the administration of catumaxomab reportedly
yields cancer cell-reactive antibodies, which clearly shows that
acquired immunity is induced (Non-patent Document 19). From this
result, antibodies having both T cell-mediated cytotoxic activity
and the Fc.gamma.R-mediated activities by cells such as NK cells or
macrophages (these antibodies are particularly referred to as
trifunctional antibodies) have received attention because a strong
antitumor effect and induction of acquired immunity can be
expected.
[0011] The trifunctional antibodies, however, bind to CD3.epsilon.
and Fc.gamma.R at the same time even in the absence of a cancer
antigen and therefore crosslink CD3.epsilon.-expressing T cells
with Fc.gamma.R-expressing cells even in a cancer cell-absent
environment, leading to production of various cytokines in large
amounts. Such cancer antigen-independent induction of production of
various cytokines restricts the current administration of the
trifunctional antibodies to an intraperitoneal route (Non-patent
Document 20). The trifunctional antibodies are very difficult to
administer systemically due to severe cytokine storm-like adverse
reactions. In fact, in the Phase I clinical trial of administering
catumaxomab systemically to non-small-cell lung cancer patients, a
very low dose of 5 .mu.g/body is the maximum tolerable dose, and
administration of a larger dose has been reported to cause various
serious adverse reactions (Non-patent Document 21).
[0012] As such, bispecific antibodies by conventional techniques
may bind to both antigens, the first antigen being the cancer
antigen (EpCAM) and the second antigen being CD3.epsilon., at the
same time when they bind to Fc.gamma.R; and therefore, in view of
their molecular structure it is impossible to avoid adverse
reactions caused by the simultaneous binding to Fc.gamma.R and the
second antigen CD3.epsilon..
[0013] Meanwhile, unlike catumaxomab, BiTE has no Fc.gamma.
receptor-binding site, and therefore it does not cross-link the
receptors expressed on T cells and cells such as NK cells and
macrophages in a cancer antigen-independent manner. Thus, it has
been demonstrated that BiTE does not cause cancer
antigen-independent cytokine induction which is observed when
catumaxomab is administered. However, since BiTE is a modified
low-molecular-weight antibody molecule without an Fc region, the
problem is that its blood half-life after administration to a
patient is significantly shorter than IgG-type antibodies
conventionally used as therapeutic antibodies. In fact, the blood
half-life of BiTE administered in vivo has been reported to be
about several hours (Non-patent Documents 22 and 23). In the
clinical trials of blinatumomab, it is administered by continuous
intravenous infusion using a minipump. This administration method
is not only extremely inconvenient for patients but also has the
potential risk of medical accidents due to device malfunction or
the like. Thus, it cannot be said that such an administration
method is desirable.
[0014] In recent years, use of an Fc region with reduced
Fc.gamma.R-binding activity has enabled maintenance of the strong
antitumor activity possessed by BiTE and the excellent safety
property of not inducing a cytokine storm in a cancer
antigen-independent manner, and has provided novel polypeptide
assemblies that have long half-lives in blood (Patent Document
1).
[0015] On the other hand, when expressing a bispecific antibody by
conventional techniques, since two types of H chains and two types
of L chains are expressed, ten combinations are conceivable. Among
them, only one of the produced combinations has the binding
specificity of interest. Therefore, to obtain the bispecific
antibody of interest, the single antibody of interest must be
purified from the ten types of antibodies, which is very
inefficient and difficult.
[0016] A method of preferentially secreting IgGs with a
heterodimeric combination of H chains, for example, a combination
of an H chain against antigen A and an H chain against antigen B,
by introducing amino acid substitutions into the IgG H-chain CH3
region has been reported as a method for solving this problem
(Patent Documents 2, 3, 4, 5, 6, 7, and Non-patent Documents 24 and
25). A method that utilizes physical disturbance, i.e., "knob" and
"hole", and a method that utilizes electric charge repulsion have
been reported as such methods.
[0017] To obtain the molecule of interest with better efficiency,
methods using L chains that can bind to two different antigens even
though the L chains have the same amino acid sequence have been
reported (Patent Documents 8 and 9). However, the antigen affinity
may decrease greatly with the use of common L chains, and it is
difficult to find common L chains that maintain antigen
affinity.
[0018] Blinatumomab as a BiTE has been approved for acute
lymphoblastic leukemia (ALL) and is also being tested in clinical
trials for blood cancer such as non-Hodgkin's lymphoma (NHL) and
chronic lymphocytic leukemia (CLL). Clinical trials of AMG330,
which is a BiTE targeting CD33, have also been started to test it
for acute myelocytic leukemia (AML). Furthermore, BiTEs against
solid tumors are under development. AMG211, which is a BiTE
targeting CEA, AMG110, which is a BiTE targeting EpCAM, AMG212,
which is a BiTE targeting PSMA, and such are undergoing in clinical
trials. However, the responsiveness of BiTEs against solid tumors
remains unproved (Non-patent Document 26).
[0019] In recent years, immunotherapeutic agents, of which
representatives are inhibitors targeting immune checkpoint
molecules such as CTLA-4, PD-1, and PD-L1, have been demonstrated
to have drug efficacy in clinical settings. However, these
pharmaceutical agents are not effective in every patient, and there
is demand for further enhancement of the drug efficacy. With
respect to combined use of multiple immunotherapies, combined use
of Nivlolumab and Ipilimumab has been found to achieve the enhanced
drag efficacy against melanoma as compared to Ipilimumab alone
(Non-patent Document 27).
CITATION LIST
Patent Documents
[0020] [Patent Document 1] WO2012/073985 [0021] [Patent Document 2]
WO96/27011 [0022] [Patent Document 3] WO2006/106905 [0023] [Patent
Document 4] WO2007/147901 [0024] [Patent Document 5] WO2009/089004
[0025] [Patent Document 6] WO2010/129304 [0026] [Patent Document 7]
WO2013/065708 [0027] [Patent Document 8] WO98/050431 [0028] [Patent
Document 9] WO2006/109592
Non-Patent Documents
[0028] [0029] [Non-patent Document 1] Nat. Biotechnol. (2005) 23,
1073-1078 [0030] [Non-patent Document 2] Eur J Pharm Biopharm.
(2005) 59 (3), 389-396 [0031] [Non-patent Document 3] Drug Des
Devel Ther (2009) 3, 7-16 [0032] [Non-patent Document 4] Clin
Cancer Res. (2010) 16 (1), 11-20 [0033] [Non-patent Document 5]
Immunol. Lett. (2002) 82, 57-65 [0034] [Non-patent Document 6] Nat.
Rev. Immunol. (2008) 8, 34-47 [0035] [Non-patent Document 7] Ann.
Rev. Immunol. (1988). 6. 251-81 [0036] [Non-patent Document 8] J.
Bio. Chem., (20001) 276, 16469-16477 [0037] [Non-patent Document 9]
Nat. Biotech., (2011) 28, 502-10 [0038] [Non-patent Document 10]
Endocr Relat Cancer (2006) 13, 45-51 [0039] [Non-patent Document
11] MAbs. (2012) Mar. 1, 4(2) [0040] [Non-patent Document 12] Nat.
Rev. (2010) 10, 301-316 [0041] [Non-patent Document 13] Peds
(2010), 23(4), 289-297 [0042] [Non-patent Document 14] J. Immunol.
(1999) Aug. 1, 163(3), 1246-52 [0043] [Non-patent Document 15]
Nature (1985) 314 (6012), 628-31 [0044] [Non-patent Document 16]
Int J Cancer (1988) 41 (4), 609-15. [0045] [Non-patent Document 17]
Proc Natl Acad Sci USA (1986) 83 (5), 1453-7 [0046] [Non-patent
Document 18] Cancer Treat Rev. (2010) Oct. 36(6), 458-67 [0047]
[Non-patent Document 19] Future Oncol. (2012) Jan. 8(1), 73-85
[0048] [Non-patent Document 20] Cancer Immunol Immunother. (2007)
56(9), 1397-406 [0049] [Non-patent Document 21] Cancer Immunol
Immunother. (2007) 56 (10), 1637-44 [0050] [Non-patent Document 22]
Cancer Immunol Immunother. (2006) 55(5), 503-14 [0051] [Non-patent
Document 23] Cancer Immunol Immunother. (2009) 58(1), 95-109 [0052]
[Non-patent Document 24] Protein Engineering. (1996) vol. 9, p.
617-621 [0053] [Non-patent Document 25] Nature Biotechnology.
(1998) vol. 16, p. 677-681 [0054] [Non-patent Document 26]
Immunological Reviews. (2016) vol. 270, p. 193-208 [0055]
[Non-patent Document 27] N Eng J Med (2015) vol. 373, p. 23-34
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0056] The present invention was achieved in view of the above
circumstances. An objective of the present invention is to provide
anticancer agents comprising as an active ingredient multispecific
antigen-binding molecules that bring T cells close to the target
cancer cells, and can treat cancer through the cytotoxic activity
of T cells against target cancer tissues containing glypican
3-expressing cells, and are molecular forms that can be produced
with high efficiency. Another objective of the present inventions
is to provide combination therapies using the multispecific
antigen-binding molecules and other pharmaceutical agents.
Means for Solving the Problems
[0057] The present inventors discovered an L chain common to a
domain comprising a glypican 3-binding antibody variable region,
and a domain comprising a T-cell receptor complex-binding antibody
variable region, where the common L chain is capable of improving
affinity to both antigens. This allows preparation of molecular
forms that can be produced with high efficiency, and further
discovery of novel multispecific antigen-binding molecules that
maintain the strong antitumor activity possessed by T-cell
redirecting antibodies such as BiTE and the excellent safety
property of not inducing a cytokine storm in a cancer
antigen-independent manner, and also have long half-lives in blood.
Furthermore, the present inventors discovered that the
multispecific antigen-binding molecules comprising common L chains
target glypican 3-expressing cancer cells and cause cytotoxicity.
Based on this discovery, the present inventors elucidated that the
multispecific antigen-binding molecules of the present invention
cause injury to cancer tissues containing glypican 3-expressing
cancer cells. The present inventors revealed anticancer agents that
comprise as an active ingredient the multispecific antigen-binding
molecule; methods for treating or preventing cancer by combined use
of the multispecific antigen-binding molecule and another
anticancer agent; multispecific antigen-binding molecules,
anticancer agents, or pharmaceutical compositions comprising a
combination of a multispecific antigen-binding molecule and an
anticancer agent, each of which is used in combination
therapies.
[0058] Specifically, the present invention provides the
following:
[1] An anticancer agent comprising as an active ingredient a
bispecific antibody of any one of (a) to (c) below that comprises
an antibody variable region having glypican 3-binding activity and
an antibody variable region having CD3-binding activity:
[0059] (a) a bispecific antibody in which CDR1, CDR2, and CDR3
comprised in the antibody variable region having glypican 3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 206, respectively; CDR1, CDR2, and CDR3 comprised in the
antibody variable region having CD3-binding activity are sequences
having at least 80% identity to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 168,
respectively; and CDR1, CDR2, and CDR3 comprised in an antibody
variable region of a common L chain are sequences having at least
80% identity to the amino acid sequences of the CDR1, CDR2, and
CDR3 regions comprised in SEQ ID NO: 223, respectively;
[0060] (b) a bispecific antibody in which the antibody variable
region having glypican 3-binding activity is a sequence having at
least 80% identity to the amino acid sequence of SEQ ID NO: 206;
the antibody variable region having CD3-binding activity is a
sequence having at least 80% identity to the amino acid sequence of
SEQ ID NO: 168; and an antibody variable region of a common L chain
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 223; and
[0061] (c) a bispecific antibody which has an antibody H chain
having glypican 3-binding activity and having at least 80% identity
to the amino acid sequence of SEQ ID NO: 385; an antibody H chain
having CD3-binding activity and having at least 80% identity to the
amino acid sequence of SEQ ID NO: 402; and common L chains having
at least 80% identity to the amino acid sequence of SEQ ID NO:
410.
[2] An anticancer agent comprising as an active ingredient a
bispecific antibody that comprises the antibody H chain of SEQ ID
NO: 385 having glypican 3-binding activity, the antibody H chain of
SEQ ID NO: 402 having CD3-binding activity, and the common L chains
of SEQ ID NO: 410. [3] The anticancer agent of [1] or [2], wherein
the cancer is a glypican 3-positive cancer. [4] The anticancer
agent of [3], wherein the glypican 3-positive cancer is a cancer in
which the number of glypican 3 antigens on cell surface per cell is
100 or more. [5] The anticancer agent of any one of [1] to [4],
wherein the cancer is any cancer selected from the group consisting
of gastric cancer, head and neck cancer, esophageal cancer, lung
cancer, liver cancer, ovary cancer, breast cancer, colon cancer,
kidney cancer, skin cancer, muscle tumor, pancreas cancer, prostate
cancer, testis cancer, uterine cancer, cholangiocarcinoma, Merkel
cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal
cancer, anus cancer, central nervous system tumor, neuroendocrine
tissue tumor, penis cancer, pleura tumor, salivary gland tumor,
vulva cancer, thymoma, and childhood cancer. [6] The anticancer
agent of any one of [1] to [5], which is for treating a patient
having cancer that is refractory to treatment with an immune
checkpoint inhibitor. [7] A pharmaceutical composition for use in
combination with another anticancer agent, the pharmaceutical
composition comprising as an active ingredient a bispecific
antibody of any one of (a) to (c) below that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity:
[0062] (a) a bispecific antibody in which CDR1, CDR2, and CDR3
comprised in the antibody variable region having glypican 3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 206, respectively; CDR1, CDR2, and CDR3 comprised in the
antibody variable region having CD3-binding activity are sequences
having at least 80% identity to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 168,
respectively; and CDR1, CDR2, and CDR3 comprised in an antibody
variable region of a common L chain are sequences having at least
80% identity to the amino acid sequences of the CDR1, CDR2, and
CDR3 regions comprised in SEQ ID NO: 223, respectively;
[0063] (b) a bispecific antibody in which the antibody variable
region having glypican 3-binding activity is a sequence having at
least 80% identity to the amino acid sequence of SEQ ID NO: 206;
the antibody variable region having CD3-binding activity is a
sequence having at least 80% identity to the amino acid sequence of
SEQ ID NO: 168; and an antibody variable region of a common L chain
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 223; and
[0064] (c) a bispecific antibody which has an antibody H chain
having glypican 3-binding activity and having at least 80% identity
to the amino acid sequence of SEQ ID NO: 385; an antibody H chain
having CD3-binding activity and having at least 80% identity to the
amino acid sequence of SEQ ID NO: 402; and common L chains having
at least 80% identity to the amino acid sequence of SEQ ID NO:
410.
[8] A pharmaceutical composition comprising as an active ingredient
a bispecific antibody that has an antibody H chain of SEQ ID NO:
385 having glypican 3-binding activity, an antibody H chain of SEQ
ID NO: 402 having CD3-binding activity, and common antibody L
chains of SEQ ID NO: 410, wherein the pharmaceutical composition is
for use in combination with another anticancer agent. [9] The
pharmaceutical composition of [7] or [8], wherein the bispecific
antibody is administered simultaneously with said another
anticancer agent. [10] The pharmaceutical composition of [7] or
[8], wherein the bispecific antibody is administered before or
after administration of said another anticancer agent. [11] The
pharmaceutical composition of any one of [7] to [10], wherein said
another anticancer agent is a chemotherapeutic agent, a T
cell-activating agonist agent, an immune checkpoint inhibitor, or
an angiogenic inhibitor. [12] The pharmaceutical composition of
[11], wherein the chemotherapeutic agent is an antimetabolite, a
plant alkaloid, or a platinum compound. [13] The pharmaceutical
composition of [11], wherein the T cell-activating agonist agent is
an agonist antibody against TNFRSF. [14] The pharmaceutical
composition of [11], wherein the immune checkpoint inhibitor is a
PD1 antibody, a PDL1 antibody, a TIM3 antibody, or an LAG3
antibody. [15] The pharmaceutical composition of [11], wherein the
angiogenic inhibitor is a VEGFR2 antibody. [16] An agent for
inducing cytotoxicity, an agent for suppressing cell proliferation,
an agent for inhibiting cell proliferation, an agent for activating
immune response, an agent for treating cancer, or an agent for
preventing cancer, which comprises the pharmaceutical composition
of any one of [7] to [15]. [17] The anticancer agent of [1],
wherein CDR1, CDR2, and CDR3 are CDR1, CDR2, and CDR3 regions based
on Kabat numbering.
[0065] The following inventions are also provided:
[2-1] An anticancer agent comprising as an active ingredient a
multispecific antigen-binding molecule that comprises:
[0066] (1) a domain comprising an antibody variable region having
glypican 3-binding activity,
[0067] (2) a domain comprising an antibody variable region having
T-cell receptor complex-binding activity, and
[0068] (3) a domain comprising an Fc region with reduced binding
activity towards an Fc.gamma. receptor, wherein the L chain
variable regions comprised in the variable region of (1) and the
variable region of (2) have a common amino acid sequence; wherein
the multispecific antigen-binding molecule has a cytotoxic activity
equivalent to or greater than that of the bispecific antibody
GPC3_ERY22_rCE115 comprising a glypican 3-binding domain comprising
SEQ ID NOs: 47 and 48, and a T-cell receptor complex-binding domain
comprising SEQ ID NOs: 49 and 50.
[2-2] The anticancer agent of [2-1], wherein the cytotoxic activity
is T-cell-dependent cytotoxic activity. [2-3] The anticancer agent
of [2-1] or [2-2], wherein the T-cell receptor complex-binding
activity is binding activity towards a T-cell receptor. [2-4] The
anticancer agent of any one of [2-1] to [2-3], wherein the T-cell
receptor complex-binding activity is binding activity towards a
CD3.epsilon. chain. [2-5] The anticancer agent of any one of [2-1]
to [2-4], wherein the antibody variable region of (1) in [1] is an
antibody variable region that comprises any one of the combinations
of H-chain CDR1, CDR2, and CDR3 selected from (a1) to (a5) below,
or an antibody variable region functionally equivalent thereto:
[0069] (a1) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 40;
[0070] (a2) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 197;
[0071] (a3) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 206;
[0072] (a4) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 211; and
[0073] (a5) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215.
[2-6] The anticancer agent of any one of [2-1] to [2-4], wherein
the antibody variable region of (2) in [2-1] is an antibody
variable region that comprises any one of the combinations of
H-chain CDR1, CDR2, and CDR3 amino acid sequences selected from
(b1) to (b15) below, or an antibody variable region functionally
equivalent thereto:
[0074] (b1) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 52;
[0075] (b2) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 103;
[0076] (b3) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 122;
[0077] (b4) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 128;
[0078] (b5) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 129;
[0079] (b6) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 132;
[0080] (b7) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 142;
[0081] (b8) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 144;
[0082] (b9) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 164;
[0083] (b10) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 168;
[0084] (b11) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 421;
[0085] (b12) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 424;
[0086] (b13) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 426;
[0087] (b14) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 429; and
[0088] (b15) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 430.
[2-7] The anticancer agent of any one of [2-1] to [2-4], wherein
the antibody variable regions of (1) and (2) in [2-1] are antibody
variable regions comprising any one of the combinations of H-chain
CDR1, CDR2, and CDR3 selected from the following (c1) to (c19), or
antibody variable regions functionally equivalent thereto:
[0089] (c1) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 40; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
(2) in [2-1] and identical to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 52;
[0090] (c2) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 40; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
(2) in [2-1] and identical to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 421;
[0091] (c3) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 40; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
(2) in [2-1] and identical to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 426;
[0092] (c4) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 40; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
(2) in [2-1] and identical to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 429;
[0093] (c5) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 40; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
(2) in [2-1] and identical to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 430;
[0094] (c6) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 197; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
(2) in [2-1] and identical to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 128;
[0095] (c7) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 206; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
(2) in [2-1] and identical to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 142;
[0096] (c8) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 206; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
(2) in [2-1] and identical to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 144;
[0097] (c9) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 206; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
(2) in [2-1] and identical to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 164;
[0098] (c10) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 206; and CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (2) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 168;
[0099] (c11) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 211; and CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (2) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 142;
[0100] (c12) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 211; and CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (2) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 144;
[0101] (c13) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 211; and CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (2) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 164;
[0102] (c14) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 211; and CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (2) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 168;
[0103] (c15) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; and CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (2) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 103;
[0104] (c16) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; and CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (2) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 122;
[0105] (c17) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; and CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (2) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 129;
[0106] (c18) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; and CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (2) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 132; and
[0107] (c19) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; and CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (2) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 424.
[2-8] The anticancer agent of any one of [2-5] to [2-7], wherein
CDR1, CDR2, and CDR3 are CDR1, CDR2, and CDR3 regions based on
Kabat numbering. [2-9] The anticancer agent of any one of [2-1] to
[2-4], wherein the antibody variable region of (1) in [2-1] is an
antibody variable region comprising any one of the H-chain variable
regions selected from (a1) to (a5) below, or an antibody variable
region functionally equivalent thereto:
[0108] (a1) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 40;
[0109] (a2) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 197;
[0110] (a3) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 206;
[0111] (a4) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 211; and
[0112] (a5) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 215.
[2-10] The anticancer agent of any one of [2-1] to [2-4], wherein
the antibody variable region of (2) in [2-1] is an antibody
variable region comprising any one of the H-chain variable regions
selected from (b1) to (b15) below, or an antibody variable region
functionally equivalent thereto:
[0113] (b1) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 52;
[0114] (b2) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 103;
[0115] (b3) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 122;
[0116] (b4) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 128;
[0117] (b5) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 129;
[0118] (b6) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 132;
[0119] (b7) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 142;
[0120] (b8) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 144;
[0121] (b9) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 164;
[0122] (b10) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 168;
[0123] (b11) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 421;
[0124] (b12) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 424;
[0125] (b13) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 426;
[0126] (b14) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 429; and
[0127] (b15) an H-chain variable region having the amino acid
sequence of SEQ ID NO: 430.
[2-11] The anticancer agent of any one of [2-1] to [2-4], wherein
the antibody variable regions of (1) and (2) in [2-1] are antibody
variable regions comprising any one of the combinations of H-chain
variable regions selected from (c1) to (c19) below, or antibody
variable regions functionally equivalent thereto:
[0128] (c1) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 40; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 52;
[0129] (c2) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 40; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 421;
[0130] (c3) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 40; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 426;
[0131] (c4) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 40; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 429;
[0132] (c5) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 40; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 430;
[0133] (c6) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 197; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 128;
[0134] (c7) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 206; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 142;
[0135] (c8) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 206; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 144;
[0136] (c9) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 206; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 164;
[0137] (c10) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 206; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 168;
[0138] (c11) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 211; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 142;
[0139] (c12) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 211; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 144;
[0140] (c13) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 211; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 164;
[0141] (c14) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 211; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 168;
[0142] (c15) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 215; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 103;
[0143] (c16) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 215; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 122;
[0144] (c17) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 215; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 129;
[0145] (c18) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 215; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 132; and
[0146] (c19) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] which has the amino acid sequence
of SEQ ID NO: 215; and an H-chain variable region comprised in the
antibody variable region of (2) in [2-1] which has the amino acid
sequence of SEQ ID NO: 424.
[2-12] The anticancer agent of any one of [2-1] to [2-11], wherein
the common L chain of [2-1] is a common L chain comprising any one
of the combinations of CDR1, CDR2, and CDR3 selected from (d1) to
(d11) below, or a common L chain functionally equivalent
thereto:
[0147] (d1) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 53;
[0148] (d2) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 223;
[0149] (d3) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 299;
[0150] (d4) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 301;
[0151] (d5) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 302;
[0152] (d6) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 304;
[0153] (d7) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 306;
[0154] (d8) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 307;
[0155] (d9) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 309;
[0156] (d10) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 310; and
[0157] (d11) CDR1, CDR2, and CDR3 identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 319.
[2-13] The anticancer agent of any one of [2-1] to [2-11], wherein
the L chain variable region of [2-1] is a variable region of any
one of the L chain amino acid sequences selected from (d1) to (d11)
below:
[0158] (d1) an L chain comprising the amino acid sequence of SEQ ID
NO: 53;
[0159] (d2) an L chain comprising the amino acid sequence of SEQ ID
NO: 223;
[0160] (d3) an L chain comprising the amino acid sequence of SEQ ID
NO: 299;
[0161] (d4) an L chain comprising the amino acid sequence of SEQ ID
NO: 301;
[0162] (d5) an L chain comprising the amino acid sequence of SEQ ID
NO: 302;
[0163] (d6) an L chain comprising the amino acid sequence of SEQ ID
NO: 304;
[0164] (d7) an L chain comprising the amino acid sequence of SEQ ID
NO: 306;
[0165] (d8) an L chain comprising the amino acid sequence of SEQ ID
NO: 307;
[0166] (d9) an L chain comprising the amino acid sequence of SEQ ID
NO: 309;
[0167] (d10) an L chain comprising the amino acid sequence of SEQ
ID NO: 310; and
[0168] (d11) an L chain comprising the amino acid sequence of SEQ
ID NO: 319.
[2-14] The anticancer agent of any one of [2-1] to [2-4], wherein
the antibody variable regions of (1) and (2) of [2-1] and the
common L chain variable region are antibody variable regions
comprising any one of the combinations of H-chain CDR1, CDR2, and
CDR3 and L-chain CDR1, CDR2, and CDR3 selected from (e1) to (e25)
below, or antibody variable regions functionally equivalent
thereto:
[0169] (e1) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 197; CDR1,
CDR2, and CDR3 comprised in the antibody variable region of (2) in
[2-1] and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 128; and CDR1, CDR2, and
CDR3 comprised in the antibody variable region of the common L
chain and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 53;
[0170] (e2) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 197; CDR1,
CDR2, and CDR3 comprised in the antibody variable region of (2) in
[2-1] and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 128; and CDR1, CDR2, and
CDR3 comprised in the antibody variable region of the common L
chain and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 299;
[0171] (e3) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 197; CDR1,
CDR2, and CDR3 comprised in the antibody variable region of (2) in
[2-1] and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 128; and CDR1, CDR2, and
CDR3 comprised in the antibody variable region of the common L
chain and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 310;
[0172] (e4) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 197; CDR1,
CDR2, and CDR3 comprised in the antibody variable region of (2) in
[2-1] and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 128; and CDR1, CDR2, and
CDR3 comprised in the antibody variable region of the common L
chain and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 319;
[0173] (e5) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 206; CDR1,
CDR2, and CDR3 comprised in the antibody variable region of (2) in
[2-1] and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 142; and CDR1, CDR2, and
CDR3 comprised in the antibody variable region of the common L
chain and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 223;
[0174] (e6) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 206; CDR1,
CDR2, and CDR3 comprised in the antibody variable region of (2) in
[2-1] and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 144; and CDR1, CDR2, and
CDR3 comprised in the antibody variable region of the common L
chain and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 223;
[0175] (e7) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 206; CDR1,
CDR2, and CDR3 comprised in the antibody variable region of (2) in
[2-1] and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 164; and CDR1, CDR2, and
CDR3 comprised in the antibody variable region of the common L
chain and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 223;
[0176] (e8) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 206; CDR1,
CDR2, and CDR3 comprised in the antibody variable region of (2) in
[2-1] and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 168; and CDR1, CDR2, and
CDR3 comprised in the antibody variable region of the common L
chain and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 223;
[0177] (e9) CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (1) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 211; CDR1,
CDR2, and CDR3 comprised in the antibody variable region of (2) in
[2-1] and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 142; and CDR1, CDR2, and
CDR3 comprised in the antibody variable region of the common L
chain and identical to the amino acid sequences of the CDR1, CDR2,
and CDR3 regions comprised in SEQ ID NO: 223;
[0178] (e10) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 211; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 142; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 299;
[0179] (e11) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 211; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 144; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 223;
[0180] (e12) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 211; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 164; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 223;
[0181] (e13) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 211; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 168; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 223;
[0182] (e14) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 103; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 53;
[0183] (e15) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 103; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 299;
[0184] (e16) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 103; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 301;
[0185] (e17) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 103; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 302;
[0186] (e18) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 103; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 304;
[0187] (e19) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 103; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 306;
[0188] (e20) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 103; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 307;
[0189] (e21) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 103; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 309;
[0190] (e22) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 122; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 53;
[0191] (e23) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 129; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 53;
[0192] (e24) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 132; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 53; and
[0193] (e25) CDR1, CDR2, and CDR3 comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 215; CDR1, CDR2, and CDR3 comprised in the antibody variable
region of (2) in [2-1] and identical to the amino acid sequences of
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 424; and
CDR1, CDR2, and CDR3 comprised in the antibody variable region of
the common L chain and identical to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 53.
[2-15] The anticancer agent of any one of [2-1] to [2-4], wherein
the antibody variable regions of (1) and (2) of [2-1] and the
common L chain variable region are antibody variable regions
comprising any one of the combinations of variable regions selected
from (f1) to (f26) below, or antibody variable regions functionally
equivalent thereto:
[0194] (f1) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 197; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 128; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 53;
[0195] (f2) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 197; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 128; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 299;
[0196] (f3) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 197; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 128; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 310;
[0197] (f4) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 197; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 128; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 319;
[0198] (f5) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 206; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 142; and an antibody variable
region of the common L chain and identical to the amino acid
sequence of the variable region comprised in SEQ ID NO: 223;
[0199] (f6) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 206; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 144; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 223;
[0200] (f7) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 206; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 164; and an antibody variable
region of the common L chain and identical to the amino acid
sequence of the variable region comprised in SEQ ID NO: 223;
[0201] (f8) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 206; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 168; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 223;
[0202] (f9) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 211; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 142; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 223;
[0203] (f10) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 211; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 142; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 299;
[0204] (f11) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 211; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 144; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 223;
[0205] (f12) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 211; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 164; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 223;
[0206] (f13) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 211; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 168; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 223;
[0207] (f14) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 215; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 103; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 53;
[0208] (f15) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 215; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 103; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 299;
[0209] (f16) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 215; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 103; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 301;
[0210] (f17) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 215; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 103; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 302;
[0211] (f18) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 215; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 103; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 304;
[0212] (f19) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 215; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 103; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 306;
[0213] (f20) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 215; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 103; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 307;
[0214] (f21) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 215; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 103; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 309;
[0215] (f22) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 215; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 122; and an antibody variable
region of the common L chain identical to the amino acid sequence
of the variable region comprised in SEQ ID NO: 53;
[0216] (f23) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 215; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 129; and an antibody variable
region of the common L chain and identical to the amino acid
sequence of the variable region comprised in SEQ ID NO: 53;
[0217] (f24) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 215; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 132; and an antibody variable
region of the common L chain and identical to the amino acid
sequence of the variable region comprised in SEQ ID NO: 53;
[0218] (f25) an H-chain variable region comprised in the antibody
variable region of (1) in [2-1] and identical to the amino acid
sequence of SEQ ID NO: 215; an H-chain variable region comprised in
the antibody variable region of (2) in [2-1] and identical to the
amino acid sequence of SEQ ID NO: 424; and an antibody variable
region of the common L chain and identical to the amino acid
sequence of the variable region comprised in SEQ ID NO: 53; and
[0219] (f26) multispecific antigen-binding molecule that binds to
an epitope overlapping with each of the epitopes on glypican 3 and
T-cell receptor complex bound by the multispecific antigen-binding
molecule of any one of (f1) to (f25), and which has a common L
chain.
[2-16] The anticancer agent of any one of [2-1] to [2-15], wherein
the Fc region of (3) in [2-1] is an Fc region with an amino acid
mutation at any of the Fc region-constituting amino acids of SEQ ID
NOs: 23 to 26 (IgG1 to IgG4). [2-17] The anticancer agent of
[2-16], wherein the Fc region of (3) in [2-1] is an Fc region with
mutation of at least one amino acid selected from the following
amino acid positions specified by EU numbering: position 220,
position 226, position 229, position 231, position 232, position
233, position 234, position 235, position 236, position 237,
position 238, position 239, position 240, position 264, position
265, position 266, position 267, position 269, position 270,
position 295, position 296, position 297, position 298, position
299, position 300, position 325, position 327, position 328,
position 329, position 330, position 331, and position 332. [2-18]
The anticancer agent of [2-16], wherein the Fc region of (3) in
[2-1] is an Fc region comprising at least one amino acid selected
from the following amino acids specified by EU numbering: Arg at
amino acid position 234, Ala or Arg at amino acid position 235, Lys
at amino acid position 239, and Ala at amino acid position 297.
[2-19] The anticancer agent of any one of [2-16] to [2-18], wherein
the Fc region of (3) in [2-1] further comprises an amino acid
mutation for promoting formation of a heterodimeric Fc region.
[2-20] The anticancer agent of [2-19], wherein the heterodimeric Fc
region is the amino acid sequence combination of (g1) or (g2)
below:
[0220] (g1) a combination of an amino acid sequence identical to
the Fc region of a constant region comprising the amino acid
sequence of SEQ ID NO: 57, and an amino acid sequence identical to
the Fc region of a constant region comprising the amino acid
sequence of SEQ ID NO: 58; and
[0221] (g2) a combination of an amino acid sequence identical to
the Fc region of a constant region comprising the amino acid
sequence of SEQ ID NO: 60 or 62, and an amino acid sequence
identical to the Fc region of a constant region comprising the
amino acid sequence of SEQ ID NO: 61.
[2-21] The anticancer agent of any one of [2-1] to [2-20], wherein
the multispecific antigen-binding molecule is a bispecific
antibody. [2-22] An anticancer agent comprising as an active
ingredient a bispecific antibody of any one of (h1) to (h25)
below:
[0222] (h1) a bispecific antibody having an antibody H chain having
glypican 3-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 215
and a constant region having the amino acid sequence of SEQ ID NO:
61; an antibody H chain having T-cell receptor complex-binding
activity that comprises an antibody H-chain variable region having
the amino acid sequence of SEQ ID NO: 424 and a constant region
having the amino acid sequence of SEQ ID NO: 60 or 62; and a common
antibody L chain having the amino acid sequence of SEQ ID NO:
53;
[0223] (h2) a bispecific antibody having an antibody H chain having
glypican 3-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 215
and a constant region having the amino acid sequence of SEQ ID NO:
61; an antibody H chain having T-cell receptor complex-binding
activity that comprises an antibody H-chain variable region having
the amino acid sequence of SEQ ID NO: 103 and a constant region
having the amino acid sequence of SEQ ID NO: 60 or 62; and a common
antibody L chain having the amino acid sequence of SEQ ID NO:
53;
[0224] (h3) a bispecific antibody having an antibody H chain having
glypican 3-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 215
and a constant region having the amino acid sequence of SEQ ID NO:
61; an antibody H chain having T-cell receptor complex-binding
activity that comprises an antibody H-chain variable region having
the amino acid sequence of SEQ ID NO: 103 and a constant region
having the amino acid sequence of SEQ ID NO: 60 or 62; and a common
antibody L chain having the amino acid sequence of SEQ ID NO:
299;
[0225] (h4) a bispecific antibody having an antibody H chain having
glypican 3-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 215
and a constant region having the amino acid sequence of SEQ ID NO:
61; an antibody H chain having T-cell receptor complex-binding
activity that comprises an antibody H-chain variable region having
the amino acid sequence of SEQ ID NO: 103 and a constant region
having the amino acid sequence of SEQ ID NO: 60 or 62; and a common
antibody L chain having the amino acid sequence of SEQ ID NO:
301;
[0226] (h5) a bispecific antibody having an antibody H chain having
glypican 3-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 215
and a constant region having the amino acid sequence of SEQ ID NO:
61; an antibody H chain having T-cell receptor complex-binding
activity that comprises an antibody H-chain variable region having
the amino acid sequence of SEQ ID NO: 103 and a constant region
having the amino acid sequence of SEQ ID NO: 60 or 62; and a common
antibody L chain having the amino acid sequence of SEQ ID NO:
302;
[0227] (h6) a bispecific antibody having an antibody H chain having
glypican 3-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 215
and a constant region having the amino acid sequence of SEQ ID NO:
61; an antibody H chain having T-cell receptor complex-binding
activity that comprises an antibody H-chain variable region having
the amino acid sequence of SEQ ID NO: 103 and a constant region
having the amino acid sequence of SEQ ID NO: 60 or 62; and a common
antibody L chain having the amino acid sequence of SEQ ID NO:
304;
[0228] (h7) a bispecific antibody having an antibody H chain having
glypican 3-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 215
and a constant region having the amino acid sequence of SEQ ID NO:
61; an antibody H chain having T-cell receptor complex-binding
activity that comprises an antibody H-chain variable region having
the amino acid sequence of SEQ ID NO: 103 and a constant region
having the amino acid sequence of SEQ ID NO: 60 or 62; and a common
antibody L chain having the amino acid sequence of SEQ ID NO:
306;
[0229] (h8) a bispecific antibody having an antibody H chain having
glypican 3-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 215
and a constant region having the amino acid sequence of SEQ ID NO:
61; an antibody H chain having T-cell receptor complex-binding
activity that comprises an antibody H-chain variable region having
the amino acid sequence of SEQ ID NO: 103 and a constant region
having the amino acid sequence of SEQ ID NO: 60 or 62; and a common
antibody L chain having the amino acid sequence of SEQ ID NO:
307;
[0230] (h9) a bispecific antibody having an antibody H chain having
glypican 3-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 215
and a constant region having the amino acid sequence of SEQ ID NO:
61; an antibody H chain having T-cell receptor complex-binding
activity that comprises an antibody H-chain variable region having
the amino acid sequence of SEQ ID NO: 103 and a constant region
having the amino acid sequence of SEQ ID NO: 60 or 62; and a common
antibody L chain having the amino acid sequence of SEQ ID NO:
309;
[0231] (h10) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 215 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 122
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 53;
[0232] (h11) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 215 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 129
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 53;
[0233] (h12) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 215 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 132
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 53;
[0234] (h13) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 197 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 128
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 299;
[0235] (h14) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 197 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 128
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 310;
[0236] (h15) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 197 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 128
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 319;
[0237] (h16) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 197 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 128
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 53;
[0238] (h17) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 211 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 142
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 299;
[0239] (h18) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 211 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 142
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 223;
[0240] (h19) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 211 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 144
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 223;
[0241] (h20) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 206 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 144
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 223;
[0242] (h21) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 206 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 142
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 223;
[0243] (h22) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 206 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 164
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 223;
[0244] (h23) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 206 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 168
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 223;
[0245] (h24) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 211 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 164
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 223; and
[0246] (h25) a bispecific antibody having an antibody H chain
having glypican 3-binding activity that comprises an antibody
H-chain variable region having the amino acid sequence of SEQ ID
NO: 211 and a constant region having the amino acid sequence of SEQ
ID NO: 61; an antibody H chain having T-cell receptor
complex-binding activity that comprises an antibody H-chain
variable region having the amino acid sequence of SEQ ID NO: 168
and a constant region having the amino acid sequence of SEQ ID NO:
60 or 62; and a common antibody L chain having the amino acid
sequence of SEQ ID NO: 223.
[0247] Furthermore, the present invention relates to an anticancer
agent comprising as an active ingredient a multispecific
antigen-binding molecule which comprises the following domains:
[0248] (3-1) a domain comprising an antibody variable region having
glypican 3-binding activity;
[0249] (3-2) a domain comprising an antibody variable region having
T-cell receptor complex-binding activity; wherein the L-chain
variable regions contained in the variable regions of (3-1) and
(3-2) have a commonly shared amino acid sequence. The present
invention also relates to an anticancer agent comprising as an
active ingredient the domain of (3-1), which is more specifically a
domain that comprises antibody heavy-chain and/or light-chain
variable regions having glypican 3-binding activity, and which is
comprised in the multispecific antigen-binding molecule. The
present invention additionally relates to an anticancer agent
comprising as an active ingredient the domain of (3-2), which is
more specifically a domain that comprises an antibody variable
region having T-cell receptor complex-binding activity, and which
is comprised in the multispecific antigen-binding molecule. Details
of the domains of (3-1) and (3-2) may include those described in
[2-1] to [2-22] mentioned above. The multispecific antigen-binding
molecule may be a bispecific antibody. Furthermore, the
multispecific antigen-binding molecule may further comprise a
domain comprising an Fc region, and the Fc region may have a
reduced Fc.gamma. receptor-binding activity. Details of the domain
comprising an Fc region may include those described in [2-1] to
[2-22] mentioned above. Furthermore, the present invention relates
to an anticancer agent comprising the multispecific antigen-binding
molecule and a pharmaceutically acceptable carrier. The anticancer
agent may induce cytotoxicity, the cytotoxicity may be
T-cell-dependent cellular cytotoxicity, and the agent may be for
administration to a patient in need of the multispecific
antigen-binding molecule.
[0250] The present invention also provides an anticancer agent
comprising as an active ingredient a multispecific antigen-binding
molecule that binds to epitopes overlapping and/or competing with
epitopes on each of glypican 3 and T-cell receptor complex bound by
the multispecific antigen-binding molecule of any one of (e1) to
(e25) of [2-14] mentioned above, and a multispecific
antigen-binding molecule that binds to epitopes overlapping and/or
competing with epitopes on each of glypican 3 and T-cell receptor
complex bound by the multispecific antigen-binding molecule of any
one of (f1) to (f25) of [2-15].
[0251] Regarding (g1) and (g2) of [2-20] mentioned above, of the
two Fc regions, the former Fc region may be included in the
antibody H chain having glypican 3-binding activity and the latter
Fc region may be included in the antibody H chain having T-cell
receptor complex-binding activity; or the former Fc region may be
included in the antibody H chain having T-cell receptor
complex-binding activity and the latter Fc region may be included
in the antibody H chain having glypican 3-binding activity.
[0252] The present invention also provides anticancer agents
comprising as an active ingredient a bispecific antibody that binds
to epitopes overlapping and/or competing with epitopes on each of
glypican 3 and T-cell receptor complex bound by a bispecific
antibody which has the antibody H chain of SEQ ID NO: 385 having
glypican 3-binding activity, the antibody H chain of SEQ ID NO: 402
having CD3-binding activity, and the common antibody L chains of
SEQ ID NO: 410.
[0253] The following inventions are also provided:
[4-1] A pharmaceutical composition comprising another anticancer
agent as an active ingredient, the pharmaceutical composition being
used in combination with a bispecific antibody of any one of (a) to
(c) below:
[0254] (a) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein CDR1, CDR2,
and CDR3 comprised in the antibody variable region having glypican
3-binding activity are sequences having at least 80% identity to
the amino acid sequences of the CDR1, CDR2, and CDR3 regions
comprised in SEQ ID NO: 206, respectively; CDR1, CDR2, and CDR3
comprised in the antibody variable region having CD3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 168, respectively; and CDR1, CDR2, and CDR3 comprised in an
antibody variable region of a common L chain are sequences having
at least 80% identity to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 223,
respectively;
[0255] (b) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the antibody
variable region having glypican 3-binding activity is a sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 206; the antibody variable region having CD3-binding activity
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 168; and an antibody variable region of a
common L chain is a sequence having at least 80% identity to the
amino acid sequence of SEQ ID NO: 223; and
[0256] (c) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the bispecific
antibody has an antibody H chain having glypican 3-binding activity
and having at least 80% identity to the amino acid sequence of SEQ
ID NO: 385; an antibody H chain having CD3-binding activity and
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 402; and antibody common L chains having at least 80% identity
to the amino acid sequence of SEQ ID NO: 410.
[4-2] The pharmaceutical composition of [4-1], wherein said another
anticancer agent is administered simultaneously with the bispecific
antibody. [4-3] The pharmaceutical composition of [4-1], wherein
said another anticancer agent is administered before or after
administration of the bispecific antibody. [4-4] A pharmaceutical
composition for treating or preventing cancer, comprising a
combination of a bispecific antibody of any one of (a) to (c) below
and another anticancer agent:
[0257] (a) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein CDR1, CDR2,
and CDR3 comprised in the antibody variable region having glypican
3-binding activity are sequences having at least 80% identity to
the amino acid sequences of the CDR1, CDR2, and CDR3 regions
comprised in SEQ ID NO: 206, respectively; CDR1, CDR2, and CDR3
comprised in the antibody variable region having CD3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 168, respectively; and CDR1, CDR2, and CDR3 comprised in an
antibody variable region of a common L chain are sequences having
at least 80% identity to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 223,
respectively;
[0258] (b) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the antibody
variable region having glypican 3-binding activity is a sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 206; the antibody variable region having CD3-binding activity
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 168; and an antibody variable region of a
common L chain is a sequence having at least 80% identity to the
amino acid sequence of SEQ ID NO: 223; and
[0259] (c) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the bispecific
antibody has an antibody H chain having glypican 3-binding activity
and having at least 80% identity to the amino acid sequence of SEQ
ID NO: 385; an antibody H chain having CD3-binding activity and
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 402; and antibody common L chains having at least 80% identity
to the amino acid sequence of SEQ ID NO: 410.
[4-5] The pharmaceutical composition of [4-4], which is a
combination preparation. [4-6] The pharmaceutical composition of
[4-4], wherein the bispecific antibody and said another anticancer
agent are administered separately. [4-7] The pharmaceutical
composition of [4-6], wherein the bispecific antibody and said
another anticancer agent are administered simultaneously or
sequentially. [4-8] The pharmaceutical composition of any one of
[4-1] to [4-7], wherein said another anticancer agent is a
chemotherapeutic agent, a T cell-activating agonist agent, an
immune checkpoint inhibitor, or an angiogenic inhibitor. [4-9] The
pharmaceutical composition of any one of [4-1] to [4-8], which is
for treating or preventing any cancer selected from the group
consisting of gastric cancer, head and neck cancer, esophageal
cancer, lung cancer, liver cancer, ovary cancer, breast cancer,
colon cancer, kidney cancer, skin cancer, muscle tumor, pancreas
cancer, prostate cancer, testis cancer, uterine cancer,
cholangiocarcinoma, Merkel cell carcinoma, bladder cancer, thyroid
cancer, schwannoma, adrenal cancer, anus cancer, central nervous
system tumor, neuroendocrine tissue tumor, penis cancer, pleura
tumor, salivary gland tumor, vulva cancer, thymoma, and childhood
cancer. [4-10] An agent for inducing cytotoxicity, an agent for
suppressing cell proliferation, an agent for inhibiting cell
proliferation, an agent for activating immune response, an agent
for treating cancer, or an agent for preventing cancer, which
comprises the pharmaceutical composition of any one of [4-1] to
[4-9].
[0260] The following inventions are also provided:
[4-11] A combination of a bispecific antibody of any one of (a) to
(c) below and another anticancer agent:
[0261] (a) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein CDR1, CDR2,
and CDR3 comprised in the antibody variable region having glypican
3-binding activity are sequences having at least 80% identity to
the amino acid sequences of the CDR1, CDR2, and CDR3 regions
comprised in SEQ ID NO: 206, respectively; CDR1, CDR2, and CDR3
comprised in the antibody variable region having CD3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 168, respectively; and CDR1, CDR2, and CDR3 comprised in an
antibody variable region of a common L chain are sequences having
at least 80% identity to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 223,
respectively;
[0262] (b) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the antibody
variable region having glypican 3-binding activity is a sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 206; the antibody variable region having CD3-binding activity
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 168; and an antibody variable region of a
common L chain is a sequence having at least 80% identity to the
amino acid sequence of SEQ ID NO: 223; and
[0263] (c) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the bispecific
antibody has an antibody H chain having glypican 3-binding activity
and having at least 80% identity to the amino acid sequence of SEQ
ID NO: 385; an antibody H chain having CD3-binding activity and
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 402; and antibody common L chains having at least 80% identity
to the amino acid sequence of SEQ ID NO: 410.
[4-12] The combination of [4-11], wherein the bispecific antibody
is administered simultaneously with said another anticancer agent.
[4-13] The combination of [4-11], wherein the bispecific antibody
is administered before or after administration of said another
anticancer agent. [4-14] The combination of any one of [4-11] to
[4-13], wherein said another anticancer agent is a chemotherapeutic
agent, a T cell-activating agonist agent, an immune checkpoint
inhibitor, or an angiogenic inhibitor. [4-15] The combination of
any one of [4-11] to [4-14], which is for treating or preventing
any cancer selected from the group consisting of gastric cancer,
head and neck cancer, esophageal cancer, lung cancer, liver cancer,
ovary cancer, breast cancer, colon cancer, kidney cancer, skin
cancer, muscle tumor, pancreas cancer, prostate cancer, testis
cancer, uterine cancer, cholangiocarcinoma, Merkel cell carcinoma,
bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anus
cancer, central nervous system tumor, neuroendocrine tissue tumor,
penis cancer, pleura tumor, salivary gland tumor, vulva cancer,
thymoma, and childhood cancer. [4-16] An agent for inducing
cytotoxicity, an agent for suppressing cell proliferation, an agent
for inhibiting cell proliferation, an agent for activating immune
response, an agent for treating cancer, or an agent for preventing
cancer, which comprises the combination of any one of [4-11] to
[4-15].
[0264] The following inventions are also provided:
[4-17] A method for inducing cytotoxicity, for suppressing cell
proliferation, for inhibiting cell proliferation, for activating
immune response, for treating cancer, or for preventing cancer in
an individual, comprising administering an effective amount of a
bispecific antibody of any one of (a) to (c) below and an effective
amount of another anticancer agent:
[0265] (a) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein CDR1, CDR2,
and CDR3 comprised in the antibody variable region having glypican
3-binding activity are sequences having at least 80% identity to
the amino acid sequences of the CDR1, CDR2, and CDR3 regions
comprised in SEQ ID NO: 206, respectively; CDR1, CDR2, and CDR3
comprised in the antibody variable region having CD3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 168, respectively; and CDR1, CDR2, and CDR3 comprised in an
antibody variable region of a common L chain are sequences having
at least 80% identity to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 223,
respectively;
[0266] (b) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the antibody
variable region having glypican 3-binding activity is a sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 206; the antibody variable region having CD3-binding activity
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 168; and an antibody variable region of a
common L chain is a sequence having at least 80% identity to the
amino acid sequence of SEQ ID NO: 223; and
[0267] (c) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the bispecific
antibody has an antibody H chain having glypican 3-binding activity
and having at least 80% identity to the amino acid sequence of SEQ
ID NO: 385; an antibody H chain having CD3-binding activity and
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 402; and antibody common L chains having at least 80% identity
to the amino acid sequence of SEQ ID NO: 410.
[4-18] A method for inducing cytotoxicity, for suppressing cell
proliferation, for inhibiting cell proliferation, for activating
immune response, for treating cancer, or for preventing cancer in
an individual with combined use of a bispecific antibody of any one
of (a) to (c) below, comprising administering to the individual an
effective amount of another anticancer agent:
[0268] (a) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein CDR1, CDR2,
and CDR3 comprised in the antibody variable region having glypican
3-binding activity are sequences having at least 80% identity to
the amino acid sequences of the CDR1, CDR2, and CDR3 regions
comprised in SEQ ID NO: 206, respectively; CDR1, CDR2, and CDR3
comprised in the antibody variable region having CD3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 168, respectively; and CDR1, CDR2, and CDR3 comprised in an
antibody variable region of a common L chain are sequences having
at least 80% identity to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 223,
respectively;
[0269] (b) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the antibody
variable region having glypican 3-binding activity is a sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 206; the antibody variable region having CD3-binding activity
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 168; and an antibody variable region of a
common L chain is a sequence having at least 80% identity to the
amino acid sequence of SEQ ID NO: 223; and
[0270] (c) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the bispecific
antibody has an antibody H chain having glypican 3-binding activity
and having at least 80% identity to the amino acid sequence of SEQ
ID NO: 385; an antibody H chain having CD3-binding activity and
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 402; and antibody common L chains having at least 80% identity
to the amino acid sequence of SEQ ID NO: 410.
[4-19] A method for inducing cytotoxicity, for suppressing cell
proliferation, for inhibiting cell proliferation, for activating
immune response, for treating cancer, or for preventing cancer in
an individual with combined use of another anticancer agent,
comprising administering to the individual an effective amount of a
bispecific antibody of any one of (a) to (c) below;
[0271] (a) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein CDR1, CDR2,
and CDR3 comprised in the antibody variable region having glypican
3-binding activity are sequences having at least 80% identity to
the amino acid sequences of the CDR1, CDR2, and CDR3 regions
comprised in SEQ ID NO: 206, respectively; CDR1, CDR2, and CDR3
comprised in the antibody variable region having CD3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 168, respectively; and CDR1, CDR2, and CDR3 comprised in an
antibody variable region of a common L chain are sequences having
at least 80% identity to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 223,
respectively;
[0272] (b) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the antibody
variable region having glypican 3-binding activity is a sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 206; the antibody variable region having CD3-binding activity
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 168; and an antibody variable region of a
common L chain is a sequence having at least 80% identity to the
amino acid sequence of SEQ ID NO: 223; and
[0273] (c) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the bispecific
antibody has an antibody H chain having glypican 3-binding activity
and having at least 80% identity to the amino acid sequence of SEQ
ID NO: 385; an antibody H chain having CD3-binding activity and
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 402; and antibody common L chains having at least 80% identity
to the amino acid sequence of SEQ ID NO: 410.
[4-20] A method for enhancing effects of inducing cytotoxicity,
suppressing cell proliferation, inhibiting cell proliferation,
activating immune response, treating cancer, or preventing cancer
in an individual by a bispecific antibody of any one of (a) to (c)
below, comprising administering an effective amount of another
anticancer agent to the individual:
[0274] (a) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein CDR1, CDR2,
and CDR3 comprised in the antibody variable region having glypican
3-binding activity are sequences having at least 80% identity to
the amino acid sequences of the CDR1, CDR2, and CDR3 regions
comprised in SEQ ID NO: 206, respectively; CDR1, CDR2, and CDR3
comprised in the antibody variable region having CD3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 168, respectively; and CDR1, CDR2, and CDR3 comprised in an
antibody variable region of a common L chain are sequences having
at least 80% identity to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 223,
respectively;
[0275] (b) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the antibody
variable region having glypican 3-binding activity is a sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 206; the antibody variable region having CD3-binding activity
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 168; and an antibody variable region of a
common L chain is a sequence having at least 80% identity to the
amino acid sequence of SEQ ID NO: 223; and
[0276] (c) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the bispecific
antibody has an antibody H chain having glypican 3-binding activity
and having at least 80% identity to the amino acid sequence of SEQ
ID NO: 385; an antibody H chain having CD3-binding activity and
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 402; and antibody common L chains having at least 80% identity
to the amino acid sequence of SEQ ID NO: 410.
[4-21] A method for enhancing effects of inducing cytotoxicity,
suppressing cell proliferation, inhibiting cell proliferation,
activating immune response, treating cancer, or preventing cancer
in an individual by another anticancer agent, comprising
administering an effective amount of a bispecific antibody of any
one of (a) to (c) below:
[0277] (a) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein CDR1, CDR2,
and CDR3 comprised in the antibody variable region having glypican
3-binding activity are sequences having at least 80% identity to
the amino acid sequences of the CDR1, CDR2, and CDR3 regions
comprised in SEQ ID NO: 206, respectively; CDR1, CDR2, and CDR3
comprised in the antibody variable region having CD3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 168, respectively; and CDR1, CDR2, and CDR3 comprised in an
antibody variable region of a common L chain are sequences having
at least 80% identity to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 223,
respectively;
[0278] (b) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the antibody
variable region having glypican 3-binding activity is a sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 206; the antibody variable region having CD3-binding activity
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 168; and an antibody variable region of a
common L chain is a sequence having at least 80% identity to the
amino acid sequence of SEQ ID NO: 223; and
[0279] (c) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the bispecific
antibody has an antibody H chain having glypican 3-binding activity
and having at least 80% identity to the amino acid sequence of SEQ
ID NO: 385; an antibody H chain having CD3-binding activity and
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 402; and antibody common L chains having at least 80% identity
to the amino acid sequence of SEQ ID NO: 410.
[4-22] The method of any one of [4-17] to [4-21], wherein the
bispecific antibody and said another anticancer agent are
administrated separately. [4-23] The method of any one of [4-17] to
[4-22], wherein the bispecific antibody and said another anticancer
agent are administrated simultaneously or sequentially. [4-24] The
method of any one of [4-17] to [4-23], wherein said another
anticancer agent is a chemotherapeutic agent, a T cell-activating
agonist agent, an immune checkpoint inhibitor, or an angiogenic
inhibitor. [4-25] The method of any one of [4-17] to [4-24],
wherein the cancer is any cancer selected from the group consisting
of gastric cancer, head and neck cancer, esophageal cancer, lung
cancer, liver cancer, ovary cancer, breast cancer, colon cancer,
kidney cancer, skin cancer, muscle tumor, pancreas cancer, prostate
cancer, testis cancer, uterine cancer, cholangiocarcinoma, Merkel
cell carcinoma, bladder cancer, thyroid cancer, schwannoma, adrenal
cancer, anus cancer, central nervous system tumor, neuroendocrine
tissue tumor, penis cancer, pleura tumor, salivary gland tumor,
vulva cancer, thymoma, and childhood cancer.
[0280] The following inventions are also provided:
[4-26] A kit comprising: (A) a pharmaceutical composition
comprising a bispecific antibody of any one of (a) to (c) below;
(B) a container; and (C) an instruction or a label indicating that
the bispecific antibody and at least one type of another anticancer
agent are administered in combination to an individual for treating
or preventing cancer in the individual;
[0281] (a) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein CDR1, CDR2,
and CDR3 comprised in the antibody variable region having glypican
3-binding activity are sequences having at least 80% identity to
the amino acid sequences of the CDR1, CDR2, and CDR3 regions
comprised in SEQ ID NO: 206, respectively; CDR1, CDR2, and CDR3
comprised in the antibody variable region having CD3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 168, respectively; and CDR1, CDR2, and CDR3 comprised in an
antibody variable region of a common L chain are sequences having
at least 80% identity to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 223,
respectively;
[0282] (b) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the antibody
variable region having glypican 3-binding activity is a sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 206; the antibody variable region having CD3-binding activity
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 168; and an antibody variable region of a
common L chain is a sequence having at least 80% identity to the
amino acid sequence of SEQ ID NO: 223; and
[0283] (c) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the bispecific
antibody has an antibody H chain having glypican 3-binding activity
and having at least 80% identity to the amino acid sequence of SEQ
ID NO: 385; an antibody H chain having CD3-binding activity and
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 402; and antibody common L chains having at least 80% identity
to the amino acid sequence of SEQ ID NO: 410.
[4-27] A kit comprising: (A) another anticancer agent; (B) a
container; and (C) an instruction or a label indicating that said
another anticancer agent and a pharmaceutical composition
comprising at least one type of a bispecific antibody of (a) to (c)
below are administered in combination to an individual for treating
or preventing cancer in the individual;
[0284] (a) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein CDR1, CDR2,
and CDR3 comprised in the antibody variable region having glypican
3-binding activity are sequences having at least 80% identity to
the amino acid sequences of the CDR1, CDR2, and CDR3 regions
comprised in SEQ ID NO: 206, respectively; CDR1, CDR2, and CDR3
comprised in the antibody variable region having CD3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 168, respectively; and CDR1, CDR2, and CDR3 comprised in an
antibody variable region of a common L chain are sequences having
at least 80% identity to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 223,
respectively;
[0285] (b) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the antibody
variable region having glypican 3-binding activity is a sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 206; the antibody variable region having CD3-binding activity
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 168; and an antibody variable region of a
common L chain is a sequence having at least 80% identity to the
amino acid sequence of SEQ ID NO: 223; and
[0286] (c) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the bispecific
antibody has an antibody H chain having glypican 3-binding activity
and having at least 80% identity to the amino acid sequence of SEQ
ID NO: 385; an antibody H chain having CD3-binding activity and
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 402; and antibody common L chains having at least 80% identity
to the amino acid sequence of SEQ ID NO: 410.
[4-28] A kit comprising: (A) a pharmaceutical composition
comprising a bispecific antibody of any one of (a) to (c)
below:
[0287] (a) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein CDR1, CDR2,
and CDR3 comprised in the antibody variable region having glypican
3-binding activity are sequences having at least 80% identity to
the amino acid sequences of the CDR1, CDR2, and CDR3 regions
comprised in SEQ ID NO: 206, respectively; CDR1, CDR2, and CDR3
comprised in the antibody variable region having CD3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 168, respectively; and CDR1, CDR2, and CDR3 comprised in an
antibody variable region of a common L chain are sequences having
at least 80% identity to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 223,
respectively;
[0288] (b) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the antibody
variable region having glypican 3-binding activity is a sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 206; the antibody variable region having CD3-binding activity
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 168; and an antibody variable region of a
common L chain is a sequence having at least 80% identity to the
amino acid sequence of SEQ ID NO: 223; and
[0289] (c) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the bispecific
antibody has an antibody H chain having glypican 3-binding activity
and having at least 80% identity to the amino acid sequence of SEQ
ID NO: 385; an antibody H chain having CD3-binding activity and
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 402; and antibody common L chains having at least 80% identity
to the amino acid sequence of SEQ ID NO: 410;
(B) a container; and (C) another anticancer agent. [4-29] The kit
of any one of [4-26] to [4-28], wherein the bispecific antibody is
administered simultaneously with said another anticancer agent.
[4-30] The kit of any one of [4-26] to [4-28], wherein the
bispecific antibody is administered before or after administration
of said another anticancer agent. [4-31] The kit of any one of
[4-26] to [4-30], wherein said another anticancer agent is a
chemotherapeutic agent, a T cell-activating agonist agent, an
immune checkpoint inhibitor, or an angiogenic inhibitor. [4-32] The
kit of any one of [4-26] to [4-31], wherein the cancer is any
cancer selected from the group consisting of gastric cancer, head
and neck cancer, esophageal cancer, lung cancer, liver cancer,
ovary cancer, breast cancer, colon cancer, kidney cancer, skin
cancer, muscle tumor, pancreas cancer, prostate cancer, testis
cancer, uterine cancer, cholangiocarcinoma, Merkel cell carcinoma,
bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anus
cancer, central nervous system tumor, neuroendocrine tissue tumor,
penis cancer, pleura tumor, salivary gland tumor, vulva cancer,
thymoma, and childhood cancer.
[0290] The following inventions are also provided:
[4-33] A method for inducing damage to a cancer cell or a cancer
cell-comprising tumor tissue, or a method for suppressing
proliferation of a cancer cell or growth of a cancer
cell-comprising tumor tissue, by contacting a cancer cell with a
bispecific antibody of any one of (a) to (c) below and another
anticancer agent;
[0291] (a) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein CDR1, CDR2,
and CDR3 comprised in the antibody variable region having glypican
3-binding activity are sequences having at least 80% identity to
the amino acid sequences of the CDR1, CDR2, and CDR3 regions
comprised in SEQ ID NO: 206, respectively; CDR1, CDR2, and CDR3
comprised in the antibody variable region having CD3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 168, respectively; and CDR1, CDR2, and CDR3 comprised in an
antibody variable region of a common L chain are sequences having
at least 80% identity to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 223,
respectively;
[0292] (b) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the antibody
variable region having glypican 3-binding activity is a sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 206; the antibody variable region having CD3-binding activity
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 168; and an antibody variable region of a
common L chain is a sequence having at least 80% identity to the
amino acid sequence of SEQ ID NO: 223; and
[0293] (c) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the bispecific
antibody has an antibody H chain having glypican 3-binding activity
and having at least 80% identity to the amino acid sequence of SEQ
ID NO: 385; an antibody H chain having CD3-binding activity and
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 402; and antibody common L chains having at least 80% identity
to the amino acid sequence of SEQ ID NO: 410.
[4-34] A method for assessing whether a bispecific antibody and
another anticancer agent induce damage to a cancer cell or a cancer
cell-comprising tumor tissue, or suppress proliferation of a cancer
cell or growth of a cancer cell-comprising tumor tissue, by
contacting a cancer cell with the bispecific antibody of any one of
(a) to (c) below and another anticancer agent;
[0294] (a) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein CDR1, CDR2,
and CDR3 comprised in the antibody variable region having glypican
3-binding activity are sequences having at least 80% identity to
the amino acid sequences of the CDR1, CDR2, and CDR3 regions
comprised in SEQ ID NO: 206, respectively; CDR1, CDR2, and CDR3
comprised in the antibody variable region having CD3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 168, respectively; and CDR1, CDR2, and CDR3 comprised in an
antibody variable region of a common L chain are sequences having
at least 80% identity to the amino acid sequences of the CDR1,
CDR2, and CDR3 regions comprised in SEQ ID NO: 223,
respectively;
[0295] (b) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the antibody
variable region having glypican 3-binding activity is a sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 206; the antibody variable region having CD3-binding activity
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 168; and an antibody variable region of a
common L chain is a sequence having at least 80% identity to the
amino acid sequence of SEQ ID NO: 223; and
[0296] (c) a bispecific antibody that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity, wherein the bispecific
antibody has an antibody H chain having glypican 3-binding activity
and having at least 80% identity to the amino acid sequence of SEQ
ID NO: 385; an antibody H chain having CD3-binding activity and
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 402; and antibody common L chains having at least 80% identity
to the amino acid sequence of SEQ ID NO: 410.
[4-35] The method of [4-33] or [4-34], wherein said another
anticancer agent is a chemotherapeutic agent, a T cell-activating
agonist agent, an immune checkpoint inhibitor, or an angiogenic
inhibitor. [4-36] The method of any one of [4-33] to [4-35],
wherein the cancer cell is any cancer cell selected from the group
consisting of gastric cancer, head and neck cancer, esophageal
cancer, lung cancer, liver cancer, ovary cancer, breast cancer,
colon cancer, kidney cancer, skin cancer, muscle tumor, pancreas
cancer, prostate cancer, testis cancer, uterine cancer,
cholangiocarcinoma, Merkel cell carcinoma, bladder cancer, thyroid
cancer, schwannoma, adrenal cancer, anus cancer, central nervous
system tumor, neuroendocrine tissue tumor, penis cancer, pleura
tumor, salivary gland tumor, vulva cancer, thymoma, and childhood
cancer.
[0297] The following inventions are also provided:
[5-1] The pharmaceutical composition of any one of [7] to [12] and
[4-1] to [4-9], which is for increasing and/or maintaining the
expression of CD3.epsilon. chain in an individual as compared to
administration of the bispecific antibody of any one of [7] (a) to
(c) or said another anticancer agent alone. [5-2] The
pharmaceutical composition of any one of [7] to [12] and [4-1] to
[4-9], which is for expanding T cell population in an individual as
compared to administration of the bispecific antibody of any one of
[7] (a) to (c) or said another anticancer agent alone. [5-3] The
pharmaceutical composition of [5-2], wherein the T cell population
is an activated T cell population. [5-4] The pharmaceutical
composition of any one of [7] to [12] and [4-1] to [4-9], which is
for increasing the expression of a cytokine and/or chemokine in an
individual as compared to administration of the bispecific antibody
of any one of [7] (a) to (c) or said another anticancer agent
alone. [5-5] The pharmaceutical composition of [5-4], wherein the
cytokine and/or chemokine is one or more cytokines and/or
chemokines selected from the group consisting of IFN.gamma., IL2,
IL6, IL7, IL8, IL10, IL17A, TNF, CXCL9, and CXCL10. [5-6] The
pharmaceutical composition of any one of [7] to [12] and [4-1] to
[4-9], which is for increasing the expression of a gene involved in
cell death in an individual as compared to administration of the
bispecific antibody of any one of [7] (a) to (c) or said another
anticancer agent alone. [5-7] The pharmaceutical composition of
[5-6], wherein the gene involved in cell death is one or more genes
selected from the group consisting of TNFSF10, FAS, FASL, caspase
8, and caspase 7. [5-8] The pharmaceutical composition of any one
of [7] to [12] and [4-1] to [4-9], which is for inhibiting a gene
involved in cell cycle promotion in an individual as compared to
administration of the bispecific antibody of any one of [7] (a) to
(c) or said another anticancer agent alone. [5-9] The
pharmaceutical composition of [5-8], wherein the gene involved in
cell cycle promotion is one or more genes selected from the group
consisting of PCNA, CCNA2, and CDK4. [5-10] The pharmaceutical
composition of any one of [7] to [12] and [4-1] to [4-9], which is
for increasing the expression of a gene involved in cell cycle
suppression in an individual as compared to administration of the
bispecific antibody of any one of [7] (a) to (c) or said another
anticancer agent alone. [5-11] The pharmaceutical composition of
[5-10], wherein the gene involved in cell cycle suppression is p21.
[5-12] The pharmaceutical composition of any one of [7] to [12] and
[4-1] to [4-9], which is for increasing a leukocyte marker in an
individual as compared to administration of the bispecific antibody
of any one of [7] (a) to (c) or said another anticancer agent
alone. [5-13] The pharmaceutical composition of [5-12], wherein the
leukocyte marker is CD45. [5-14] The pharmaceutical composition of
any one of [7] to [12] and [4-1] to [4-9], which is for increasing
a T cell marker and/or T cell activation marker in an individual as
compared to administration of the bispecific antibody of any one of
[7] (a) to (c) or said another anticancer agent alone. [5-15] The
pharmaceutical composition of [5-14], wherein the T cell marker
and/or T cell activation marker is one or more T cell markers
and/or T cell activation markers selected from the group consisting
of CD3, CD4, CD8a, GZB, PRF1, and IFN.gamma.. [5-16] The
pharmaceutical composition of any one of [7] to [12] and [4-1] to
[4-9], which is for increasing the expression of an immune
checkpoint gene in an individual as compared to administration of
the bispecific antibody of any one of [7] (a) to (c) or said
another anticancer agent alone. [5-17] The pharmaceutical
composition of [5-16], wherein the immune checkpoint gene is one or
more immune checkpoint genes selected from the group consisting of
PD-L1, PD-1, TIM3, LAG3, and CTLA4.
[0298] In each item of [4-1] to [4-36] above, "another anticancer
agent" refers to an anticancer agent which comprises as an active
ingredient a substance that is different from the bispecific
antibody recited in each item. Specifically, the term "another
anticancer agent" merely indicates that the invention of each item
is specified as inventions in which the other/another anticancer
agent is an anticancer agent comprising as an active ingredient a
substance that is different from the bispecific antibody, and the
invention is not limited to inventions in which the bispecific
antibody is used as an anticancer agent. For example, even when
each invention of [4-1] to [4-36] recites the term "another
anticancer agent", the invention also includes embodiments where no
other anticancer agent than the "another anticancer agent" is used,
and in this case, the invention includes embodiments where the
bispecific antibody is used as an enhancer, combination drug,
excipient, or such for the other anticancer agent.
Effects of the Invention
[0299] The present invention provides novel multispecific
antigen-binding molecules with molecular forms that can be produced
with high efficiency, which maintain the strong antitumor activity
possessed by BiTE and the excellent safety property of not causing
cancer antigen-independent induction of a cytokine storm and such,
and have long half-lives in blood. Anticancer agents which comprise
a multispecific antigen-binding molecule of the present invention
as an active ingredient and combination therapies using the
multispecific antigen-binding molecule and another anticancer agent
target cancer tissues containing glypican 3-expressing cancer cells
to cause cytotoxicity, and can treat or prevent various cancers.
The invention enables desirable treatment which has not only a high
level of safety but also reduced physical burden, and is highly
convenient for patients.
BRIEF DESCRIPTION OF THE DRAWINGS
[0300] FIG. 1A is graphs showing the cytotoxic activity of
antibody-38 when cell lines derived from various cancer types are
used as target cells.
[0301] FIG. 1B is graphs showing the cytotoxic activity of
antibody-38 when cell lines derived from various cancer types are
used as target cells (a continuation of FIG. 1A).
[0302] FIG. 1C is graphs showing the cytotoxic activity of
antibody-38 when cell lines derived from various cancer types are
used as target cells (a continuation of FIG. 1B).
[0303] FIG. 2 is graphs showing the anti-tumor activity of
antibody-38 against xenograft tumors derived from various cancer
types in a human T cell transplantation model.
[0304] FIG. 3 is a graph showing the anti-tumor activities of
antibody-39 and -40 against PC-10 xenograft tumor in a human T cell
transplantation model.
[0305] FIG. 4 is a graph showing the anti-tumor activities of
antibody-30, -31, -32, and -33 against SK-HEP-1/hGPC3 (SK-pca31a)
xenograft tumor in a human T cell transplantation model.
[0306] FIG. 5 is a graph showing the anti-tumor activity of
antibody-38 against PC-10 xenograft tumor in a humanized NOG mouse
model.
[0307] FIG. 6 is a graph showing the anti-tumor activities of
antibody-38, and anti-human GPC3 antibody, anti-mouse CTLA-4
antibody, anti-mouse PD-1 antibody, or anti-mouse PD-L1 antibody
against LLC1/hGPC3 syngeneic tumor in a human CD3.epsilon.6.gamma.
gene-modified mouse model.
[0308] FIG. 7 is graphs showing the anti-tumor activities resulting
from a single or combination of antibody, and capecitabine,
cisplatin, or paclitaxel against MKN45 xenograft tumor in a human T
cell transplantation model.
[0309] FIG. 8 is graphs showing the anti-tumor activities resulting
from a single or combination of antibody, and cisplatin or
paclitaxel against NCI-H446 xenograft tumor in a human T cell
transplantation model.
[0310] FIG. 9 is graphs showing the anti-tumor activities resulting
from a single or combination of antibody-38, and anti-mouseTIM-3
antibody, anti-mouse LAG-3 antibody, anti-mouse CD137 antibody, or
anti-mouse VEGFR2 antibody against Hepa1-6/hGPC3 syngeneic tumor in
a human CD3.epsilon.6.gamma. gene-modified mouse model.
[0311] FIG. 10 is a graph showing the anti-tumor activities
resulting from a single or combination of antibody-38 and
anti-mouse CD137 antibody against LLC1/hGPC3 syngeneic tumor in a
human CD3.epsilon.6.gamma. gene-modified mouse model.
[0312] FIG. 11 is graphs showing the anti-tumor activities
resulting from a single or combination of antibody-38, and
anti-mouse PD-1 antibody or anti-mouse PD-L1 antibody against
Hepa1-6/hGPC3 syngeneic tumor in a human CD3.epsilon. gene-modified
mouse model.
[0313] FIG. 12 shows schematic diagrams of a: ERY22 and b:
ERY27.
[0314] FIG. 13 is a graph showing the cytotoxic activities of
GPC3_ERY22_rCE115 and GPC3_ERY27_hCE115 when NCI-H446 is used as
the target cell. The filled diamond (.diamond-solid.) and the
filled triangle (.tangle-solidup.) indicate the cytotoxic activity
of GPC3_ERY22_rCE115 and GPC3_ERY27_hCE115, respectively.
[0315] FIG. 14 is a graph showing the cytotoxic activities of
GPC3_ERY22_rCE115 and GPC3_ERY27_hCE115 when PC-10 is used as the
target cell. The filled diamond (.diamond-solid.) and the filled
triangle (.tangle-solidup.) indicate the cytotoxic activity of
GPC3_ERY22_rCE115 and GPC3_ERY27_hCE115, respectively.
[0316] FIG. 15 is a graph showing the cytotoxic activities of
optimized antibodies (a) 1, 2, 3 and 4, and (b) 1, 5, 6, 7 and 8
when NCI-H446 is used as the target cell. The optimized antibodies
are described in Table 17.
[0317] FIG. 16 is a graph showing the cytotoxic activities of
optimized antibodies (a) 1, 13, 14, 15 and 16, and (b) 1, 17, 18,
19 and 20 when NCI-H446 is used as the target cell. The optimized
antibodies are described in Table 17.
[0318] FIG. 17 is a graph showing the cytotoxic activities of
optimized antibodies (a) 1, 21, 22, 23 and 24, and (b) 1, 25, 26,
27 and 28 when NCI-H446 is used as the target cell. The optimized
antibodies are described in Table 17.
[0319] FIG. 18 is a graph showing the cytotoxic activities of
optimized antibodies (a) 1, 29, 30, 31 and 32, and (b) 1, 33, 31,
32 and 30 when NCI-H446 is used as the target cell. The optimized
antibodies are described in Table 17.
[0320] FIG. 19 is a graph showing the cytotoxic activities of
optimized antibodies (a) 1, 30, 34, 33 an 35, and (b) 1, 34, 35, 36
and 37 when NCI-H446 is used as the target cell. The optimized
antibodies are described in Table 17.
[0321] FIG. 20 is a graph showing the cytotoxic activities of
optimized antibodies (a) 1, 30, 34, 35 and 37, and (b) 1, 34, 37,
30 and 38 when NCI-H446 is used as the target cell. The optimized
antibodies are described in Table 17.
[0322] FIG. 21 shows the in vivo antitumor effects of optimized
antibodies (a) 37, 34, 30 and 1, and (b) 37 and 38 when PC-10 is
used as the target cell. The optimized antibodies are described in
Table 17.
[0323] FIG. 22 shows the in vivo antitumor effects of optimized
antibodies (a) 1, 30, 34, 33 and 35, and (b) 37, 34, 30 and 1 when
NCI-H446 is used as the target cell. The optimized antibodies are
described in Table 17.
[0324] FIG. 23 shows the relationship between the amino acid
residues constituting the Fc regions of IgG1, IgG2, IgG3, and IgG4,
and the Kabat EU numbering system (herein, also referred to as EU
INDEX).
[0325] FIG. 24A shows the heavy-chain variable region sequences and
their numbering according to Kabat et al. Residues 1 to 79 of the
heavy-chain variable region sequences of CE115HA000 (SEQ ID NO:
52), TR01H040 (SEQ ID NO: 103), TR01H071 (SEQ ID NO: 132), TR01H082
(SEQ ID NO: 142), TR01H084 (SEQ ID NO: 144), TR01H109 (SEQ ID NO:
164), TR01H113 (SEQ ID NO: 168), H0000 (SEQ ID NO: 40), H0610 (SEQ
ID NO: 215), GCH054 (SEQ ID NO: 197), GCH065 (SEQ ID NO: 206), and
GCH094 (SEQ ID NO: 211) are shown.
[0326] FIG. 24B shows the heavy-chain variable region sequences and
their numbering according to Kabat et al. Residues 80 to 113 of the
heavy-chain variable region sequences of CE115HA000 (SEQ ID NO:
52), TR01H040 (SEQ ID NO: 103), TR01H071 (SEQ ID NO: 132), TR01H082
(SEQ ID NO: 142), TR01H084 (SEQ ID NO: 144), TR01H109 (SEQ ID NO:
164), TR01H113 (SEQ ID NO: 168), H0000 (SEQ ID NO: 40), H0610 (SEQ
ID NO: 215), GCH054 (SEQ ID NO: 197), GCH065 (SEQ ID NO: 206), and
GCH094 (SEQ ID NO: 211) are shown.
[0327] FIG. 25 shows the light-chain variable region sequences and
their numbering according to Kabat et al. Light-chain variable
region sequences of L0000 (SEQ ID NO: 53), L0011 (SEQ ID NO: 223),
L0201 (SEQ ID NO: 299), L0203 (SEQ ID NO: 301), L0204 (SEQ ID NO:
302), L0206 (SEQ ID NO: 304), L0208 (SEQ ID NO: 306), L0209 (SEQ ID
NO: 307), L0211 (SEQ ID NO: 309), L0212 (SEQ ID NO: 310), L0222
(SEQ ID NO: 319) and L0272 (SEQ ID NO: 359) are shown.
[0328] FIG. 26A presents the structure of a genomic DNA containing
mouse Cd3.epsilon., Cd3.delta., and Cd3.gamma. genes (1), a mouse
Cd3 gene modification vector constructed by modifying a bacterial
artificial chromosome (BAC) clone containing the whole gene region
(2), the structure of a genomic DNA in which loxP and Rox sequences
have been inserted at the target position using the above-mentioned
vector (3), and the structure of a Cd3.epsilon., Cd3.delta., and
Cd3.gamma.-gene deficient allele produced by the actions of Cre and
Dre recombinases (4).
[0329] FIG. 26B presents the structures of a BAC clone containing
human CD3.epsilon., CD3.delta., and CD3.gamma. genes (a);
5'-modifying cassette (b) and 3'-modifying cassette (c), both of
which are for modifying the BAC clone; and a human CD3 gene region
introduction vector constructed through modifications using those
above (d).
[0330] FIG. 27 presents the representative examples of PCR analyses
performed for establishing mouse Cd3 gene-modified ES cells.
[0331] FIGS. 28A and 28B present the representative examples of PCR
analyses of genotypes of ES cell clones obtained by introducing
into mouse Cd3 gene-modified ES cells the human CD3 gene region
introduction vector along with a Cre expression vector and a Dre
expression vector. FIG. 28A presents the representative examples of
PCR results that detect the deficiency of the mouse Cd3 gene
region. FIG. 28B presents the representative examples of PCR
results that detect the introduction of the human CD3 gene
region.
[0332] FIG. 29 presents the representative macroscopic photographs
of thymuses collected from each of the established lines of human
CD3 gene-substituted mice, Cd3 gene-deficient mice, wild type, and
human CD3.epsilon. gene-introduced mice. Thymuses extirpated from
12 to 13-week-old males are shown for the respective genotypes.
[0333] FIG. 30 presents the results of measuring the tissue weights
of the spleens and thymuses collected from each of the established
lines of human CD3 gene-substituted mice, Cd3 gene-deficient mice,
wild-type, and human CD3.epsilon. gene-introduced mice. Ratios of
tissue weight per body weight were calculated, and the value
obtained for each individual is plotted by a black dot and the mean
values are shown by columns.
[0334] FIG. 31 presents the results of examining by RT-PCR the
expressions of each of the human CD3 molecules and each of the
mouse Cd3 genes in each of the established lines of human CD3
gene-substituted mice, Cd3 gene-deficient mice, wild-type mice, and
human CD3.epsilon. gene-introduced (hCD3.epsilon. Tg) mice. Among
the established lines of the human CD3 gene-substituted mice,
signals specific to hCD3.epsilon., hCD3.delta., and hCD3.gamma.
were detected in line numbers 1C3 and 8I12. The signals were not
detected in line numbers 3B1 and 2A4.
[0335] FIG. 32 presents the representative examples of
immunohistological staining for CD3 performed on the thymus (A) and
spleen (B) of each established line of human CD3 gene-substituted
mice (1C3, 8I12, and 4HH3). In both tissues, staining was observed
only in the T cell zone as in the wild-type mouse. Furthermore,
staining was not observed in the Cd3 gene-deficient mice, and this
showed that the staining in the human CD3 gene-substituted mice is
due to the expression of the introduced human CD3 genes.
[0336] FIG. 33 presents the representative results of analyzing by
FACS the abundance ratio of mature T cells in the thymus of each
established line of human CD3-substituted mice.
[0337] FIG. 34 presents the results of measuring the chicken
ovoalbumin (OVA)-specific IgG1 and IgE serum concentrations in each
established line of human CD3-substituted mice immunized with OVA.
The OVA-specific serum IgG1 and IgE concentrations for each
individual are shown as a bar graph. The numbers below the bar
graph indicate the individual identification numbers.
[0338] FIG. 35A shows a result of comprehensive RNA analysis of
tumor tissues when Paclitaxel and antibody-38 are used in
combination.
[0339] FIG. 35B shows a result of comprehensive RNA analysis of
tumor tissues when Paclitaxel and antibody-38 are used in
combination (a continuation of FIG. 35-1).
[0340] FIG. 35C shows a result of comprehensive RNA analysis of
tumor tissues when Paclitaxel and antibody-38 are used in
combination (a continuation of FIG. 35-2).
[0341] FIG. 35D shows a result of comprehensive RNA analysis of
tumor tissues when Capecitabine and antibody-38 are used in
combination.
[0342] FIG. 35E shows a result of comprehensive RNA analysis of
tumor tissues when Capecitabine and antibody-38 are used in
combination (a continuation of FIG. 35-4).
[0343] FIG. 35F shows a result of comprehensive RNA analysis of
tumor tissues when Capecitabine and antibody-38 are used in
combination (a continuation of FIG. 35-5).
[0344] FIG. 36A shows a result of tumor infiltrating lymphocyte
(TIL) analysis of tumor tissues when Paclitaxel and antibody-38 are
used in combination.
[0345] FIG. 36B shows a result of tumor infiltrating lymphocyte
(TIL) analysis of tumor tissues when Cisplatin and antibody-38 are
used in combination.
MODE FOR CARRYING OUT THE INVENTION
[0346] The definitions below are provided to help understanding of
the present invention illustrated herein.
Antibody
[0347] Herein, "antibody" refers to a natural immunoglobulin or an
immunoglobulin produced by partial or complete synthesis.
Antibodies can be isolated from natural sources such as
naturally-occurring plasma and serum, or culture supernatants of
antibody-producing hybridomas. Alternatively, antibodies can be
partially or completely synthesized using techniques such as
genetic recombination. Preferred antibodies include, for example,
antibodies of an immunoglobulin isotype or subclass belonging
thereto. Known human immunoglobulins include antibodies of the
following nine classes (isotypes): IgG1, IgG2, IgG3, IgG4, IgA1,
IgA2, IgD, IgE, and IgM. Of these isotypes, antibodies of the
present invention may include IgG1, IgG2, IgG3, and IgG4.
[0348] Methods for producing an antibody with desired binding
activity are known to those skilled in the art. Below is an example
that describes a method for producing an antibody (anti-GPC3
antibody) that binds to Glypican-3 (hereinafter, also referred to
as GPC3), which belongs to the GPI-anchored receptor family (Int J
Cancer. (2003) 103(4), 455-65). Antibodies that bind to a T-cell
receptor complex can also be produced according to the example
described below.
[0349] Anti-GPC3 antibodies can be obtained as polyclonal or
monoclonal antibodies using known methods. The anti-GPC3 antibodies
preferably produced are monoclonal antibodies derived from mammals.
Such mammal-derived monoclonal antibodies include antibodies
produced by hybridomas or host cells transformed with an expression
vector carrying an antibody gene by genetic engineering
techniques.
[0350] Monoclonal antibody-producing hybridomas can be produced
using known techniques, for example, as described below.
Specifically, mammals are immunized by conventional immunization
methods using a GPC3 protein as a sensitizing antigen. Resulting
immune cells are fused with known parental cells by conventional
cell fusion methods. Then, hybridomas producing an anti-GPC3
antibody can be selected by screening for monoclonal
antibody-producing cells using conventional screening methods.
[0351] Specifically, monoclonal antibodies are prepared as
mentioned below. First, the GPC3 gene whose nucleotide sequence is
disclosed in RefSeq accession number NM_001164617.1 (SEQ ID NO: 1)
can be expressed to produce a GPC3 protein shown in RefSeq
accession number NP_001158089.1 (SEQ ID NO: 2), which will be used
as a sensitizing antigen for antibody preparation. That is, a gene
sequence encoding GPC3 is inserted into a known expression vector,
and appropriate host cells are transformed with this vector. The
desired human GPC3 protein is purified from the host cells or their
culture supernatants by known methods. For example, to prepare
soluble GPC3 from culture supernatants, amino acids at positions
564 to 580 that form the hydrophobic region corresponding to the
GPI-anchor sequence used to anchor GPC3 on the cell membrane are
deleted from the GPC3 polypeptide sequence of SEQ ID NO: 2, and
then the resulting protein is expressed instead of the GPC3 protein
of SEQ ID NO: 2. Alternatively, it is possible to use a purified
natural GPC3 protein as a sensitizing antigen.
[0352] The purified GPC3 protein can be used as a sensitizing
antigen for use in immunization of mammals. Partial peptides of
GPC3 can also be used as sensitizing antigens. In this case, the
partial peptides may also be obtained by chemical synthesis from
the human GPC3 amino acid sequence. Furthermore, they may also be
obtained by incorporating a portion of the GPC3 gene into an
expression vector and expressing it. Moreover, they may also be
obtained by degrading the GPC3 protein using proteases, but the
region and size of the GPC3 peptide used as the partial peptide are
not particularly limited to a special embodiment. As the preferred
region, any sequence from the amino acid sequence corresponding to
the amino acids at positions 524 to 563, or more preferably any
sequence from the amino acid sequence corresponding to the amino
acids at positions 537 to 563 in the amino acid sequence of SEQ ID
NO: 2 may be selected. Preferably, any sequence may be selected
from the amino acid sequence of the region not containing the amino
acid sequence corresponding to amino acids at positions 550 to 663
in the amino acid sequence of SEQ ID NO: 2. Preferably, any
sequence may be selected from the amino acid sequence corresponding
to positions 544 to 553, and more preferably, any sequence may be
selected from the amino acid sequence corresponding to positions
546 to 551 in the amino acid sequence of SEQ ID NO: 2. The number
of amino acids constituting a peptide to be used as the sensitizing
antigen is at least five or more, or preferably for example, six or
more, or seven or more. More specifically, peptides consisting of 8
to 50 residues or preferably 10 to 30 residues may be used as the
sensitizing antigen.
[0353] For sensitizing antigen, alternatively it is possible to use
a fusion protein prepared by fusing a desired partial polypeptide
or peptide of the GPC3 protein with a different polypeptide. For
example, antibody Fc fragments and peptide tags are preferably used
to produce fusion proteins to be used as sensitizing antigens.
Vectors for expression of such fusion proteins can be constructed
by fusing in frame genes encoding two or more desired polypeptide
fragments and inserting the fusion gene into an expression vector
as described above. Methods for producing fusion proteins are
described in Molecular Cloning 2nd ed. (Sambrook, J et al.,
Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab.
Press). Methods for preparing GPC3 to be used as a sensitizing
antigen, and immunization methods using GPC3 are specifically
described in WO 2003/000883, WO 2004/022754, and WO
2006/006693.
[0354] There is no particular limitation on the mammals to be
immunized with the sensitizing antigen. However, it is preferable
to select the mammals by considering their compatibility with the
parent cells to be used for cell fusion. In general, rodents such
as mice, rats, and hamsters, rabbits, and monkeys are preferably
used.
[0355] The above animals are immunized with a sensitizing antigen
by known methods. Generally performed immunization methods include,
for example, intraperitoneal or subcutaneous injection of a
sensitizing antigen into mammals. Specifically, a sensitizing
antigen is appropriately diluted with PBS (Phosphate-Buffered
Saline), physiological saline, or the like. If desired, a
conventional adjuvant such as Freund's complete adjuvant is mixed
with the antigen, and the mixture is emulsified. Then, the
sensitizing antigen is administered to a mammal several times at 4-
to 21-day intervals. Appropriate carriers may be used in
immunization with the sensitizing antigen. In particular, when a
low-molecular-weight partial peptide is used as the sensitizing
antigen, it is sometimes desirable to couple the sensitizing
antigen peptide to a carrier protein such as albumin or keyhole
limpet hemocyanin for immunization.
[0356] Alternatively, hybridomas producing a desired antibody can
be prepared using DNA immunization as mentioned below. DNA
immunization is an immunization method that confers
immunostimulation by expressing a sensitizing antigen in an animal
immunized as a result of administering a vector DNA constructed to
allow expression of an antigen protein-encoding gene in the animal.
As compared to conventional immunization methods in which a protein
antigen is administered to animals to be immunized, DNA
immunization is expected to be superior in that:
[0357] immunostimulation can be provided while retaining the
structure of a membrane protein such as GPC3; and
[0358] there is no need to purify the antigen for immunization.
[0359] In order to prepare a monoclonal antibody of the present
invention using DNA immunization, first, a DNA expressing a GPC3
protein is administered to an animal to be immunized. The
GPC3-encoding DNA can be synthesized by known methods such as PCR.
The obtained DNA is inserted into an appropriate expression vector,
and then this is administered to an animal to be immunized.
Preferably used expression vectors include, for example,
commercially-available expression vectors such as pcDNA3.1. Vectors
can be administered to an organism using conventional methods. For
example, DNA immunization is performed by using a gene gun to
introduce expression vector-coated gold particles into cells in the
body of an animal to be immunized. Antibodies that recognized GPC3
can also be produced by the methods described in WO
2003/104453.
[0360] After immunizing a mammal as described above, an increase in
the titer of a GPC3-binding antibody is confirmed in the serum.
Then, immune cells are collected from the mammal, and then
subjected to cell fusion. In particular, splenocytes are preferably
used as immune cells.
[0361] A mammalian myeloma cell is used as a cell to be fused with
the above-mentioned immunocyte. The myeloma cells preferably
comprise a suitable selection marker for screening. A selection
marker confers characteristics to cells for their survival (or
death) under a specific culture condition. Hypoxanthine-guanine
phosphoribosyltransferase deficiency (hereinafter abbreviated as
HGPRT deficiency) and thymidine kinase deficiency (hereinafter
abbreviated as TK deficiency) are known as selection markers. Cells
with HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine
sensitivity (hereinafter abbreviated as HAT sensitivity).
HAT-sensitive cells cannot synthesize DNA in a HAT selection
medium, and are thus killed. However, when the cells are fused with
normal cells, they can continue DNA synthesis using the salvage
pathway of the normal cells, and therefore they can grow even in
the HAT selection medium.
[0362] HGPRT-deficient and TK-deficient cells can be selected in a
medium containing 6-thioguanine, 8-azaguanine (hereinafter
abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal
cells are killed because they incorporate these pyrimidine analogs
into their DNA. Meanwhile, cells that are deficient in these
enzymes can survive in the selection medium, since they cannot
incorporate these pyrimidine analogs. In addition, a selection
marker referred to as G418 resistance provided by the
neomycin-resistant gene confers resistance to 2-deoxystreptamine
antibiotics (gentamycin analogs). Various types of myeloma cells
that are suitable for cell fusion are known.
[0363] For example, myeloma cells including the following cells can
be preferably used:
P3(P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550);
P3x63Ag8U.1 (Current Topics in Microbiology and Immunology
(1978)81, 1-7);
[0364] NS-1 (C. Eur. J. Immunol. (1976)6 (7), 511-519);
MPC-11 (Cell (1976) 8 (3), 405-415);
SP2/0 (Nature (1978) 276 (5685), 269-270);
[0365] FO (J. Immunol. Methods (1980) 35 (1-2), 1-21);
S194/5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323);
R210 (Nature (1979) 277 (5692), 131-133), etc.
[0366] Cell fusions between the immunocytes and myeloma cells are
essentially carried out using known methods, for example, a method
by Kohler and Milstein et al. (Methods Enzymol. (1981) 73:
3-46).
[0367] More specifically, cell fusion can be carried out, for
example, in a conventional culture medium in the presence of a cell
fusion-promoting agent. The fusion-promoting agents include, for
example, polyethylene glycol (PEG) and Sendai virus (HVJ). If
required, an auxiliary substance such as dimethyl sulfoxide is also
added to improve fusion efficiency.
[0368] The ratio of immunocytes to myeloma cells may be determined
at one's own discretion, preferably, for example, one myeloma cell
for every one to ten immunocytes. Culture media to be used for cell
fusions include, for example, media that are suitable for the
growth of myeloma cell lines, such as RPMI1640 medium and MEM
medium, and other conventional culture medium used for this type of
cell culture. In addition, serum supplements such as fetal calf
serum (FCS) may be preferably added to the culture medium.
[0369] For cell fusion, predetermined amounts of the above immune
cells and myeloma cells are mixed well in the above culture medium.
Then, a PEG solution (for example, the average molecular weight is
about 1,000 to 6,000) prewarmed to about 37.degree. C. is added
thereto at a concentration of generally 30% to 60% (w/v). This is
gently mixed to produce desired fusion cells (hybridomas). Then, an
appropriate culture medium mentioned above is gradually added to
the cells, and this is repeatedly centrifuged to remove the
supernatant. Thus, cell fusion agents and such which are
unfavorable to hybridoma growth can be removed.
[0370] The hybridomas thus obtained can be selected by culture
using a conventional selective medium, for example, HAT medium (a
culture medium containing hypoxanthine, aminopterin, and
thymidine). Cells other than the desired hybridomas (non-fused
cells) can be killed by continuing culture in the above HAT medium
for a sufficient period of time. Typically, the period is several
days to several weeks. Then, hybridomas producing the desired
antibody are screened and singly cloned by conventional limiting
dilution methods.
[0371] The hybridomas thus obtained can be selected using a
selection medium based on the selection marker possessed by the
myeloma used for cell fusion. For example, HGPRT- or TK-deficient
cells can be selected by culture using the HAT medium (a culture
medium containing hypoxanthine, aminopterin, and thymidine).
Specifically, when HAT-sensitive myeloma cells are used for cell
fusion, cells successfully fused with normal cells can selectively
proliferate in the HAT medium. Cells other than the desired
hybridomas (non-fused cells) can be killed by continuing culture in
the above HAT medium for a sufficient period of time. Specifically,
desired hybridomas can be selected by culture for generally several
days to several weeks. Then, hybridomas producing the desired
antibody may be screened and singly cloned by conventional limiting
dilution methods.
[0372] Desired antibodies can be preferably selected and singly
cloned by screening known methods based on antigen/antibody
reaction. For example, a GPC3-binding monoclonal antibody can bind
to GPC3 expressed on the cell surface. Such a monoclonal antibody
can be screened by fluorescence activated cell sorting (FACS). FACS
is a system that assesses the binding of an antibody to cell
surface by analyzing cells contacted with a fluorescent antibody
using laser beam, and measuring the fluorescence emitted from
individual cells.
[0373] To screen for hybridomas that produce a monoclonal antibody
of the present invention by FACS, GPC3-expressing cells are first
prepared. Cells preferably used for screening are mammalian cells
in which GPC3 is forcedly expressed. As control, the activity of an
antibody to bind to cell-surface GPC3 can be selectively detected
using non-transformed mammalian cells as host cells. Specifically,
hybridomas producing an anti-GPC3 monoclonal antibody can be
isolated by selecting hybridomas that produce an antibody which
binds to cells forced to express GPC3, but not to host cells.
[0374] Alternatively, the activity of an antibody to bind to
immobilized GPC3-expressing cells can be assessed based on the
principle of ELISA. For example, GPC3-expressing cells are
immobilized to the wells of an ELISA plate. Culture supernatants of
hybridomas are contacted with the immobilized cells in the wells,
and antibodies that bind to the immobilized cells are detected.
When the monoclonal antibodies are derived from mouse, antibodies
bound to the cells can be detected using an anti-mouse
immunoglobulin antibody. Hybridomas producing a desired antibody
having the antigen-binding ability are selected by the above
screening, and they can be cloned by a limiting dilution method or
the like.
[0375] Monoclonal antibody-producing hybridomas thus prepared can
be passaged in a conventional culture medium, and stored in liquid
nitrogen for a long period.
[0376] The above hybridomas are cultured by a conventional method,
and desired monoclonal antibodies can be prepared from the culture
supernatants. Alternatively, the hybridomas are administered to and
grown in compatible mammals, and monoclonal antibodies are prepared
from the ascites. The former method is suitable for preparing
antibodies with high purity.
[0377] Antibodies encoded by antibody genes that are cloned from
antibody-producing cells such as the above hybridomas can also be
preferably used. A cloned antibody gene is inserted into an
appropriate vector, and this is introduced into a host to express
the antibody encoded by the gene. Methods for isolating antibody
genes, inserting the genes into vectors, and transforming host
cells have already been established, for example, by Vandamme et
al. (Eur. J. Biochem. (1990) 192(3), 767-775). Methods for
producing recombinant antibodies are also known as described
below.
[0378] For example, a cDNA encoding the variable region (V region)
of an anti-GPC3 antibody is prepared from hybridoma cells
expressing the anti-GPC3 antibody. For this purpose, total RNA is
first extracted from hybridomas. Methods used for extracting mRNAs
from cells include, for example:
[0379] the guanidine ultracentrifugation method (Biochemistry
(1979) 18(24), 5294-5299), and
[0380] the AGPC method (Anal. Biochem. (1987) 162(1), 156-159)
[0381] Extracted mRNAs can be purified using the mRNA Purification
Kit (GE Healthcare Bioscience) or such. Alternatively, kits for
extracting total mRNA directly from cells, such as the QuickPrep
mRNA Purification Kit (GE Healthcare Bioscience), are also
commercially available. mRNAs can be prepared from hybridomas using
such kits. cDNAs encoding the antibody V region can be synthesized
from the prepared mRNAs using a reverse transcriptase. cDNAs can be
synthesized using the AMV Reverse Transcriptase First-strand cDNA
Synthesis Kit (Seikagaku Co.) or such. Furthermore, the SMART RACE
cDNA amplification kit (Clontech) and the PCR-based 5'-RACE method
(Proc. Natl. Acad. Sci. USA (1988) 85(23), 8998-9002; Nucleic Acids
Res. (1989) 17(8), 2919-2932) can be appropriately used to
synthesize and amplify cDNAs. In such a cDNA synthesis process,
appropriate restriction enzyme sites described below may be
introduced into both ends of a cDNA.
[0382] The cDNA fragment of interest is purified from the resulting
PCR product, and then this is ligated to a vector DNA. A
recombinant vector is thus constructed, and introduced into E. coli
or such. After colony selection, the desired recombinant vector can
be prepared from the colony-forming E. coli. Then, whether the
recombinant vector has the cDNA nucleotide sequence of interest is
tested by a known method such as the dideoxy nucleotide chain
termination method.
[0383] The 5'-RACE method which uses primers to amplify the
variable region gene is conveniently used for isolating the gene
encoding the variable region. First, a 5'-RACE cDNA library is
constructed by cDNA synthesis using RNAs extracted from hybridoma
cells as a template. A commercially available kit such as the SMART
RACE cDNA amplification kit is appropriately used to synthesize the
5'-RACE cDNA library.
[0384] The antibody gene is amplified by PCR using the prepared
5'-RACE cDNA library as a template. Primers for amplifying the
mouse antibody gene can be designed based on known antibody gene
sequences. The nucleotide sequences of the primers vary depending
on the immunoglobulin subclass. Therefore, it is preferable that
the subclass is determined in advance using a commercially
available kit such as the Iso Strip mouse monoclonal antibody
isotyping kit (Roche Diagnostics).
[0385] Specifically, for example, primers that allow amplification
of genes encoding .gamma.1, .gamma.2a, .gamma.2b, and .gamma.3
heavy chains and x and a light chains can be used to isolate mouse
IgG-encoding genes. In general, a primer that anneals to a constant
region site close to the variable region is used as a 3'-side
primer to amplify an IgG variable region gene. Meanwhile, a primer
attached to a 5' RACE cDNA library construction kit is used as a
5'-side primer.
[0386] PCR products thus amplified are used to reconstruct
immunoglobulins composed of a combination of heavy and light
chains. A desired antibody can be selected using the GPC3-binding
activity of a reconstructed immunoglobulin as an indicator. For
example, when the objective is to isolate an antibody against GPC3,
it is more preferred that the binding of the antibody to GPC3 is
specific. A GPC3-binding antibody can be screened, for example, by
the following steps:
[0387] (1) contacting a GPC3-expressing cell with an antibody
comprising the V region encoded by a cDNA isolated from a
hybridoma;
[0388] (2) detecting the binding of the antibody to the
GPC3-expressing cell; and
[0389] (3) selecting an antibody that binds to the GPC3-expressing
cell.
[0390] Methods for detecting the binding of an antibody to
GPC3-expressing cells are known. Specifically, the binding of an
antibody to GPC3-expressing cells can be detected by the
above-described techniques such as FACS. Immobilized samples of
GPC3-expressing cells are appropriately used to assess the binding
activity of an antibody.
[0391] Preferred antibody screening methods that use the binding
activity as an indicator also include panning methods using phage
vectors. Screening methods using phage vectors are advantageous
when the antibody genes are isolated from heavy-chain and
light-chain subclass libraries from a polyclonal
antibody-expressing cell population. Genes encoding the heavy-chain
and light-chain variable regions can be linked by an appropriate
linker sequence to form a single-chain Fv (scFv). Phages presenting
scFv on their surface can be produced by inserting a gene encoding
scFv into a phage vector. The phages are contacted with an antigen
of interest. Then, a DNA encoding scFv having the binding activity
of interest can be isolated by collecting phages bound to the
antigen. This process can be repeated as necessary to enrich scFv
having a desired binding activity.
[0392] After isolation of the cDNA encoding the V region of the
anti-GPC3 antibody of interest, the cDNA is digested with
restriction enzymes that recognize the restriction sites introduced
into both ends of the cDNA. Preferred restriction enzymes recognize
and cleave a nucleotide sequence that occurs in the nucleotide
sequence of the antibody gene at a low frequency. Furthermore, a
restriction site for an enzyme that produces a cohesive end is
preferably introduced into a vector to insert a single-copy
digested fragment in the correct orientation. The cDNA encoding the
V region of the anti-GPC3 antibody is digested as described above,
and this is inserted into an appropriate expression vector to
construct an antibody expression vector. In this case, if a gene
encoding the antibody constant region (C region) and a gene
encoding the above V region are fused in-frame, a chimeric antibody
is obtained. Herein, "chimeric antibody" means that the origin of
the constant region is different from that of the variable region.
Thus, in addition to mouse/human heterochimeric antibodies,
human/human allochimeric antibodies are included in the chimeric
antibodies of the present invention. A chimeric antibody expression
vector can be constructed by inserting the above V region gene into
an expression vector that already has the constant region.
Specifically, for example, a recognition sequence for a restriction
enzyme that excises the above V region gene can be appropriately
placed on the 5' side of an expression vector carrying a DNA
encoding a desired antibody constant region (C region). A chimeric
antibody expression vector is constructed by fusing in frame the
two genes digested with the same combination of restriction
enzymes.
[0393] To produce an anti-GPC3 monoclonal antibody, antibody genes
are inserted into an expression vector so that the genes are
expressed under the control of an expression regulatory region. The
expression regulatory region for antibody expression includes, for
example, enhancers and promoters. Furthermore, an appropriate
signal sequence may be attached to the amino terminus so that the
expressed antibody is secreted to the outside of cells. In the
Reference Examples described below, a peptide having the amino acid
sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) is used as a signal
sequence. Meanwhile, other appropriate signal sequences may be
attached. The expressed polypeptide is cleaved at the carboxyl
terminus of the above sequence, and the resulting polypeptide is
secreted to the outside of cells as a mature polypeptide. Then,
appropriate host cells are transformed with the expression vector,
and recombinant cells expressing the anti-GPC3 antibody-encoding
DNA are obtained.
[0394] DNAs encoding the antibody heavy chain (H chain) and light
chain (L chain) are separately inserted into different expression
vectors to express the antibody gene. An antibody molecule having
the H and L chains can be expressed by co-transfecting the same
host cell with vectors into which the H-chain and L-chain genes are
respectively inserted. Alternatively, host cells can be transformed
with a single expression vector into which DNAs encoding the H and
L chains are inserted (see WO 94/11523).
[0395] There are various known host cell/expression vector
combinations for antibody preparation by introducing isolated
antibody genes into appropriate hosts. All of these expression
systems are applicable to isolation of domains including antibody
variable regions of the present invention. Appropriate eukaryotic
cells used as host cells include animal cells, plant cells, and
fungal cells. Specifically, the animal cells include, for example,
the following cells.
[0396] (1) mammalian cells: CHO, COS, myeloma, baby hamster kidney
(BHK), HeLa, Vero, or such;
[0397] (2) amphibian cells: Xenopus oocytes, or such; and
[0398] (3) insect cells: sf9, sf21, Tn5, or such.
[0399] In addition, as a plant cell, an antibody gene expression
system using cells derived from the Nicotiana genus such as
Nicotiana tabacum is known. Callus cultured cells can be
appropriately used to transform plant cells.
[0400] Furthermore, the following cells can be used as fungal
cells:
yeasts: the Saccharomyces genus such as Saccharomyces cerevisiae,
and the Pichia genus such as Pichia pastoris; and filamentous
fungi: the Aspergillus genus such as Aspergillus niger.
[0401] Furthermore, antibody gene expression systems that utilize
prokaryotic cells are also known. For example, when using bacterial
cells, E. coli cells, Bacillus subtilis cells, and such can
suitably be utilized in the present invention. Expression vectors
carrying the antibody genes of interest are introduced into these
cells by transfection. The transfected cells are cultured in vitro,
and the desired antibody can be prepared from the culture of
transformed cells.
[0402] In addition to the above-described host cells, transgenic
animals can also be used to produce a recombinant antibody. That
is, the antibody can be obtained from an animal into which the gene
encoding the antibody of interest is introduced. For example, the
antibody gene can be constructed as a fusion gene by inserting in
frame into a gene that encodes a protein produced specifically in
milk. Goat .beta.-casein or such can be used, for example, as the
protein secreted in milk. DNA fragments containing the fused gene
inserted with the antibody gene is injected into a goat embryo, and
then this embryo is introduced into a female goat. Desired
antibodies can be obtained as a protein fused with the milk protein
from milk produced by the transgenic goat born from the
embryo-recipient goat (or progeny thereof). In addition, to
increase the volume of milk containing the desired antibody
produced by the transgenic goat, hormones can be administered to
the transgenic goat as necessary (Ebert, K. M. et al.,
Bio/Technology (1994) 12 (7), 699-702).
[0403] When an antigen-binding molecule described herein is
administered to human, a domain derived from a genetically
recombinant antibody that has been artificially modified to reduce
the heterologous antigenicity against human and such, can be
appropriately used as the domain of the antigen-binding molecule
including an antibody variable region. Such genetically recombinant
antibodies include, for example, humanized antibodies. These
modified antibodies are appropriately produced by known
methods.
[0404] An antibody variable region used to produce a domain of an
antigen-binding molecule including an antibody variable region
described herein is generally formed by three
complementarity-determining regions (CDRs) that are separated by
four framework regions (FRs). CDR is a region that substantially
determines the binding specificity of an antibody. The amino acid
sequences of CDRs are highly diverse. On the other hand, the
FR-forming amino acid sequences often have high identity even among
antibodies with different binding specificities. Therefore,
generally, the binding specificity of a certain antibody can be
introduced to another antibody by CDR grafting.
[0405] A humanized antibody is also called a reshaped human
antibody. Specifically, humanized antibodies prepared by grafting
the CDR of a non-human animal antibody such as a mouse antibody to
a human antibody and such are known. Common genetic engineering
techniques for obtaining humanized antibodies are also known.
Specifically, for example, overlap extension PCR is known as a
method for grafting a mouse antibody CDR to a human FR. In overlap
extension PCR, a nucleotide sequence encoding a mouse antibody CDR
to be grafted is added to primers for synthesizing a human antibody
FR. Primers are prepared for each of the four FRs. It is generally
considered that when grafting a mouse CDR to a human FR, selecting
a human FR that has high identity to a mouse FR is advantageous for
maintaining the CDR function. That is, it is generally preferable
to use a human FR comprising an amino acid sequence which has high
identity to the amino acid sequence of the FR adjacent to the mouse
CDR to be grafted.
[0406] Nucleotide sequences to be ligated are designed so that they
will be connected to each other in frame. Human FRs are
individually synthesized using the respective primers. As a result,
products in which the mouse CDR-encoding DNA is attached to the
individual FR-encoding DNAs are obtained. Nucleotide sequences
encoding the mouse CDR of each product are designed so that they
overlap with each other. Then, complementary strand synthesis
reaction is conducted to anneal the overlapping CDR regions of the
products synthesized using a human antibody gene as template. Human
FRs are ligated via the mouse CDR sequences by this reaction.
[0407] The full length V region gene, in which three CDRs and four
FRs are ultimately ligated, is amplified using primers that anneal
to its 5'- or 3'-end, which are added with suitable restriction
enzyme recognition sequences. An expression vector for humanized
antibody can be produced by inserting the DNA obtained as described
above and a DNA that encodes a human antibody C region into an
expression vector so that they will ligate in frame. After the
recombinant vector is transfected into a host to establish
recombinant cells, the recombinant cells are cultured, and the DNA
encoding the humanized antibody is expressed to produce the
humanized antibody in the cell culture (see, European Patent
Publication No. EP 239400 and International Patent Publication No.
WO 1996/002576).
[0408] By qualitatively or quantitatively measuring and evaluating
the antigen-binding activity of the humanized antibody produced as
described above, one can suitably select human antibody FRs that
allow CDRs to form a favorable antigen-binding site when ligated
through the CDRs. Amino acid residues in FRs may be substituted as
necessary, so that the CDRs of a reshaped human antibody form an
appropriate antigen-binding site. For example, amino acid sequence
mutations can be introduced into FRs by applying the PCR method
used for grafting a mouse CDR into a human FR. More specifically,
partial nucleotide sequence mutations can be introduced into
primers that anneal to the FR. Nucleotide sequence mutations are
introduced into the FRs synthesized by using such primers. Mutant
FR sequences having the desired characteristics can be selected by
measuring and evaluating the activity of the amino acid-substituted
mutant antibody to bind to the antigen by the above-mentioned
method (Sato, K. et al., Cancer Res. (1993) 53: 851-856).
[0409] Alternatively, desired human antibodies can be obtained by
immunizing transgenic animals having the entire repertoire of human
antibody genes (see International Patent Publication Nos. WO
1993/012227; WO 1992/003918; WO 1994/002602; WO 1994/025585; WO
1996/034096; WO 1996/033735) by DNA immunization.
[0410] Furthermore, techniques for preparing human antibodies by
panning using human antibody libraries are also known. For example,
the V region of a human antibody is expressed as a single-chain
antibody (scFv) on phage surface by the phage display method.
Phages expressing an scFv that binds to the antigen can be
selected. The DNA sequence encoding the human antibody V region
that binds to the antigen can be determined by analyzing the genes
of selected phages. The DNA sequence of the scFv that binds to the
antigen is determined. An expression vector can be prepared by
fusing the V region sequence in frame with the C region sequence of
a desired human antibody, and inserting this into an appropriate
expression vector. The expression vector is introduced into cells
appropriate for expression such as those described above. The human
antibody can be produced by expressing the human antibody-encoding
gene in the cells. These methods are already known (see
International Patent Publication Nos. WO 1992/001047; WO
1992/020791; WO 1993/006213; WO 1993/011236; WO 1993/019172; WO
1995/001438; WO 1995/015388).
A Domain Comprising an Antibody Variable Region Having Glypican 3
(GPC3)-Binding Activity
[0411] Herein, the phrase "a domain comprising an antibody variable
region having glypican 3 (GPC3)-binding activity" refers to an
antibody portion that comprises a region that specifically binds to
the above-mentioned GPC3 protein, or to all or a portion of a
partial peptide of the GPC3 protein, and is also complementary
thereto. Domains comprising an antibody variable region may be
provided from variable domains of one or a plurality of antibodies.
Preferably, domains comprising an antibody variable region comprise
antibody light-chain and heavy-chain variable regions (VL and VH).
Suitable examples of such domains comprising antibody variable
regions include "single chain Fv (scFv)", "single chain antibody",
"Fv", "single chain Fv 2 (scFv2)", "Fab", "F(ab')2", etc.
A Domain Comprising an Antibody Variable Region Having T-Cell
Receptor Complex-Binding Activity
[0412] Herein, the phrase "a domain comprising an antibody variable
region having T-cell receptor complex-binding activity" refers to a
T-cell receptor complex-binding antibody portion that comprises a
region that specifically binds to all or a portion of a T-cell
receptor complex and is also complementary thereto. The T-cell
receptor complex may be a T-cell receptor itself, or an adaptor
molecule constituting a T-cell receptor complex along with a T-cell
receptor. CD3 is suitable as an adaptor molecule.
A Domain Comprising an Antibody Variable Region that has T-Cell
Receptor-Binding Activity
[0413] Herein, the phrase "a domain comprising an antibody variable
region having T-cell receptor-binding activity" refers to a T-cell
receptor-binding antibody portion produced by including a region
that specifically binds to all or a portion of a T-cell receptor
and is also complementary thereto.
[0414] The portion of a T cell receptor to which the domain of the
present invention binds may be a variable region or a constant
region, but an epitope present in the constant region is preferred.
Examples of the constant region sequence include the T cell
receptor .alpha. chain of RefSeq Accession No. CAA26636.1 (SEQ ID
NO: 4), the T cell receptor .beta. chain of RefSeq Accession No.
C25777 (SEQ ID NO: 5), the T cell receptor .gamma.1 chain of RefSeq
Accession No. A26659 (SEQ ID NO: 6), the T cell receptor .gamma.2
chain of RefSeq Accession No. AAB63312.1 (SEQ ID NO: 7), and the T
cell receptor S chain of RefSeq Accession No. AAA61033.1 (SEQ ID
NO: 8).
A Domain Comprising an Antibody Variable Region that has
CD3-Binding Activity
[0415] Herein, the phrase "a domain comprising an antibody variable
region that has CD3-binding activity" refers to a CD3-binding
antibody portion produced by including a region that specifically
binds to all or a portion of CD3 and is also complementary thereto.
Preferably, the domain comprises the light-chain and heavy-chain
variable regions (VL and VH) of an anti-CD3 antibody. Suitable
examples of such a domain include "single chain Fv (scFv)", "single
chain antibody", "Fv", "single chain Fv 2 (scFv2)", "Fab",
"F(ab')2", etc.
[0416] The domain comprising an antibody variable region that has
CD3-binding activity of the present invention may be any
epitope-binding domain as long as the epitope exists in the
.gamma.-chain, 6-chain, or s-chain sequence that constitutes human
CD3. In the present invention, preferably, a domain comprising an
anti-CD3 antibody light-chain variable region (VL) and an anti-CD3
antibody heavy-chain variable region (VH) that bind to an epitope
present in the extracellular region of the s chain of the human CD3
complex is suitably used. Besides the anti-CD3 antibody light chain
variable region (VL) and anti-CD3 antibody heavy chain variable
region (VH) described in the Reference Examples, various known
CD3-binding domains containing a CD3-binding antibody light chain
variable region (VL) and a CD3-binding antibody heavy chain
variable region (VH), and those of the OKT3 antibody (Proc. Natl.
Acad. Sci. USA (1980) 77, 4914-4917) are suitably used as such
domains. One may appropriately use an antibody variable
region-containing domain derived from the anti-CD3 antibody having
desired properties, which is obtained by immunizing a desired
animal by the above-mentioned method using the .gamma.-chain,
.delta.-chain, or .epsilon.-chain constituting the human CD3. Human
antibodies and properly humanized antibodies as described above may
be appropriately used as the anti-CD3 antibody to give rise to the
domain containing the antibody variable region having CD3-binding
activity. Regarding the structure of the .gamma.-chain, 6-chain, or
s-chain constituting CD3, their polynucleotide sequences are shown
in SEQ ID NOs: 9 (NM_000073.2), 10 (NM_000732.4), and 11
(NM_000733.3), and their polypeptide sequences are shown in SEQ ID
NOs: 12 (NP_000064.1), 13 (NP_000723.1), and 14 (NP_000724.1) (the
RefSeq accession number is shown in parentheses).
[0417] Antibody variable region-containing domains in antigen
binding molecules of the present invention may bind to the same
epitope. Herein, the same epitope may be present in a protein
comprising the amino acid sequence of SEQ ID NO: 2 or 14.
Alternatively, antibody variable region-containing domains in
antigen binding molecules of the present invention may bind to
different epitopes, respectively. Herein, the different epitopes
may be present in a protein comprising the amino acid sequence of
SEQ ID NO: 2 or 14.
Specific
[0418] The term "specific" means that one of molecules involved in
specific binding does not show any significant binding to molecules
other than a single or a number of binding partner molecules.
Furthermore, the term is also used when a domain containing an
antibody variable region is specific to a particular epitope among
multiple epitopes in an antigen. When an epitope bound by a domain
containing an antibody variable region is included in a number of
different antigens, antigen-binding molecules comprising the
antibody variable region-containing domain can bind to various
antigens that have the epitope.
Epitope
[0419] "Epitope" means an antigenic determinant in an antigen, and
refers to an antigen site to which a domain of an antigen-binding
molecule including an antibody variable region disclosed herein
binds. Thus, for example, the epitope can be defined according to
its structure. Alternatively, the epitope may be defined according
to the antigen-binding activity of an antigen-binding molecule that
recognizes the epitope. When the antigen is a peptide or
polypeptide, the epitope can be specified by the amino acid
residues forming the epitope. Alternatively, when the epitope is a
sugar chain, the epitope can be specified by its specific sugar
chain structure.
[0420] A linear epitope is an epitope that contains an epitope
whose primary amino acid sequence is recognized. Such a linear
epitope typically contains at least three and most commonly at
least five, for example, about 8 to 10 or 6 to 20 amino acids in
its specific sequence.
[0421] In contrast to the linear epitope, "conformational epitope"
is an epitope in which the primary amino acid sequence containing
the epitope is not the only determinant of the recognized epitope
(for example, the primary amino acid sequence of a conformational
epitope is not necessarily recognized by an epitope-defining
antibody). Conformational epitopes may contain a greater number of
amino acids compared to linear epitopes. A conformational
epitope-recognizing antibody recognizes the three-dimensional
structure of a peptide or protein. For example, when a protein
molecule folds and forms a three-dimensional structure, amino acids
and/or polypeptide main chains that form a conformational epitope
become aligned, and the epitope is made recognizable by the
antibody. Methods for determining epitope conformations include,
for example, X ray crystallography, two-dimensional nuclear
magnetic resonance, site-specific spin labeling, and electron
paramagnetic resonance, but are not limited thereto. See, for
example, Epitope Mapping Protocols in Methods in Molecular Biology
(1996), Vol. 66, Morris (ed.).
[0422] A method for confirming binding to an epitope by a test
antigen-binding molecule comprising a domain that contains an
antibody variable region having GPC3-binding activity is
exemplified below, and a method for confirming binding to an
epitope by a test antigen-binding molecule comprising a domain that
contains an antibody variable region having T-cell receptor
complex-binding activity may also be performed suitably according
to the examples below.
[0423] For example, recognition of a linear epitope present in the
GPC3 molecule by a test antigen-binding molecule comprising a
domain that contains an antibody variable region having
GPC3-binding activity can be confirmed below. A linear peptide
comprising the amino acid sequence constituting the extracellular
domain of GPC3 is synthesized for the above-mentioned objective.
The peptide may be synthesized chemically. Alternatively, it can be
obtained by genetic engineering methods using a region in the cDNA
of GPC3 that encodes an amino acid sequence corresponding to the
extracellular domain. Next, the binding activity between a linear
peptide comprising the amino acid sequence constituting the
extracellular domain and the test antigen-binding molecule
comprising a domain that contains an antibody variable region
having GPC3-binding activity is evaluated. For example, ELISA which
uses an immobilized linear peptide as the antigen may enable
evaluation of the binding activity of the antigen-binding molecule
towards the peptide. Alternatively, binding activity towards the
linear peptide may be elucidated based on the level of inhibition
caused by the linear peptide in the binding of the antigen-binding
molecule to GPC3-expressing cells. These tests may elucidate the
binding activity of the antigen-binding molecules toward the linear
peptide.
[0424] Furthermore, recognition of the three-dimensional structure
of the epitope by a test antigen-binding molecule comprising a
domain that contains an antibody variable region having
GPC3-binding activity can be confirmed below. GPC3-expressing cells
are prepared for the above-mentioned objective. For example, when
the test antigen-binding molecule comprising a domain that contains
an antibody variable region having GPC3-binding activity contacts
GPC3-expressing cells, it binds strongly to the cells, but on the
other hand, there are cases when the antigen-binding molecule does
not substantially bind to the immobilized linear peptide comprising
the amino acid sequence constituting the extracellular domain of
GPC3. In these cases, "does not substantially bind" refers to a
binding activity of 80% or less, generally 50% or less, preferably
30% or less, and particularly preferably 15% or less relative to
the binding activity towards human GPC3-expressing cells.
[0425] Methods for assaying the binding activity of a test
antigen-binding molecule containing a GPC3 antigen-binding domain
towards GPC3-expressing cells include, for example, the methods
described in Antibodies: A Laboratory Manual (Ed Harlow, David
Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically,
the assessment can be performed based on the principle of ELISA or
fluorescence activated cell sorting (FACS) using GPC3-expressing
cells as antigen.
[0426] In the ELISA format, the binding activity of a test
antigen-binding molecule containing a GPC3 antigen-binding domain
towards GPC3-expressing cells can be assessed quantitatively by
comparing the levels of signal generated by enzymatic reaction.
Specifically, a test antigen-binding molecule is added to an ELISA
plate onto which GPC3-expressing cells are immobilized. Then, the
test antigen-binding molecule bound to the cells is detected using
an enzyme-labeled antibody that recognizes the test antigen-binding
molecule. Alternatively, when FACS is used, a dilution series of a
test antigen-binding molecule is prepared, and the antibody binding
titer for GPC3-expressing cells can be determined to compare the
binding activity of the test antigen-binding molecule towards
GPC3-expressing cells.
[0427] The binding of a test antigen-binding molecule towards an
antigen expressed on the surface of cells suspended in buffer or
the like can be detected using a flow cytometer. Known flow
cytometers include, for example, the following devices:
FACSCanto.TM. II
FACSAria.TM.
FACSArray.TM.
FACSVantage.TM. SE
[0428] FACSCalibur.TM. (all are trade names of BD Biosciences)
EPICS ALTRA HyPerSort
Cytomics FC 500
EPICS XL-MCL ADC EPICS XL ADC
[0429] Cell Lab Quanta/Cell Lab Quanta SC (all are trade names of
Beckman Coulter)
[0430] Preferable methods for assaying the binding activity of a
test antigen-binding molecule containing a GPC3 antigen-binding
domain towards an antigen include, for example, the following
method. First, GPC3-expressing cells are reacted with a test
antigen-binding molecule, and then this is stained with an
FITC-labeled secondary antibody that recognizes the polypeptide
complex. The test antigen-binding molecule is appropriately diluted
with a suitable buffer to prepare the complex at a desired
concentration. For example, the complex can be used at a
concentration within the range of 10 .mu.g/ml to 10 ng/ml. Then,
the fluorescence intensity and cell count are determined using
FACSCalibur (BD). The fluorescence intensity obtained by analysis
using the CELL QUEST Software (BD), i.e., the Geometric Mean value,
reflects the quantity of antibody bound to cells. That is, the
binding activity of a test antigen-binding molecule, which is
represented by the quantity of the test antigen-binding molecule
bound, can be determined by measuring the Geometric Mean value.
[0431] Whether a test antigen-binding molecule containing a GPC3
antigen-binding domain shares a common epitope with another
antigen-binding molecule can be assessed based on the competition
between the two complexes for the same epitope. The competition
between antigen-binding molecules can be detected by cross-blocking
assay or the like. For example, the competitive ELISA assay is a
preferred cross-blocking assay.
[0432] Specifically, in cross-blocking assay, the GPC3 protein
immobilized to the wells of a microtiter plate is pre-incubated in
the presence or absence of a candidate competitor antigen-binding
molecule, and then a test antigen-binding molecule is added
thereto. The quantity of test antigen-binding molecule bound to the
GPC3 protein in the wells is indirectly correlated with the binding
ability of a candidate competitor antigen-binding molecule that
competes for the binding to the same epitope. That is, the greater
the affinity of the competitor antigen-binding molecule for the
same epitope, the lower the binding activity of the test
antigen-binding molecule towards the GPC3 protein-coated wells.
[0433] The quantity of the test antigen-binding molecule bound to
the wells via the GPC3 protein can be readily determined by
labeling the antigen-binding molecule in advance. For example, a
biotin-labeled antigen-binding molecule is measured using an
avidin/peroxidase conjugate and appropriate substrate. In
particular, cross-blocking assay that uses enzyme labels such as
peroxidase is called "competitive ELISA assay". The antigen-binding
molecule can also be labeled with other labeling substances that
enable detection or measurement. Specifically, radiolabels,
fluorescent labels, and such are known.
[0434] When the candidate competitor antigen-binding molecule can
block the binding by a test antigen-binding molecule containing a
GPC3 antigen-binding domain by at least 20%, preferably at least 20
to 50%, and more preferably at least 50% compared to the binding
activity in a control experiment conducted in the absence of the
competitor antigen-binding molecule, the test antigen-binding
molecule is determined to substantially bind to the same epitope
bound by the competitor antigen-binding molecule, or compete for
the binding to the same epitope.
[0435] When the structure of an epitope bound by a test
antigen-binding molecule containing a GPC3 antigen-binding domain
has already been identified, whether the test and control
antigen-binding molecules share a common epitope can be assessed by
comparing the binding activities of the two antigen-binding
molecules towards a peptide prepared by introducing amino acid
mutations into the peptide forming the epitope.
[0436] To measure the above binding activities, for example, the
binding activities of test and control antigen-binding molecules
towards a linear peptide into which a mutation is introduced are
compared in the above ELISA format. Besides the ELISA methods, the
binding activity towards the mutant peptide bound to a column can
be determined by flowing test and control antigen-binding molecules
in the column, and then quantifying the antigen-binding molecule
eluted in the elution solution. Methods for adsorbing a mutant
peptide to a column, for example, in the form of a GST fusion
peptide, are known.
[0437] Alternatively, when the identified epitope is a
conformational epitope, whether test and control antigen-binding
molecules share a common epitope can be assessed by the following
method. First, GPC3-expressing cells and cells expressing GPC3 with
a mutation introduced into the epitope are prepared. The test and
control antigen-binding molecules are added to a cell suspension
prepared by suspending these cells in an appropriate buffer such as
PBS. Then, the cell suspensions are appropriately washed with a
buffer, and an FITC-labeled antibody that recognizes the test and
control antigen-binding molecules is added thereto. The
fluorescence intensity and number of cells stained with the labeled
antibody are determined using FACSCalibur (BD). The test and
control polypeptide complexes are appropriately diluted using a
suitable buffer, and used at desired concentrations. For example,
they may be used at a concentration within the range of 10 .mu.g/ml
to 10 ng/ml. The fluorescence intensity determined by analysis
using the CELL QUEST Software (BD), i.e., the Geometric Mean value,
reflects the quantity of labeled antibody bound to cells. That is,
the binding activities of the test and control antigen-binding
molecules, which are represented by the quantity of labeled
antibody bound, can be determined by measuring the Geometric Mean
value.
[0438] In the above method, whether an antigen-binding molecule
does "not substantially bind to cells expressing mutant GPC3" can
be assessed, for example, by the following method. First, the test
and control antigen-binding molecules bound to cells expressing
mutant GPC3 are stained with a labeled antibody. Then, the
fluorescence intensity of the cells is determined. When FACSCalibur
is used for fluorescence detection by flow cytometry, the
determined fluorescence intensity can be analyzed using the CELL
QUEST Software. From the Geometric Mean values in the presence and
absence of the antigen-binding molecule, the comparison value
(AGeo-Mean) can be calculated according to the following formula to
determine the ratio of increase in fluorescence intensity as a
result of the binding by the antigen-binding molecule.
.DELTA.Geo-Mean=Geo-Mean (in the presence of the antigen-binding
molecule)/Geo-Mean (in the absence of the antigen-binding
molecule)
[0439] The Geometric Mean comparison value (AGeo-Mean value for the
mutant GPC3 molecule) determined by the above analysis, which
reflects the quantity of a test antigen-binding molecule bound to
cells expressing mutant GPC3, is compared to the AGeo-Mean
comparison value that reflects the quantity of the test
antigen-binding molecule bound to GPC3-expressing cells. In this
case, the concentrations of the test antigen-binding molecule used
to determine the AGeo-Mean comparison values for GPC3-expressing
cells and cells expressing mutant GPC3 are particularly preferably
adjusted to be equal or substantially equal. An antigen-binding
molecule that has been confirmed to recognize an epitope in GPC3 is
used as a control antigen-binding molecule.
[0440] If the .DELTA.Geo-Mean comparison value of a test
antigen-binding molecule for cells expressing mutant GPC3 is
smaller than the .DELTA.Geo-Mean comparison value of the test
antigen-binding molecule for GPC3-expressing cells by at least 80%,
preferably 50%, more preferably 30%, and particularly preferably
15%, then the test polypeptide complex "does not substantially bind
to cells expressing mutant GPC3". The formula for determining the
Geo-Mean (Geometric Mean) value is described in the CELL QUEST
Software User's Guide (BD biosciences). When the comparison shows
that the comparison values are substantially equivalent, the
epitope for the test and control antigen-binding molecules can be
determined to be the same.
Variable Fragment (Fv)
[0441] Herein, the term "variable fragment (Fv)" refers to the
minimum unit of an antibody-derived antigen-binding domain that is
composed of a pair of the antibody light chain variable region (VL)
and antibody heavy chain variable region (VH). In 1988, Skerra and
Pluckthun found that homogeneous and active antibodies can be
prepared from the E. coli periplasm fraction by inserting an
antibody gene downstream of a bacterial signal sequence and
inducing expression of the gene in E. coli (Science (1988)
240(4855), 1038-1041). In the Fv prepared from the periplasm
fraction, VH associates with VL in a manner so as to bind to an
antigen.
[0442] Herein, Fv preferably includes, for example, a pair of Fv
which is an antigen-binding molecule or such comprising:
(1) a bivalent antigen-binding domain which is a bivalent scFv,
wherein one monovalent scFv of the bivalent scFv is linked to one
polypeptide forming an Fc domain by a heavy-chain Fv fragment
forming a CD3-binding domain, and the other monovalent scFv is
linked to the other polypeptide forming an Fc domain by a
light-chain Fv fragment forming a CD3-binding domain; (2) a domain
comprising an Fc domain that has no Fc.gamma. receptor-binding
activity, and which is derived from amino acids forming the Fc
domain of IgG1, IgG2a, IgG3, or IgG4; and (3) at least a monovalent
CD3-binding domain, wherein the light-chain and heavy-chain Fv
fragments associate to form a CD3-binding domain such that it can
bind to the CD3 antigen. scFv, Single-Chain Antibody, and
Sc(Fv)2
[0443] Herein, the terms "scFv", "single-chain antibody", and
"sc(Fv)2" all refer to an antibody fragment of a single polypeptide
chain that contains variable regions derived from the heavy and
light chains, but not the constant region. In general, a
single-chain antibody also contains a polypeptide linker between
the VH and VL domains, which enables formation of a desired
structure that is thought to allow antigen binding. The
single-chain antibody is discussed in detail by Pluckthun in "The
Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and
Moore, eds., Springer-Verlag, New York, 269-315 (1994)". See also
International Patent Publication WO 1988/001649; U.S. Pat. Nos.
4,946,778 and 5,260,203. In a particular embodiment, the
single-chain antibody can be bispecific and/or humanized.
[0444] scFv is an antigen-binding domain in which VH and VL forming
Fv are linked together by a peptide linker (Proc. Natl. Acad. Sci.
U.S.A. (1988) 85(16), 5879-5883). VH and VL can be retained in
close proximity by the peptide linker.
[0445] sc(Fv)2 is a single-chain antibody in which four variable
regions of two VL and two VH are linked by linkers such as peptide
linkers to form a single chain (J Immunol. Methods (1999) 231(1-2),
177-189). The two VH and two VL may be derived from different
monoclonal antibodies. Such sc(Fv)2 preferably includes, for
example, a bispecific sc(Fv)2 that recognizes two epitopes present
in a single antigen as disclosed in the Journal of Immunology
(1994) 152(11), 5368-5374. sc(Fv)2 can be produced by methods known
to those skilled in the art. For example, sc(Fv)2 can be produced
by linking scFv by a linker such as a peptide linker.
[0446] Herein, the form of an antigen-binding domain forming an
sc(Fv)2 include an antibody in which the two VH units and two VL
units are arranged in the order of VH, VL, VH, and VL
([VH]-linker-[VL]-linker-[VH]-linker-[VL]) beginning from the N
terminus of a single-chain polypeptide. The order of the two VH
units and two VL units is not limited to the above form, and they
may be arranged in any order. Example order of the form is listed
below.
[VL]-linker-[VH]-linker-[VH]-linker-[VL]
[VH]-linker-[VL]-linker-[VL]-linker-[VH]
[VH]-linker-[VH]-linker-[VL]-linker-[VL]
[VL]-linker-[VL]-linker-[VH]-linker-[VH]
[VL]-linker-[VH]-linker-[VL]-linker-[VH]
[0447] The molecular form of sc(Fv)2 is also described in detail in
WO 2006/132352. According to these descriptions, those skilled in
the art can appropriately prepare desired sc(Fv)2 to produce the
antigen-binding molecules disclosed herein.
[0448] Furthermore, the antigen-binding molecules of the present
invention may be conjugated with a carrier polymer such as PEG or
an organic compound such as an anticancer agent. Alternatively, a
sugar chain addition sequence is preferably inserted into the
polypeptide complexes such that the sugar chain produces a desired
effect.
[0449] The linkers to be used for linking the variable regions of
an antibody comprise arbitrary peptide linkers that can be
introduced by genetic engineering, and synthetic linkers disclosed
in, for example, Protein Engineering, 9(3), 299-305, 1996. However,
peptide linkers are preferred in the present invention. The length
of the peptide linkers is not particularly limited, and can be
suitably selected by those skilled in the art according to the
purpose. The length is preferably five amino acids or more (without
particular limitation, the upper limit is generally 30 amino acids
or less, preferably 20 amino acids or less), and particularly
preferably 15 amino acids. When sc(Fv)2 contains three peptide
linkers, their length may be all the same or different.
[0450] For example, such peptide linkers include:
TABLE-US-00001 Ser Gly .cndot. Ser Gly .cndot. Gly .cndot. Ser Ser
.cndot. Gly .cndot. Gly (SEQ ID NO: 15) Gly .cndot. Gly .cndot. Gly
.cndot. Ser (SEQ ID NO: 16) Ser .cndot. Gly .cndot. Gly .cndot. Gly
(SEQ ID NO: 17) Gly .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Ser
(SEQ ID NO: 18) Ser .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Gly
(SEQ ID NO: 19) Gly .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Gly
.cndot. Ser (SEQ ID NO: 20) Ser .cndot. Gly .cndot. Gly .cndot. Gly
.cndot. Gly .cndot. Gly (SEQ ID NO: 21) Gly .cndot. Gly .cndot. Gly
.cndot. Gly .cndot. Gly .cndot. Gly .cndot. Ser (SEQ ID NO: 22) Ser
.cndot. Gly .cndot. Gly .cndot. Gly .cndot. Gly .cndot. Gly .cndot.
Gly (SEQ ID NO: 17) (Gly .cndot. Gly .cndot. Gly .cndot. Gly
.cndot. Ser)n (SEQ ID NO: 18) (Ser .cndot. Gly .cndot. Gly .cndot.
Gly .cndot. Gly)n
where n is an integer of 1 or larger. The length or sequences of
peptide linkers can be selected accordingly by those skilled in the
art depending on the purpose.
[0451] Synthetic linkers (chemical crosslinking agents) are
routinely used to crosslink peptides, and for example: [0452]
N-hydroxy succinimide (NHS), [0453] disuccinimidyl suberate (DSS),
[0454] bis(sulfosuccinimidyl) suberate (BS.sup.3), [0455]
dithiobis(succinimidyl propionate) (DSP), [0456]
dithiobis(sulfosuccinimidyl propionate) (DTSSP), [0457] ethylene
glycol bis(succinimidyl succinate) (EGS), [0458] ethylene glycol
bis(sulfosuccinimidyl succinate) (sulfo-EGS), [0459] disuccinimidyl
tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), [0460]
bis[2-(succinimidoxycarbonyloxy)ethyl] sulfone (BSOCOES), [0461]
and bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl] sulfone
(sulfo-BSOCOES). These crosslinking agents are commercially
available.
[0462] In general, three linkers are required to link four antibody
variable regions together. The linkers to be used may be of the
same type or different types.
Fab, F(ab')2, and Fab'
[0463] "Fab" consists of a single light chain, and a CH1 domain and
variable region from a single heavy chain. The heavy chain of Fab
molecule cannot form disulfide bonds with another heavy chain
molecule.
[0464] "F(ab')2" or "Fab'" is produced by treating an
immunoglobulin (monoclonal antibody) with a protease such as pepsin
and papain, and refers to an antibody fragment generated by
digesting an immunoglobulin (monoclonal antibody) at near the
disulfide bonds present between the hinge regions in each of the
two H chains. For example, papain cleaves IgG upstream of the
disulfide bonds present between the hinge regions in each of the
two H chains to generate two homologous antibody fragments, in
which an L chain comprising VL (L-chain variable region) and CL
(L-chain constant region) is linked to an H-chain fragment
comprising VH (H-chain variable region) and CH.gamma.1 (.gamma.1
region in an H-chain constant region) via a disulfide bond at their
C-terminal regions. Each of these two homologous antibody fragments
is called Fab'.
[0465] "F(ab')2" consists of two light chains and two heavy chains
comprising the constant region of a CH1 domain and a portion of CH2
domains so that disulfide bonds are formed between the two heavy
chains. The F(ab')2 forming an antigen-binding molecule disclosed
herein can be preferably produced as follows. A whole monoclonal
antibody or such comprising a desired antigen-binding domain is
partially digested with a protease such as pepsin; and Fc fragments
are removed by adsorption onto a Protein A column. The protease is
not particularly limited, as long as it can cleave the whole
antibody in a selective manner to produce F(ab')2 under an
appropriate setup enzyme reaction condition such as pH. Such
proteases include, for example, pepsin and ficin.
Fc Domain
[0466] An Fc domain that forms an antigen-binding molecule
disclosed herein can be preferably produced in the following
manner. An antibody such as a monoclonal antibody is partially
digested with a protease such as pepsin. Then, the resulting
fragment is adsorbed onto a Protein A or Protein G column, and
eluted with an appropriate elution buffer. The protease is not
particularly limited, as long as it can cleave antibodies such as
monoclonal antibodies under an appropriate setup enzyme reaction
condition such as pH. Such proteases include, for example, pepsin
and ficin.
[0467] The antigen-binding molecules described herein comprise an
Fc domain with reduced Fc.gamma. receptor-binding activity, which
includes amino acids forming the Fc domain of IgG1, IgG2, IgG3, or
IgG4.
[0468] Antibody isotype is determined according to the structure of
the constant region. Constant regions of the isotypes IgG1, IgG2,
IgG3, and IgG4 are called C.gamma.1, C.gamma.2, C.gamma.3, and
C.gamma.4, respectively. The amino acid sequences of Fc domain
polypeptides forming human C.gamma.1, C.gamma.2, C.gamma.3, and
C.gamma.4 are exemplified in SEQ ID NO: 23, 24, 25, and 26,
respectively. The relationship between amino acid residues forming
each amino acid sequence and Kabat's EU numbering (herein also
referred to as EU INDEX) are shown in FIG. 23.
[0469] The Fc domain refers to the region besides F(ab')2 which
comprises two light chains and two heavy chains comprising a
portion of the constant region that comprises a CH1 domain and a
region between the CH1 and CH2 domains so that disulfide bonds are
formed between the two heavy chains. The Fc domain forming an
antigen-binding molecule disclosed herein can be preferably
produced as follows. A monoclonal IgG1, IgG2, IgG3, or IgG4
antibody or the like is partially digested with a protease such as
pepsin, followed by elution of the fraction adsorbed onto a Protein
A column. The protease is not particularly limited, as long as it
can cleave the whole antibody in a selective manner to produce
F(ab')2 in an appropriate setup enzyme reaction condition such as
pH. Such proteases include, for example, pepsin and ficin.
Fc.gamma. Receptor
[0470] Fc.gamma. receptor refers to a receptor capable of binding
to the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4
antibodies, and includes all members belonging to the family of
proteins substantially encoded by an Fc.gamma. receptor gene.
Inhuman, the family includes Fc.gamma.RI (CD64) including isoforms
Fc.gamma.RIa, Fc.gamma.RIb and Fc.gamma.RIc; Fc.gamma.RII (CD32)
including isoforms Fc.gamma.RIIa (including allotype H131 and
R131), Fc.gamma.RIIb (including Fc.gamma.RIIb-1 and
Fc.gamma.RIIb-2), and Fc.gamma.RIIc; and Fc.gamma.RIII (CD16)
including isoform Fc.gamma.RIIIa (including allotype V158 and F158)
and Fc.gamma.RIIIb (including allotype Fc.gamma.RIIIb-NA1 and
Fc.gamma.RIIIb-NA2); as well as all unidentified human Fc.gamma.Rs,
Fc.gamma.R isoforms, and allotypes thereof. However, Fc.gamma.
receptor is not limited to these examples. Without being limited
thereto, Fc.gamma.R includes those derived from humans, mice, rats,
rabbits, and monkeys. Fc.gamma.R may be derived from any organisms.
Mouse Fc.gamma.R includes, without being limited to, Fc.gamma.RI
(CD64), Fc.gamma.RII (CD32), Fc.gamma.RIII (CD16), and
Fc.gamma.RIII-2 (CD16-2), as well as all unidentified mouse
Fc.gamma.Rs, Fc.gamma.R isoforms, and allotypes thereof. Such
preferred Fc.gamma. receptors include, for example, human
Fc.gamma.RI (CD64), Fc.gamma.RIIA (CD32), Fc.gamma.RIIB (CD32),
Fc.gamma.RIIIA (CD16), and/or Fc.gamma.RIIIB (CD16). The
polynucleotide sequence and amino acid sequence of Fc.gamma.RI are
shown in SEQ ID NOs: 27 (NM_000566.3) and 28 (NP_000557.1),
respectively; the polynucleotide sequence and amino acid sequence
of Fc.gamma.RIIA are shown in SEQ ID NOs: 29 (BC020823.1) and 30
(AAH20823.1), respectively; the polynucleotide sequence and amino
acid sequence of Fc.gamma.RIIB are shown in SEQ ID NOs: 31
(BC146678.1) and 32 (AAI46679.1), respectively; the polynucleotide
sequence and amino acid sequence of Fc.gamma.RIIIA are shown in SEQ
ID NOs: 33 (BC033678.1) and 34 (AAH33678.1), respectively; and the
polynucleotide sequence and amino acid sequence of Fc.gamma.RIIIB
are shown in SEQ ID NOs: 35 (BC128562.1) and 36 (AAI28563.1),
respectively (RefSeq accession number is shown in each
parentheses). Whether an Fc.gamma. receptor has binding activity to
the Fc domain of a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody
can be assessed by ALPHA screen (Amplified Luminescent Proximity
Homogeneous Assay), surface plasmon resonance (SPR)-based BIACORE
method, and others (Proc. Natl. Acad. Sci. USA (2006) 103(11),
4005-4010), in addition to the above-described FACS and ELISA
formats.
[0471] Meanwhile, "Fc ligand" or "effector ligand" refers to a
molecule and preferably a polypeptide that binds to an antibody Fc
domain, forming an Fc/Fc ligand complex. The molecule may be
derived from any organisms. The binding of an Fc ligand to Fc
preferably induces one or more effector functions. Such Fc ligands
include, but are not limited to, Fc receptors, Fc.gamma.R,
Fc.alpha.R, Fc.epsilon.R, FcRn, C1q, and C3, mannan-binding lectin,
mannose receptor, Staphylococcus Protein A, Staphylococcus Protein
G, and viral Fc.gamma.Rs. The Fc ligands also include Fc receptor
homologs (FcRH) (Davis et al., (2002) Immunological Reviews 190,
123-136), which are a family of Fc receptors homologous to
Fc.gamma.R. The Fc ligands also include unidentified molecules that
bind to Fc.
Fc.gamma. Receptor-Binding Activity
[0472] The impaired binding activity of Fc domain to any of the
Fc.gamma. receptors Fc.gamma.I, Fc.gamma.IIA, Fc.gamma.IIB,
Fc.gamma.IIIA, and/or Fc.gamma.IIIB can be assessed by using the
above-described FACS and ELISA formats as well as ALPHA screen
(Amplified Luminescent Proximity Homogeneous Assay) and surface
plasmon resonance (SPR)-based BIACORE method (Proc. Natl. Acad.
Sci. USA (2006) 103(11), 4005-4010).
[0473] ALPHA screen is performed by the ALPHA technology based on
the principle described below using two types of beads: donor and
acceptor beads. A luminescent signal is detected only when
molecules linked to the donor beads interact biologically with
molecules linked to the acceptor beads and when the two beads are
located in close proximity. Excited by laser beam, the
photosensitizer in a donor bead converts oxygen around the bead
into excited singlet oxygen. When the singlet oxygen diffuses
around the donor beads and reaches the acceptor beads located in
close proximity, a chemiluminescent reaction within the acceptor
beads is induced. This reaction ultimately results in light
emission. If molecules linked to the donor beads do not interact
with molecules linked to the acceptor beads, the singlet oxygen
produced by donor beads do not reach the acceptor beads and
chemiluminescent reaction does not occur.
[0474] For example, a biotin-labeled antigen-binding molecule is
immobilized to the donor beads and glutathione S-transferase
(GST)-tagged Fc.gamma. receptor is immobilized to the acceptor
beads. In the absence of an antigen-binding molecule comprising a
competitive mutant Fc domain, Fc.gamma. receptor interacts with an
antigen-binding molecule comprising a wild-type Fc domain, inducing
a signal of 520 to 620 nm as a result. The antigen-binding molecule
having a non-tagged mutant Fc domain competes with the
antigen-binding molecule comprising a wild-type Fc domain for the
interaction with Fc.gamma. receptor. The relative binding affinity
can be determined by quantifying the reduction of fluorescence as a
result of competition. Methods for biotinylating antigen-binding
molecules such as antibodies using Sulfo-NHS-biotin or the like are
known. Appropriate methods for adding the GST tag to an Fc.gamma.
receptor include methods that involve fusing polypeptides encoding
Fc.gamma. and GST in-frame, expressing the fused gene using cells
introduced with a vector carrying the gene, and then purifying
using a glutathione column. The induced signal can be preferably
analyzed, for example, by fitting to a one-site competition model
based on nonlinear regression analysis using software such as
GRAPHPAD PRISM (GraphPad; San Diego).
[0475] One of the substances for observing their interaction is
immobilized as a ligand onto the gold thin layer of a sensor chip.
When light is shed on the rear surface of the sensor chip so that
total reflection occurs at the interface between the gold thin
layer and glass, the intensity of reflected light is partially
reduced at a certain site (SPR signal). The other substance for
observing their interaction is injected as an analyte onto the
surface of the sensor chip. The mass of immobilized ligand molecule
increases when the analyte binds to the ligand. This alters the
refraction index of solvent on the surface of the sensor chip. The
change in refraction index causes a positional shift of SPR signal
(conversely, the dissociation shifts the signal back to the
original position). In the Biacore system, the amount of shift
described above (i.e., the change of mass on the sensor chip
surface) is plotted on the vertical axis, and thus the change of
mass over time is shown as measured data (sensorgram). Kinetic
parameters (association rate constant (ka) and dissociation rate
constant (kd)) are determined from the curve of sensorgram, and
affinity (KD) is determined from the ratio between these two
constants. Inhibition assay is preferably used in the BIACORE
methods. Examples of such inhibition assay are described in Proc.
Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.
[0476] Herein, "Fc.gamma. receptor-binding activity is reduced"
means, for example, that based on the above-described analysis
method the competitive activity of a test antigen-binding molecule
is 50% or less, preferably 45% or less, 40% or less, 35% or less,
30% or less, 20% or less, or 15% or less, and particularly
preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or
less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less
than the competitive activity of a control antigen-binding
molecule.
[0477] Antigen-binding molecules comprising the Fc domain of a
monoclonal IgG1, IgG2, IgG3, or IgG4 antibody can be appropriately
used as control antigen-binding molecules. The Fc domain structures
are shown in SEQ ID NOs: 37 (A is added to the N terminus of RefSeq
accession number AAC82527.1), 38 (A is added to the N terminus of
RefSeq accession number AAB59393.1), 25 (A is added to the N
terminus of RefSeq accession number CAA27268.1), and 39 (A is added
to the N terminus of RefSeq accession number AAB59394.1).
Furthermore, when an antigen-binding molecule comprising an Fc
domain mutant of an antibody of a particular isotype is used as a
test substance, the effect of the mutation of the mutant on the
Fc.gamma. receptor-binding activity is assessed using as a control
an antigen-binding molecule comprising an Fc domain of the same
isotype. As described above, antigen-binding molecules comprising
an Fc domain mutant whose Fc.gamma. receptor-binding activity has
been judged to be reduced are appropriately prepared.
[0478] Such known mutants include, for example, mutants having a
deletion of amino acids 231A-238S (EU numbering) (WO 2009/011941),
as well as mutants C226S, C229S, P238S, (C220S) (J. Rheumatol
(2007) 34, 11); C226S and C229S (Hum. Antibod. Hybridomas (1990)
1(1), 47-54); C226S, C229S, E233P, L234V, and L235A (Blood (2007)
109, 1185-1192).
[0479] Specifically, the preferred antigen-binding molecules
include those comprising an Fc domain with a substitution of the
amino acid at position 220, 226, 229, 231, 232, 233, 234, 235, 236,
237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297,
298, 299, 300, 325, 327, 328, 329, 330, 331, or 332 (EU numbering)
in the amino acids forming the Fc domain of an antibody of a
particular isotype. The isotype of antibody from which the Fc
domain originates is not particularly limited, and it is possible
to use an appropriate Fc domain derived from a monoclonal IgG1,
IgG2, IgG3, or IgG4 antibody. It is preferable to use Fc domains
derived from IgG1 antibodies.
[0480] The preferred antigen-binding molecules include, for
example, those comprising an Fc domain which has any one of the
substitutions shown below, whose positions are specified according
to EU numbering (each number represents the position of an amino
acid residue in the EU numbering; and the one-letter amino acid
symbol before the number represents the amino acid residue before
substitution, while the one-letter amino acid symbol after the
number represents the amino acid residue before the substitution)
in the amino acids forming the Fc domain of IgG1 antibody:
[0481] (a) L234F, L235E, P331S;
[0482] (b) C226S, C229S, P238S;
[0483] (c) C226S, C229S;
[0484] (d) C226S, C229S, E233P, L234V, L235A;
[0485] (e) L234A, L235A or L235R, N297A;
[0486] (f) L235A or L235R, S239K, N297A
as well as those having an Fc domain which has a deletion of the
amino acid sequence at positions 231 to 238.
[0487] Furthermore, the preferred antigen-binding molecules also
include those comprising an Fc domain that has any one of the
substitutions shown below, whose positions are specified according
to EU numbering in the amino acids forming the Fc domain of an IgG2
antibody:
[0488] (g) H268Q, V309L, A330S, and P331S;
[0489] (h) V234A;
[0490] (i) G237A;
[0491] (j) V234A and G237A;
[0492] (k) A235E and G237A;
[0493] (l) V234A, A235E, and G237A. Each number represents the
position of an amino acid residue in EU numbering; and the
one-letter amino acid symbol before the number represents the amino
acid residue before substitution, while the one-letter amino acid
symbol after the number represents the amino acid residue before
the substitution.
[0494] Furthermore, the preferred antigen-binding molecules also
include those comprising an Fc domain that has any one of the
substitutions shown below, whose positions are specified according
to EU numbering in the amino acids forming the Fc domain of an IgG3
antibody:
[0495] (m) F241A;
[0496] (n) D265A;
[0497] (o) V264A.
Each number represents the position of an amino acid residue in EU
numbering; and the one-letter amino acid symbol before the number
represents the amino acid residue before substitution, while the
one-letter amino acid symbol after the number represents the amino
acid residue before the substitution.
[0498] Furthermore, the preferred antigen-binding molecules also
include those comprising an Fc domain that has any one of the
substitutions shown below, whose positions are specified according
to EU numbering in the amino acids forming the Fc domain of an IgG4
antibody:
[0499] (p) L235A, G237A, and E318A;
[0500] (q) L235E;
[0501] (r) F234A and L235A.
Each number represents the position of an amino acid residue in EU
numbering; and the one-letter amino acid symbol before the number
represents the amino acid residue before substitution, while the
one-letter amino acid symbol after the number represents the amino
acid residue before the substitution.
[0502] The other preferred antigen-binding molecules include, for
example, those comprising an Fc domain in which any amino acid at
position 233, 234, 235, 236, 237, 327, 330, or 331 (EU numbering)
in the amino acids forming the Fc domain of an IgG1 antibody is
substituted with an amino acid of the corresponding position in EU
numbering in the corresponding IgG2 or IgG4.
[0503] The preferred antigen-binding molecules also include, for
example, those comprising an Fc domain in which any one or more of
the amino acids at positions 234, 235, and 297 (EU numbering) in
the amino acids forming the Fc domain of an IgG1 antibody is
substituted with other amino acids. The type of amino acid after
substitution is not particularly limited; however, the
antigen-binding molecules comprising an Fc domain in which any one
or more of the amino acids at positions 234, 235, and 297 are
substituted with alanine are particularly preferred.
[0504] The preferred antigen-binding molecules also include, for
example, those comprising an Fc domain in which an amino acid at
position 265 (EU numbering) in the amino acids forming the Fc
domain of an IgG1 antibody is substituted with another amino acid.
The type of amino acid after substitution is not particularly
limited; however, antigen-binding molecules comprising an Fc domain
in which an amino acid at position 265 is substituted with alanine
are particularly preferred.
Multispecific Antigen-Binding Molecules
[0505] Examples of a preferred embodiment of the "multispecific
antigen-binding molecule" of the present invention include
multispecific antibodies. When an Fc region with reduced Fc.gamma.
receptor-binding activity is used as the multispecific antibody Fc
region, an Fc region derived from the multispecific antibody may be
used appropriately. Bispecific antibodies are particularly
preferred as the multispecific antibodies of the present invention.
In this case, a bispecific antibody is an antibody having two
different specificities. IgG-type bispecific antibodies can be
secreted from a hybrid hybridoma (quadroma) produced by fusing two
types of hybridomas that produce IgG antibodies (Milstein et al.,
Nature (1983) 305, 537-540).
[0506] Furthermore, IgG-type bispecific antibodies are secreted by
introducing the genes of L chains and H chains constituting the two
types of IgGs of interest, i.e. a total of four genes, into cells,
and co-expressing them. However, the number of combinations of H
and L chains of IgG that can be produced by these methods is
theoretically ten combinations. Accordingly, it is difficult to
purify an IgG comprising the desired combination of H and L chains
from ten types of IgGs. Furthermore, theoretically the amount of
secretion of the IgG having the desired combination will decrease
remarkably, and therefore large-scale culturing will be necessary,
and production costs will increase further.
[0507] Therefore, techniques for promoting the association among H
chains and between L and H chains having the desired combinations
can be applied to the multispecific antigen-binding molecules of
the present invention.
[0508] For example, techniques for suppressing undesired H-chain
association by introducing electrostatic repulsion at the interface
of the second constant region or the third constant region of the
antibody H chain (CH2 or CH3) can be applied to multispecific
antibody association (WO2006/106905).
[0509] In the technique of suppressing unintended H-chain
association by introducing electrostatic repulsion at the interface
of CH2 or CH3, examples of amino acid residues in contact at the
interface of the other constant region of the H chain include
regions corresponding to the residues at EU numbering positions
356, 439, 357, 370, 399, and 409 in the CH3 region.
[0510] More specifically, examples include an antibody comprising
two types of H-chain CH3 regions, in which one to three pairs of
amino acid residues in the first H-chain CH3 region, selected from
the pairs of amino acid residues indicated in (1) to (3) below,
carry the same type of charge: (1) amino acid residues comprised in
the H chain CH3 region at EU numbering positions 356 and 439; (2)
amino acid residues comprised in the H-chain CH3 region at EU
numbering positions 357 and 370; and (3) amino acid residues
comprised in the H-chain CH3 region at EU numbering positions 399
and 409.
[0511] Furthermore, the antibody may be an antibody in which pairs
of the amino acid residues in the second H-chain CH3 region which
is different from the first H-chain CH3 region mentioned above, are
selected from the aforementioned pairs of amino acid residues of
(1) to (3), wherein the one to three pairs of amino acid residues
that correspond to the aforementioned pairs of amino acid residues
of (1) to (3) carrying the same type of charges in the first
H-chain CH3 region mentioned above carry opposite charges from the
corresponding amino acid residues in the first H-chain CH3 region
mentioned above.
[0512] Each of the amino acid residues indicated in (1) to (3)
above come close to each other during association. Those skilled in
the art can find out positions that correspond to the
above-mentioned amino acid residues of (1) to (3) in a desired
H-chain CH3 region or H-chain constant region by homology modeling
and such using commercially available software, and amino acid
residues of these positions can be appropriately subjected to
modification.
[0513] In the antibodies mentioned above, "charged amino acid
residues" are preferably selected, for example, from amino acid
residues included in either one of the following groups:
[0514] (a) glutamic acid (E) and aspartic acid (D); and
[0515] (b) lysine (K), arginine (R), and histidine (H).
[0516] In the above-mentioned antibodies, the phrase "carrying the
same charge" means, for example, that all of the two or more amino
acid residues are selected from the amino acid residues included in
either one of groups (a) and (b) mentioned above. The phrase
"carrying opposite charges" means, for example, that when at least
one of the amino acid residues among two or more amino acid
residues is selected from the amino acid residues included in
either one of groups (a) and (b) mentioned above, the remaining
amino acid residues are selected from the amino acid residues
included in the other group.
[0517] In a preferred embodiment, the antibodies mentioned above
may have their first H-chain CH3 region and second H-chain CH3
region crosslinked by disulfide bonds.
[0518] In the present invention, amino acid residues subjected to
modification are not limited to the above-mentioned amino acid
residues of the antibody variable regions or the antibody constant
regions. Those skilled in the art can identify the amino acid
residues that form an interface in mutant polypeptides or
heteromultimers by homology modeling and such using commercially
available software; and amino acid residues of these positions can
then be subjected to modification so as to regulate the
association.
[0519] Other known techniques can also be used for the association
of multispecific antibodies of the present invention. Fc
region-containing polypeptides comprising different amino acids can
be efficiently associated with each other by substituting an amino
acid side chain present in one of the H-chain Fc regions of the
antibody with a larger side chain (knob), and substituting an amino
acid side chain present in the corresponding Fc region of the other
H chain with a smaller side chain (hole) to allow placement of the
knob within the hole (WO1996/027011; Ridgway J B et al., Protein
Engineering (1996) 9, 617-621; Merchant A. M. et al. Nature
Biotechnology (1998) 16, 677-681; and US20130336973).
[0520] In addition, other known techniques can also be used for
formation of multispecific antibodies of the present invention.
Association of polypeptides having different sequences can be
induced efficiently by complementary association of CH3 using a
strand-exchange engineered domain CH3 produced by changing part of
one of the H-chain CH3s of an antibody to a corresponding
IgA-derived sequence and introducing a corresponding IgA-derived
sequence into the complementary portion of the other H-chain CH3
(Protein Engineering Design & Selection, 23; 195-202, 2010).
This known technique can also be used to efficiently form
multispecific antibodies of interest.
[0521] In addition, technologies for antibody production using
association of antibody CH1 and CL and association of VH and VL as
described in WO 2011/028952, WO2014/018572, and Nat Biotechnol.
2014 February; 32(2):191-8; technologies for producing bispecific
antibodies using separately prepared monoclonal antibodies in
combination (Fab Arm Exchange) as described in WO2008/119353 and
WO2011/131746; technologies for regulating association between
antibody heavy-chain CH3s as described in WO2012/058768 and
WO2013/063702; technologies for producing bispecific antibodies
composed of two types of light chains and one type of heavy chain
as described in WO2012/023053; technologies for producing
bispecific antibodies using two bacterial cell strains that
individually express one of the chains of an antibody comprising a
single H chain and a single L chain as described by Christoph et
al. (Nature Biotechnology Vol. 31, p 753-758 (2013)); and such may
be used for the formation of multispecific antibodies.
[0522] An embodiment of multispecific antibody formation includes
methods for obtaining bispecific antibodies by mixing two types of
monoclonal antibodies in the presence of a reducing agent to cleave
the disulfide bonds in the core hinge region, followed by
re-association for heterodimerization (FAE) as described above.
Meanwhile, introduction of electrostatic interactions at the
interacting interface of the CH3 region (WO2006/106905) can induce
even more efficient heterodimerization during the re-association
(WO2015/046467). In FAE using naturally-occurring IgG,
re-association takes place randomly; and thus theoretically,
bispecific antibodies can only be obtained at 50% efficiency;
however, in this method, bispecific antibodies can be produced in
high yield.
[0523] Alternatively, even when a multispecific antibody of
interest cannot be formed efficiently, a multispecific antibody of
the present invention can be obtained by separating and purifying
the multispecific antibody of interest from the produced
antibodies. For example, a method for enabling purification of two
types of homomeric forms and the heteromeric antibody of interest
by ion-exchange chromatography by imparting a difference in
isoelectric points by introducing amino acid substitutions into the
variable regions of the two types of H chains has been reported
(WO2007114325). To date, as a method for purifying heteromeric
antibodies, methods using Protein A to purify a heterodimeric
antibody comprising a mouse IgG2a H chain that binds to Protein A
and a rat IgG2b H chain that does not bind to Protein A have been
reported (WO98050431 and WO95033844). Furthermore, a heterodimeric
antibody can be purified efficiently on its own by using H chains
comprising substitution of amino acid residues at EU numbering
positions 435 and 436, which is the IgG-Protein A binding site,
with Tyr, His, or such which are amino acids that yield a different
Protein A affinity, or using H chains with a different protein A
affinity obtained according to the method of Reference Example 9,
to change the interaction of each of the H chains with Protein A,
and then using a Protein A column.
[0524] Alternatively, a common L chain that can provide binding
ability to a plurality of different H chains can be obtained and
used as the common L chain of a multispecific antibody. Efficient
expression of a multispecific IgG can be achieved by introducing
the genes of such a common L chain and a plurality of different H
chains into cells to express the IgG (Nature Biotechnology (1998)
16, 677-681). A method for selecting a common L chain that shows a
strong binding ability to any of the different H chains can also be
used when selecting the common H chain (WO 2004/065611).
[0525] Furthermore, an Fc region whose Fc region C-terminal
heterogeneity has been improved can be appropriately used as an Fc
region of the present invention. More specifically, the present
invention provides Fc regions produced by deleting glycine at
position 446 and lysine at position 447 as specified by EU
numbering from the amino acid sequences of two polypeptides
constituting an Fc region derived from IgG1, IgG2, IgG3, or
IgG4.
[0526] A plurality, such as two or more, of these technologies can
be used in combination. Furthermore, these technologies can be
appropriately and separately applied to the two H chains to be
associated. Furthermore, these techniques can be used in
combination with the above-mentioned Fc region which has reduced
binding activity to an Fc.gamma. receptor. Furthermore, an
antigen-binding molecule of the present invention may be a molecule
produced separately so that it has the same amino acid sequence,
based on the antigen-binding molecule subjected to the
above-described modifications.
[0527] Anon-limiting embodiment of the present invention provides
anticancer agents comprising as an active ingredient a bispecific
antibody of any one of (a) to (c) below that comprises an antibody
variable region having glypican 3-binding activity and an antibody
variable region having CD3-binding activity:
[0528] (a) a bispecific antibody in which CDR1, CDR2, and CDR3
comprised in the antibody variable region having glypican 3-binding
activity are sequences having at least 80% identity to the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 206, respectively; CDR1, CDR2, and CDR3 comprised in the
antibody variable region having CD3-binding activity are sequences
having at least 80% identity to the amino acid sequences of the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 168,
respectively; and CDR1, CDR2, and CDR3 comprised in an antibody
variable region of a common L chain are sequences having at least
80% identity to the amino acid sequences of the CDR1, CDR2, and
CDR3 regions comprised in SEQ ID NO: 223, respectively;
[0529] (b) a bispecific antibody in which the antibody variable
region having glypican 3-binding activity is a sequence having at
least 80% identity to the amino acid sequence of SEQ ID NO: 206;
the antibody variable region having CD3-binding activity is a
sequence having at least 80% identity to the amino acid sequence of
SEQ ID NO: 168; and an antibody variable region of a common L chain
is a sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 223; and
[0530] (c) a bispecific antibody which has an antibody H chain
having glypican 3-binding activity and having at least 80% identity
to the amino acid sequence of SEQ ID NO: 385; an antibody H chain
having CD3-binding activity and having at least 80% identity to the
amino acid sequence of SEQ ID NO: 402; and common L chains having
at least 80% identity to the amino acid sequence of SEQ ID NO:
410.
[0531] In the bispecific antibody of any one of (a) to (c) above,
the amino acid sequence identity of each specified heavy chain and
light chain CDR1, CDR2, and CDR3, heavy chain variable region,
light chain variable region, whole heavy chain, and whole light
chain is preferably at least 75%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, or 90%, or higher, and more preferably at least
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher.
[0532] Herein, sequence identity is calculated as the percentage of
residues identical to those in the original amino acid sequence of
heavy chain or light chain variable region determined after the
sequences are aligned and gaps are appropriately introduced as
necessary to maximize the sequence identity.
[0533] In the bispecific antibody of any one of (a) to (c) above,
as long as the antibody has glypican 3- or CD3-binding activity,
one or more amino acids may be substituted, deleted, added, and/or
inserted in the amino acid sequences of heavy chain and light chain
CDR1, CDR2, and CDR3, and heavy chain variable region, light chain
variable region, whole heavy chain, and whole light chain. Methods
well known to those skilled in the art for preparing such amino
acid sequence in which one or more amino acids are substituted,
deleted, added, and/or inserted include a method of introducing
mutations into proteins. For example, those skilled in the art can
prepare mutants that are functionally equivalent to the heavy chain
or light chain variable region of the bispecific antibody which has
the antibody H chain of SEQ ID NO: 385 having glypican 3-binding
activity, the antibody H chain of SEQ ID NO: 402 having CD3-binding
activity, and the antibody common L chains of SEQ ID NO: 410 by
appropriately introducing mutations into the amino acid sequence of
an antibody having glypican 3- or CD3-binding activity using
site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T,
Ogasahara, Y, and Nakagawa, M. (1995) An
oligodeoxyribonucleotide-directed dual amber method for
site-directed mutagenesis. Gene 152, 271-275; Zoller, M J, and
Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA
fragments cloned into M13 vectors. Methods Enzymol. 100, 468-500;
Kramer, W, Drutsa, V, Jansen, H W, Kramer, B, Pflugfelder, M, and
Fritz, H J (1984) The gapped duplex DNA approach to
oligonucleotide-directed mutation construction. Nucleic Acids Res.
12, 9441-9456; Kramer W, and Fritz H J (1987)
Oligonucleotide-directed construction of mutations via gapped
duplex DNA Methods. Enzymol. 154, 350-367; Kunkel, T A (1985) Rapid
and efficient site-specific mutagenesis without phenotypic
selection. Proc Natl Acad Sci USA. 82, 488-492) or such. In the
present invention, the phrase "functionally equivalent" means that
the binding affinities for an antigen are equivalent, or
alternatively, it means that the cytotoxic activities against
glypican 3-expressing cells or tissues containing these cells are
equivalent when it is used as a multispecific antigen-binding
molecule. The binding affinity and cytotoxic activity can be
measured based on the description herein. The details are described
herein below.
[0534] The number of amino acids to be altered is not limited, and
is, for example, 40 or less, 30 or less, 20 or less, preferably 18
or less, 16 or less, 15 or less, 12 or less, 10 or less, 9 or less,
8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less,
or 2 or less.
[0535] When an amino acid residue is altered, the amino acid is
preferably mutated into a different amino acid(s) that conserves
the properties of the amino acid side chain. Examples of amino acid
side chain properties are: hydrophobic amino acids (A, I, L, M, F,
P, W, Y, and V), hydrophilic amino acids (R, D, N, C, E, Q, G, H,
K, S, and T), amino acids having aliphatic side chains (G, A, V, L,
I, and P), amino acids having hydroxyl group-containing side chains
(S, T, and Y), amino acids having sulfur-containing side chains (C
and M), amino acids having carboxylic acid- and amide-containing
side chains (D, N, E, and Q), amino acids having basic side chains
(R, K, and H), and amino acids having aromatic side chains (H, F,
Y, and W) (amino acids are represented by one-letter codes in
parentheses). Amino acid substitutions within each of these groups
are referred to as conservative substitutions. It is already known
that a polypeptide having a modified amino acid sequence in which
one or more amino acid residues in a given amino acid sequence are
deleted, added, and/or substituted with other amino acids can
retain the biological activity (Mark, D. F. et al., Proc. Natl.
Acad. Sci. USA (1984)81:5662-6; Zoller, M. J. and Smith, M.,
Nucleic Acids Res. (1982)10:6487-500; Wang, A. et al., Science
(1984) 224:1431-3; Dalbadie-McFarland, G. et al., Proc. Natl. Acad.
Sci. USA (1982)79:6409-13).
[0536] Alternatively, an amino acid sequence having glypican 3- or
CD3-binding activity that has a substitution, deletion, addition,
and/or insertion of one or more amino acids in the amino acid
sequence of the CDR region, heavy chain variable region, light
chain variable region, whole heavy chain region, or whole light
chain region can be obtained from nucleic acids that hybridize
under stringent conditions with nucleic acids comprising the
nucleotide sequence encoding the amino acid sequence of the CDR
region, heavy chain variable region, light chain variable region,
whole heavy chain region, or whole light chain region. Stringent
hybridization conditions for isolating a nucleic acid that
hybridizes under stringent conditions with a nucleic acid
comprising a nucleotide sequence encoding the amino acid sequence
of the CDR region, heavy chain variable region, light chain
variable region, whole heavy chain region, or whole light chain
region include, for example, the conditions of 6 M urea, 0.4% SDS,
0.5.times.SSC, and 37.degree. C., or hybridization conditions with
a stringency equivalent thereto. Isolation of nucleic acids with a
much higher homology can be expected with more stringent
conditions, for example, the conditions of 6 M urea, 0.4% SDS,
0.1.times.SSC, and 42.degree. C. The washing conditions following
the hybridization are, for example, washing with 0.5.times.SSC
(1.times.SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and
0.1% SDS at 60.degree. C., more preferably washing with
0.2.times.SSC and 0.1% SDS at 60.degree. C., even more preferably
washing with 0.2.times.SSC and 0.1% SDS at 62.degree. C., yet even
more preferably washing with 0.2.times.SSC and 0.1% SDS at
65.degree. C., and sill more preferably washing with 0.1.times.SSC
and 0.1% SDS at 65.degree. C. The washing time and the number of
washes may be appropriately adjusted, for example, a 20-minute wash
may be performed three times. The sequences of the isolated nucleic
acids can be determined by known methods described below. The
overall nucleotide sequence homology of the isolated nucleic acid
is at least 50% or higher, preferably 70% or higher, 75% or higher,
80% or higher, 85% or higher, and more preferably 90% or higher
(for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
higher) sequence identity.
[0537] Instead of the above-described methods using hybridization
techniques, gene amplification methods, for example, polymerase
chain reaction (PCR), using primers synthesized based on the
information of a nucleotide sequence encoding the amino acid
sequence of the CDR region, heavy chain variable region, light
chain variable region, whole heavy chain region, or whole light
chain region can also be employed to isolate a nucleic acid that
hybridizes under stringent conditions with a nucleic acid
comprising a nucleotide sequence encoding the amino acid sequence
of the region.
[0538] The identity of one nucleotide sequence or amino acid
sequence to another can be determined using algorithm BLAST by
Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90:5873-7).
Programs called BLASTN and BLASTX were developed based on this
algorithm (Altschul et al., J. Mol. Biol. (1990) 215:403-10).
Parameters for the analysis of nucleotide sequences by BLASTN based
on BLAST include, for example, score=100 and wordlength=12. On the
other hand, parameters for the analysis of amino acid sequences by
BLASTX based on BLAST include, for example, score=50 and
wordlength=3. Default parameters for each program are used when
using the BLAST and Gapped BLAST programs. Specific techniques for
such analyses are known (see the website of the National Center for
Biotechnology Information (NCBI), Basic Local Alignment Search Tool
(BLAST); http://www.ncbi.nlm.nih.gov).
[0539] A non-limiting embodiment of the present invention provides
anticancer agents comprising as an active ingredient a bispecific
antibody that has the two H chains and common L chains of the
antibody of sample number 38 (Table 17) described in Examples and
Reference Examples. A non-limiting embodiment of the present
invention also provides anticancer agents comprising as an active
ingredient a bispecific antibody that has the variable regions of
the two H chains and common L chains of the antibody of sample
number 38 and has glypican 3- and CD3-binding activities.
Anon-limiting embodiment of the present invention also provides
anticancer agents comprising as an active ingredient a bispecific
antibody that has the respective amino acid sequences of CDR1,
CDR2, and CDR3 regions comprised in the two H chains and common L
chains of the antibody of sample number 38 and has glypican 3- and
CD3-binding activities.
[0540] Anon-limiting embodiment of the present invention provides
anticancer agents comprising as an active ingredient a bispecific
antibody that has the antibody H chain of SEQ ID NO: 385 having
glypican 3-binding activity, the antibody H chain of SEQ ID NO: 402
having CD3-binding activity, and the antibody common L chains of
SEQ ID NO: 410.
[0541] A non-limiting embodiment of the present invention provides
anticancer agents comprising as an active ingredient a bispecific
antibody in which CDR1, CDR2, and CDR3 comprised in the antibody
variable region having glypican 3-binding activity are the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 206, respectively; CDR1, CDR2, and CDR3 comprised in the
antibody variable region having CD3-binding activity are the amino
acid sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ
ID NO: 168, respectively; and CDR1, CDR2, and CDR3 comprised in the
antibody variable region of the common L chain are the amino acid
sequences of the CDR1, CDR2, and CDR3 regions comprised in SEQ ID
NO: 223, respectively.
[0542] The amino acid sequences of CDR1, CDR2, and CDR3 comprised
in the antibody variable region can be identified by methods known
to those skilled in the art, and such methods are not particularly
limited. The CDR1, CDR2, and CDR3 regions may be those based on
numbering known to those skilled in the art, for example, Kabat,
Chothia, or Contact numbering, or may be those based on other
criteria. In a non-limiting embodiment of the present invention,
the CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 206
include the regions of amino acid sequences of positions 31 to 35
(SEQ ID NO: 456), positions 50 to 66 (SEQ ID NO: 457), and
positions 99 to 104 (SEQ ID NO: 458), respectively (Kabat
numbering); the regions of amino acid sequences of positions 26 to
32 (SEQ ID NO: 459), positions 52 to 57 (SEQ ID NO: 460), and
positions 97 to 104 (SEQ ID NO: 461), respectively (Chothia
numbering); and the regions of amino acid sequences of positions 30
to 35 (SEQ ID NO: 462), positions 47 to 59 (SEQ ID NO: 463), and
positions 97 to 103 (SEQ ID NO: 464), respectively (Contact
numbering); however, the CDR regions may be those based on other
criteria.
[0543] In a non-limiting embodiment of the present invention, the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 168 include
the regions of amino acid sequences of positions 31 to 35 (SEQ ID
NO: 465), positions 50 to 68 (SEQ ID NO: 466), and positions 101 to
111 (SEQ ID NO: 467), respectively (Kabat numbering); the regions
of amino acid sequences of positions 26 to 32 (SEQ ID NO: 468),
positions 52 to 59 (SEQ ID NO: 469), and positions 99 to 111 (SEQ
ID NO: 470), respectively (Chothia numbering); and the regions of
amino acid sequences of positions 30 to 35 (SEQ ID NO: 471),
positions 47 to 61 (SEQ ID NO: 472), and positions 99 to 110 (SEQ
ID NO: 473), respectively (Contact numbering); however, the CDR
regions may be those based on other criteria.
[0544] In a non-limiting embodiment of the present invention, the
CDR1, CDR2, and CDR3 regions comprised in SEQ ID NO: 223 include
the regions of amino acid sequences of positions 24 to 39 (SEQ ID
NO: 474), positions 55 to 61 (SEQ ID NO: 475), and positions 94 to
102 (SEQ ID NO: 476), respectively (Kabat numbering and Chothia
numbering); and the regions of amino acid sequences of positions 35
to 41 (SEQ ID NO: 477), positions 51 to 56 (SEQ ID NO: 478), and
positions 94 to 101 (SEQ ID NO: 479), respectively (Contact
numbering); however, the CDR regions may be those based on other
criteria (see FIGS. 24-1, 24-2, and 25).
[0545] A non-limiting embodiment of the present invention provides
anticancer agents comprising as an active ingredient a bispecific
antibody in which the antibody variable region having glypican
3-binding activity is the amino acid sequence of SEQ ID NO: 206;
the antibody variable region having CD3-binding activity is the
amino acid sequence of SEQ ID NO: 168; and the antibody variable
region of the common L chain is the amino acid sequence of SEQ ID
NO: 223.
[0546] An appropriate multispecific antigen-binding molecule
comprised in the anticancer agent or the pharmaceutical composition
of the present invention comprises
[0547] (1) a domain comprising an antibody variable region having
glypican 3-binding activity; (2) a domain comprising an antibody
variable region having T-cell receptor complex-binding activity;
and
[0548] (3) a domain comprising an Fc region with reduced Fc.gamma.
receptor-binding activity mentioned above, without limitation to
its structure.
[0549] In the present invention, each of the above-mentioned
domains can be linked directly by peptide bonds. For example, when
using F(ab').sub.2 as the domain comprising an antibody variable
region of (1) and (2), and these Fc regions as the domain
comprising an Fc region with reduced Fc.gamma. receptor-binding
activity of (3), the polypeptides formed by linking the antibody
variable region-containing domains of (1) and (2) and the Fc
region-containing domain of (3) by peptide bonds will form an
antibody structure. Such antibodies can be produced by purification
from the above-mentioned hybridoma culture medium, and also by
purifying antibodies from the culture medium of desired host cells
that stably carry polynucleotides encoding the polypeptides
constituting the antibody.
[0550] Examples of a preferred antibody H-chain variable region
comprised in the anticancer agent or the pharmaceutical composition
of the present invention contained in the antibody variable region
having glypican 3-binding activity comprises the antibody H-chain
variable regions of Table 1, or antibody H-chain variable regions
having CDR sequences whose CDR1, CDR2, and CDR3 amino acid
sequences are the same as the CDR1, CDR2, and CDR3 amino acid
sequences contained in the H-chain variable regions of Table 1, or
antibody H-chain variable regions which are functionally equivalent
to the above-mentioned variable regions.
TABLE-US-00002 TABLE 1 Sequence Name SEQ ID NO: H0000 40 GCH003 170
GCH005 171 GCH006 172 GCH007 173 GCH008 174 GCH010 175 GCH012 176
GCH013 177 GCH014 178 GCH015 179 GCH016 180 GCH019 181 GCH022 182
GCH023 183 GCH025 184 GCH026 185 GCH027 186 GCH029 187 GCH032 188
GCH034 189 GCH035 190 GCH039 191 GCH040 192 GCH042 193 GCH043 194
GCH045 195 GCH053 196 GCH054 197 GCH055 198 GCH056 199 GCH057 200
GCH059 201 GCH060 202 GCH061 203 GCH062 204 GCH064 205 GCH065 206
GCH066 207 GCH067 208 GCH068 209 GCH073 210 GCH094 211 GCH098 212
GCH099 213 GCH100 214 H0610 215
[0551] Examples of a preferred antibody variable region having
T-cell receptor complex-binding activity which is comprised in the
anticancer agent or the pharmaceutical composition of the present
invention include antibody variable regions having T-cell
receptor-binding activity. Of the T-cell receptors, CD3 is
preferred, and CD3.epsilon. is particularly preferred. Examples of
an antibody H-chain variable region contained in such antibody
variable regions include the antibody H-chain variable regions of
Table 2, antibody H-chain variable regions having CDR sequences
whose CDR1, CDR2, and CDR3 amino acid sequences are the same as the
CDR1, CDR2, and CDR3 amino acid sequences contained in the antibody
H-chain variable regions of Table 2, and antibody H-chain variable
regions that are functionally equivalent to the above-mentioned
variable regions.
TABLE-US-00003 TABLE 2 Sequence Name SEQ ID NO: hCE115HA 52
CE115HA177 64 CE115HA178 65 CE115HA179 66 CE115HA180 67 hCE115HAa
68 TR01H006 69 TR01H007 70 TR01H008 71 TR01H009 72 TR01H010 73
TR01H011 74 TR01H012 75 TR01H013 76 TR01H014 77 TR01H015 78
TR01H016 79 TR01H017 80 TR01H018 81 TR01H019 82 TR01H020 83
TR01H021 84 TR01H022 85 TR01H023 86 TR01H024 87 TR01H025 88
TR01H026 89 TR01H027 90 TR01H028 91 TR01H029 92 TR01H030 93
TR01H031 94 TR01H032 95 TR01H033 96 TR01H034 97 TR01H035 98
TR01H036 99 TR01H037 100 TR01H038 101 TR01H039 102 TR01H040 103
TR01H041 104 TR01H042 105 TR01H043 106 TR01H044 107 TR01H045 108
TR01H046 109 TR01H047 110 TR01H048 111 TR01H049 112 TR01H050 113
TR01H051 114 TR01H052 115 TR01H053 116 TR01H054 117 TR01H055 118
TR01H056 119 TR01H057 120 TR01H058 121 TR01H061 122 TR01H062 123
TR01H063 124 TR01H064 125 TR01H065 126 TR01H066 127 TR01H067 128
TR01H068 129 TR01H069 130 TR01H070 131 TR01H071 132 TR01H072 133
TR01H073 134 TR01H074 135 TR01H075 136 TR01H076 137 TR01H077 138
TR01H079 139 TR01H080 140 TR01H081 141 TR01H082 142 TR01H083 143
TR01H084 144 TR01H090 145 TR01H091 146 TR01H092 147 TR01H093 148
TR01H094 149 TR01H095 150 TR01H096 151 TR01H097 152 TR01H098 153
TR01H099 154 TR01H100 155 TR01H101 156 TR01H102 157 TR01H103 158
TR01H104 159 TR01H105 160 TR01H106 161 TR01H107 162 TR01H108 163
TR01H109 164 TR01H110 165 TR01H111 166 TR01H112 167 TR01H113 168
TR01H114 169 TR01H001 420 TR01H002 421 TR01H003 422 TR01H004 423
rCE115H 424 CE115HA121 425 CE115HA122 426 CE115HA124 427 CE115HA192
428 CE115HA236 429 CE115HA251 430 CE115HA252 431
[0552] The relationship between the CDR regions of the amino acid
residues constituting the antibody H chain amino acid sequence and
Kabat numbering is as shown in FIG. 24 (FIGS. 24-1 and 24-2).
[0553] For the antibody L-chain variable regions contained in the
antibody variable region having glypican 3-binding activity and the
antibody variable region having T-cell receptor complex-binding
activity which are comprised in the anticancer agent or the
pharmaceutical composition of the present invention, it is
preferable to obtain a common L chain that may provide a binding
activity to the H chain having glypican 3-binding activity and a
binding activity to the H chain having T-cell receptor complex, and
to use this as the common L-chain variable region of the
multispecific antigen-binding molecule.
[0554] Examples of the common L-chain variable region to be used in
the present invention include the L-chain variable regions of Table
3, antibody L-chain variable regions having CDR sequences whose
CDR1, CDR2, and CDR3 amino acid sequences are the same as the CDR1,
CDR2, and CDR3 amino acid sequences contained in the antibody
L-chain variable regions of Table 3, and antibody L-chain variable
regions that are functionally equivalent to the above-mentioned
variable regions.
TABLE-US-00004 TABLE 3 Sequence Name SEQ ID NO: L0000 53 L0002 217
L0003 218 L0006 219 L0007 220 L0008 221 L0009 222 L0011 223 L0012
224 L0013 225 L0014 226 L0015 227 L0016 228 L0032 229 L0038 230
L0039 231 L0041 232 L0042 233 L0043 234 L0044 235 L0045 236 L0046
237 L0047 238 L0062 239 L0063 240 L0064 241 L0065 242 L0066 243
L0069 244 L0075 245 L0079 246 L0082 247 L0085 248 L0089 249 L0090
250 L0091 251 L0093 252 L0104 253 L0106 254 L0107 255 L0109 256
L0113 257 L0115 258 L0117 259 L0120 260 L0122 261 L0123 262 L0124
263 L0125 264 L0126 265 L0127 266 L0129 267 L0132 268 L0134 269
L0136 270 L0137 271 L0138 272 L0139 273 L0140 274 L0141 275 L0143
276 L0144 277 L0145 278 L0147 279 L0148 280 L0149 281 L0151 282
L0152 283 L0154 284 L0155 285 L0157 286 L0160 287 L0161 288 L0163
289 L0167 290 L0168 291 L0173 292 L0175 293 L0180 294 L0181 295
L0186 296 L0187 297 L0200 298 L0201 299 L0202 300 L0203 301 L0204
302 L0205 303 L0206 304 L0207 305 L0208 306 L0209 307 L0210 308
L0211 309 L0212 310 L0213 311 L0214 312 L0215 313 L0216 314 L0217
315 L0218 316 L0219 317 L0220 318 L0222 319 L0223 320 L0224 321
L0226 322 L0227 323 L0228 324 L0229 325 L0230 326 L0231 327 L0232
328 L0233 329 L0234 330 L0235 331 L0236 332 L0237 333 L0238 334
L0239 335 L0240 336 L0241 337 L0242 338 L0243 339 L0246 340 L0247
341 L0248 342 L0249 343 L0250 344 L0258 345 L0259 346 L0260 347
L0261 348 L0262 349 L0263 350 L0264 351 L0265 352 L0266 353 L0267
354 L0268 355 L0269 356 L0270 357 L0271 358 L0272 359
[0555] The relationship between the CDR regions of the amino acid
residues constituting the antibody L-chain amino acid sequence and
Kabat numbering is as shown in FIG. 25.
[0556] In the present invention, the phrase "functionally
equivalent" means that the binding affinities for an antigen are
equivalent, or alternatively, it means that the cytotoxic
activities against glypican 3-expressing cells or tissues
containing these cells are equivalent when it is used as a
multispecific antigen-binding molecule. The binding affinity and
cytotoxic activity can be measured based on the description herein.
The cells used for measurement of cytotoxic activity may be the
desired GPC3-expressing cells or a desired tissue containing these
cells, and for example, PC-10 or NCI-H446 which are GPC3-expressing
human cancer cell lines can be used. Regarding the antibody
constant regions, the phrase may mean that the decreases in
Fc.gamma. receptor-binding activity are equivalent.
[0557] For example, an antibody H-chain variable region
functionally equivalent to the antibody H chain variable region
described herein (i.e., the original H chain variable region) means
that this region has the same binding affinity when it is combined
with the antibody L-chain variable region described herein which
forms a pair with the original H chain, or alternatively that the
region has the same cytotoxic activity towards glypican
3-expressing cells or a tissue containing these cells when used for
a multispecific antigen-binding molecule. Furthermore, an antibody
L-chain variable region functionally equivalent to the antibody
L-chain variable region described herein (i.e., the original
L-chain variable region) means that this region has the same
binding affinity when it is combined with the antibody H-chain
variable region described herein which forms a pair with the
original L chain, or alternatively that the region has the same
cytotoxic activity towards glypican 3-expressing cells or a tissue
containing these cells when used for a multispecific
antigen-binding molecule.
[0558] The term "equivalent" does not necessarily have to mean the
same degree of activity, and the activity may be enhanced.
Specifically, for antigen-binding affinity, examples include the
case where the value (KD value/parent KD value) obtained by
comparison to the binding affinity of the antibody variable region
serving as the control (parent KD value) is 1.5 or less. The value
of KD value/parent KD value is preferably 1.3 or less, more
preferably 1.2 or less, 1.1 or less, 1.0 or less, 0.9 or less, 0.8
or less, 0.7 or less, 0.6 or less, or 0.5 or less. While there is
no lower limit, examples include 10.sup.-1, 10.sup.-2, 10.sup.-3,
10.sup.-4, 10.sup.-5, or 10.sup.-6. More specifically, in the
present invention, the value of KD value/parent KD value is
preferably 10.sup.-6 to 1.5.times.10.sup.-0, more preferably
10.sup.-6 to 10.sup.-1, even more preferably 10.sup.-6 to
10.sup.-2, and yet even more preferably 10.sup.-6 to 10.sup.-3. For
cytotoxic activity, examples include the case where the value (cell
proliferation inhibition rate/parent cell proliferation inhibition
rate) obtained by comparison to the cell proliferation inhibition
rate of the multispecific antigen-binding molecule serving as the
control (parent cell proliferation inhibition rate) is 0.7 or more.
The concentration of the added multispecific antigen-binding
molecule can be determined appropriately, but is preferably, for
example, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM; and preferably,
measurements are taken at 0.05 nM or 0.1 nM. The value for cell
proliferation inhibition rate/parent cell proliferation inhibition
rate is preferably 0.8 or higher, more preferably 0.9 or higher,
1.0 or higher, 1.2 or higher, 1.5 or higher, 2 or higher, 3 or
higher, 5 or higher, 10 or higher, or 20 or higher. While there is
no upper limit, the value may be 10, 10.sup.2, 10.sup.3, 10.sup.4,
10.sup.5, or 10.sup.6.
[0559] Furthermore, for cytotoxic activity, examples include the
case where the value (concentration for 50% inhibition of cell
proliferation/parent concentration for 50% inhibition of cell
proliferation) obtained by comparison to the concentration of the
original multispecific antigen-binding molecule for 50% inhibition
of cell proliferation (parent concentration for 50% inhibition of
cell proliferation) is 1.5 or less. Concentration for 50% growth
inhibition refers to the concentration of the multispecific
antigen-binding molecule necessary for reducing the cell
proliferation rate to one half compared to when the multispecific
antigen-binding molecule is not added. The value of "concentration
for 50% inhibition of cell proliferation/parent concentration for
50% inhibition of cell proliferation" is preferably 1.3 or less,
more preferably 1.2 or less, 1.1 or less, 1.0 or less, 0.9 or less,
0.8 or less, 0.7 or less, 0.6 or less, or 0.5 or less. While there
is no lower limit, the value may be, for example, 10.sup.-1,
10.sup.-2, 10.sup.-3, 10.sup.-4, 10.sup.-5, or 10.sup.-6.
Specifically, the value is preferably 10.sup.-6 to
1.5.times.10.sup.-0, more preferably 10.sup.-6 to 10.sup.-1, even
more preferably 10.sup.-6 to 10.sup.-2, and yet even more
preferably 10.sup.-6 to 10.sup.-3.
[0560] Regarding the domain comprising an antibody variable region
having GPC3-binding activity, the KD value towards GPC3 (for
example, human GPC3) may be, for example, 5.times.10.sup.-9 M or
less, preferably 4.times.10.sup.-9 M or less, such as
3.times.10.sup.-9 M or less, 2.times.10.sup.-9 M or less,
1.times.10.sup.-9 M or less, 8.times.10.sup.-10 M or less,
5.times.10.sup.-10 M or less, 4.times.10.sup.-10 M or less,
3.times.10.sup.-10 M or less, 2.times.10.sup.-10 M or less,
1.times.10.sup.-10 M or less, 8.times.10.sup.-11 M or less,
5.times.10.sup.-11 M or less, 4.times.10.sup.-11 M or less,
3.times.10.sup.-11 M or less, 2.times.10.sup.-11 M or less,
1.times.10.sup.-11 M or less, 8.times.10.sup.-11 M or less,
5.times.10.sup.-11 M or less, 4.times.10.sup.-4 M or less,
3.times.10.sup.-11 M or less, 2.times.10.sup.-11 M or less,
1.times.10.sup.-11 M or less, 8.times.10.sup.-13 M or less,
5.times.10.sup.-13 M or less, 4.times.10.sup.-13 M or less,
3.times.10.sup.-13 M or less, 2.times.10.sup.-13 M or less, or
1.times.10.sup.-13 M or less.
[0561] Regarding the domain comprising an antibody variable region
having T-cell receptor complex-binding activity, the KD value
towards a human T-cell receptor complex such as a human T cell
receptor, or more specifically for example human CD3.epsilon. may
be, for example, 2.times.10.sup.-7 M or less, preferably
1.5.times.10.sup.-7 M or less, such as 1.4.times.10.sup.-7 M or
less, 1.3.times.10.sup.-7 M or less, 1.2.times.10.sup.-7 M or less,
1.times.10.sup.-7 M or less, 3.times.10.sup.-8 M or less,
2.times.10.sup.-8 M or less, 1.times.10.sup.-8 M or less,
8.times.10.sup.-9 M or less, 5.times.10.sup.-9 M or less,
4.times.10.sup.-9 M or less, 3.times.10.sup.-9 M or less,
2.times.10.sup.-9 M or less, 1.times.10.sup.-9 M or less,
8.times.10.sup.-10 M or less, 5.times.10.sup.-10 M or less,
4.times.10.sup.-10 M or less, 3.times.10.sup.-10 M or less,
2.times.10.sup.-10 M or less, 1.times.10.sup.-10 M or less,
8.times.10.sup.-11 M or less, 5.times.10.sup.-11 M or less,
4.times.10.sup.-11 M or less, 3.times.10.sup.-11 M or less,
2.times.10.sup.-11 M or less, 1.times.10.sup.-11 M or less,
8.times.10.sup.-11 M or less, 5.times.10.sup.-12 M or less,
4.times.10.sup.-12 M or less, 3.times.10.sup.-12 M or less,
2.times.10.sup.-12 M or less, or 1.times.10.sup.-12 M or less.
[0562] The multispecific antigen-binding molecules of the present
invention preferably have KD values toward human GPC3 and human
T-cell receptor complex (for example, human CD3.epsilon. chain)
that are 5.times.10.sup.-9 M or less and 2.times.10.sup.-7 M or
less, respectively, and more preferably 1.times.10.sup.-9 M or less
and 5.times.10.sup.-8 M or less, respectively.
[0563] In the present invention, antibody variable regions that are
"functionally equivalent" are not particularly limited as long as
they are antibody H-chain and/or antibody L-chain variable regions
that satisfy the above-described conditions. Examples of such
antibody variable regions include regions produced by introducing
substitution, deletion, addition, and/or insertion of one or more
amino acids (for example, 1, 2, 3, 4, 5, or 10 amino acids) into
the amino acid sequences of the variable regions of Tables 1 to 3
mentioned above. A method well known to those skilled in the art
for introducing one or more amino-acid substitutions, deletions,
additions, and/or insertions into an amino acid sequence is a
method of introducing mutations into proteins. For example, those
skilled in the art can prepare variable regions that are
functionally equivalent to the antibody variable regions having the
above-mentioned functions by appropriately introducing mutations
into amino acid sequences using methods such as site-directed
mutagenesis (Hashimoto-Gotoh, T., Mizuno, T., Ogasahara, Y., and
Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber
method for site-directed mutagenesis. Gene 152, 271-275; Zoller, M.
J., and Smith, M. (1983) Oligonucleotide-directed mutagenesis of
DNA fragments cloned into M13 vectors. Methods Enzymol. 100,
468-500; Kramer, W., Drutsa, V., Jansen, H. W., Kramer, B.,
Pflugfelder, M., and Fritz, H. J. (1984). The gapped duplex DNA
approach to oligonucleotide-directed mutation construction. Nucleic
Acids Res. 12, 9441-9456; Kramer, W., and Fritz, H. J. (1987)
Oligonucleotide-directed construction of mutations via gapped
duplex DNA Methods. Enzymol. 154, 350-367; and Kunkel, T. A. (1985)
Rapid and efficient site-specific mutagenesis without phenotypic
selection. Proc Natl Acad. Sci. USA. 82, 488-492).
[0564] When an amino acid residue is altered, the amino acid is
preferably mutated into a different amino acid(s) that conserves
the properties of the amino acid side-chain as mentioned above.
Examples of amino-acid side chain properties are: hydrophobic amino
acids (A, I, L, M, F, P, W, Y, and V), hydrophilic amino acids (R,
D, N, C, E, Q, G, H, K, S, and T), amino acids containing aliphatic
side chains (G, A, V, L, I, and P), amino acids containing hydroxyl
group-containing side chains (S, T, and Y), amino acids containing
sulfur atom-containing side chains (C and M), amino acids
containing carboxylic acid- and amide-containing side chains (D, N,
E, and Q), amino acids containing basic side chains (R, K, and H),
and amino acids containing aromatic side chains (H, F, Y, and W)
(amino acids are represented by one-letter codes in parentheses).
Amino acid substitutions within each of these groups are called
conservative substitutions. It is already known that a polypeptide
containing a modified amino acid sequence in which one or more
amino acid residues in a given amino acid sequence are deleted,
added, and/or substituted with other amino acids can retain the
original biological activity (Mark, D. F. et al., Proc. Natl. Acad.
Sci. USA; (1984) 81: 5662-6; Zoller, M. J. and Smith, M., Nucleic
Acids Res. (1982) 10: 6487-500; Wang, A. et al., Science (1984)
224: 1431-3; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci.
USA (1982) 79: 6409-13). Variable regions of the present invention
containing such amino acid modifications have an amino acid
sequence identity of at least 70%, more preferably at least 75%,
even more preferably at least 80%, still more preferably at least
85%, yet more preferably at least 90%, and most preferably at least
95%, with the amino acid sequence of the CDR sequences, FR
sequences, or whole variable regions of the variable region prior
to modification. Herein, sequence identity is defined as the
percentage of residues identical to those in the original amino
acid sequence of the H-chain variable region or L-chain variable
region determined after the sequences are aligned, and gaps are
appropriately introduced to maximize the sequence identity as
necessary. The identity of amino acid sequences can be determined
by the method described below.
[0565] Furthermore, a "functionally equivalent antibody variable
region" can be obtained, for example, from nucleic acids that
hybridize under stringent conditions with nucleic acids comprising
a nucleotide sequence encoding the amino acid sequence of a
variable region in Tables 1 to 3 mentioned above. Stringent
hybridization conditions for isolating a nucleic acid that
hybridizes under stringent conditions with a nucleic acid
comprising a nucleotide sequence encoding the amino acid sequence
of a variable region include, for example, the conditions of 6 M
urea, 0.4% SDS, 0.5.times.SSC, and 37.degree. C., or hybridization
conditions with a stringency equivalent thereto as mentioned above.
Isolation of nucleic acids with a much higher homology can be
expected with more stringent conditions, for example, the
conditions of 6 M urea, 0.4% SDS, 0.1.times.SSC, and 42.degree. C.
The washing conditions following the hybridization are, for
example, washing using 0.5.times.SSC (1.times.SSC is 0.15 M NaCl
and 0.015 M sodium citrate at pH7.0) and 0.1% SDS at 60.degree. C.,
more preferably washing using 0.2.times.SSC and 0.1% SDS at
60.degree. C., even more preferably washing using 0.2.times.SSC and
0.1% SDS at 62.degree. C., yet even more preferably washing using
0.2.times.SSC and 0.1% SDS at 65.degree. C., and still more
preferably washing using 0.1.times.SSC and 0.1% SDS at 65.degree.
C. as mentioned above. The sequences of the isolated nucleic acids
can be determined by the known methods described below. The overall
nucleotide sequence homology of the isolated nucleic acid is at
least 50% or higher, preferably 70% or higher, and more preferably
90% or higher (for example, 95%, 96%, 97%, 98%, 99%, or higher)
sequence identity.
[0566] Nucleic acids that hybridize under stringent conditions to a
nucleic acid comprising a nucleotide sequence encoding the amino
acid sequence of a variable region can also be isolated by using,
instead of the above-described methods using hybridization
techniques, gene amplification methods such as polymerase chain
reaction (PCR) that uses primers synthesized based on information
of the nucleotide sequence encoding the variable-region amino acid
sequence.
[0567] The identity of one nucleotide sequence or amino acid
sequence to another can be determined using the algorithm BLAST, by
Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90: 5873-7)
as mentioned above. Programs called BLASTN and BLASTX were
developed based on this algorithm (Altschul et al., J. Mol. Biol.
(1990) 215: 403-10). To analyze nucleotide sequences according to
BLASTN based on BLAST, the parameters are set, for example, as
score=100 and wordlength=12. On the other hand, parameters used for
the analysis of amino acid sequences by BLASTX based on BLAST
include, for example, score=50 and wordlength=3. Default parameters
for each program are used when using the BLAST and Gapped BLAST
programs. Specific techniques for such analyses are known in the
art (see the website of the National Center for Biotechnology
Information (NCBI), Basic Local Alignment Search Tool (BLAST);
http://www.ncbi.nlm.nih.gov).
[0568] The combination of the antibody variable region having
glypican 3-binding activity and the antibody variable region having
T-cell receptor complex binding activity as comprised in the
multispecific antigen-binding molecule which is comprised in the
anticancer agent or the pharmaceutical composition of the present
invention is not particularly limited as long as it has the
above-described activities. However, in the present invention, the
cytotoxic activity of the multispecific antigen-binding molecule is
preferably equivalent to or greater than that of the bispecific
antibody GPC3_ERY22_rCE115 described in Reference Example 3. Here,
the term "equivalent" does not necessarily have to mean the same
degree of activity as described above, and the activity may be
enhanced. Being equivalent to GPC3_ERY22_rCE115 is, for example,
when the value of (cell proliferation inhibition rate/cell
proliferation inhibition rate (GPC3_ERY22_rCE115)) relative to the
cell proliferation inhibition rate of GPC3_ERY22_rCE115 (cell
proliferation inhibition rate (GPC3_ERY22_rCE115)) is 0.7 or
greater, preferably 0.8 or greater, 0.9 or greater, 1.0 or greater,
1.2 or greater, 1.5 or greater, 2 or greater, 3 or greater, 5 or
greater, 10 or greater, or 20 or greater. While there is no upper
limit, the value may be, for example, 10, 102, 103, 104, 10', or
10.sup.6. The concentration of the multispecific antigen-binding
molecule to be added can be determined appropriately, but is
preferably, for example, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM;
and preferably, measurements are taken at 0.05 nM or 0.1 nM.
[0569] Furthermore, examples include the case where the value
(concentration for 50% inhibition of cell
proliferation/concentration for 50% inhibition of cell
proliferation (GPC3_ERY22_rCE115)) obtained by comparison to the
concentration for 50% inhibition of growth of GPC3_ERY22_rCE115
cells (concentration for 50% inhibition of cell proliferation
(GPC3_ERY22_rCE115)) is 1.5 or less. The value for "concentration
for 50% inhibition of cell proliferation/concentration for 50%
inhibition of cell proliferation (GPC3_ERY22_rCE115)" is preferably
1.3 or less, more preferably 1.2 or less, 1.1 or less, 1.0 or less,
0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, or 0.5 or less.
While there is no lower limit, the value may be for example,
10.sup.-1, 10.sup.--2, 10.sup.-1, 10.sup.-4, 10.sup.-5, or
10.sup.-6. Specifically, the value is preferably 10.sup.-6 to
1.5.times.10.sup.-0, more preferably 10.sup.-6 to 10.sup.-1, even
more preferably 10.sup.-6 to 10.sup.-2, and yet even more
preferably 10.sup.-6 to 10.sup.-3.
[0570] The preferred specific KD values for human GPC3 and human T
cell receptor complex (for example, human CD3.epsilon. chain) are
also as indicated above. Desired cells showing GPC3 expression or
desired tissues containing these cells may be used for the cells,
and for example, PC-10 or NCI-H446 which are GPC3-expressing human
cancer cell lines can be used.
[0571] Examples of such a combination of the antibody variable
region having glypican 3-binding activity and the antibody variable
region having T-cell receptor complex binding activity, include the
combinations of antibody H-chain variable regions shown in Table 4,
combinations of antibody H-chain variable regions having CDR
sequences whose CDR1, CDR2, and CDR3 amino acid sequences are the
same as the CDR1, CDR2, and CDR3 amino acid sequences carried by
the antibody H-chain variable regions of Table 4, and combinations
of antibody H-chain variable regions functionally equivalent to
these variable regions. Here, "functionally equivalent" has the
same meaning described above.
TABLE-US-00005 TABLE 4 GPC3 side/T cell receptor complex side SEQ
ID NO: H0000/hCE115HA 40/52 H0000/CE115HA251 40/500
H0000/CE115HA236 40/429 H0000/TR01H002 40/421 H0000/CE115HA122
40/426 H0610/rCE115H 215/424 H0610/TR01H040 215/103 H0610/TR01H061
215/122 H0610/TR01H068 215/129 H0610/TR01H071 215/132
GCH054/TR01H067 197/128 GCH094/TR01H082 211/142 GCH094/TR01H084
211/144 GCH065/TR01H084 206/144 GCH065/TR01H082 206/142
GCH094/TR01H109 211/164 GCH065/TR01H109 206/164 GCH094/TR01H113
211/168 GCH065/TR01H113 206/168
[0572] A preferred common L chain for such combinations of an
antibody variable region having glypican 3-binding activity and an
antibody variable region having T-cell receptor complex binding
activity includes, for example, L0000, L0011, L0201, L0203, L0204,
L0206, L0208, L0209, L0211, L0212, L0222, and a common L chain
having CDR sequences (CDR1, CDR2, and CDR3 amino acid sequences)
identical to the CDR1, CDR2, and CDR3 amino acid sequences as in
the above common L chain. Specific combinations include, for
example, the combinations of antibody H-chain variable regions and
a common L chain shown in Table 5, combinations of antibody
variable regions having CDR sequences (CDR1, CDR2, and CDR3 amino
acid sequences) identical to the amino acid sequences of CDR1,
CDR2, and CDR3 carried by the antibody variable regions and a
common L chain of Table 5, and combinations of antibody H-chain
variable regions and a common L chain functionally equivalent to
these variable regions. Here, "functionally equivalent" has the
same meaning as described above.
TABLE-US-00006 TABLE 5 GPC3 side/T cell receptor complex
side/common L chain SEQ ID NO: H0610/rCE115H/L0000 215/424/53
H0610/TR01H040/L0000 215/103/53 H0610/TR01H040/L0201 215/103/299
H0610/TR01H040/L0203 215/103/301 H0610/TR01H040/L0204 215/103/302
H0610/TR01H040/L0206 215/103/304 H0610/TR01H040/L0208 215/103/306
H0610/TR01H040/L0209 215/103/307 H0610/TR01H040/L0211 215/103/309
H0610/TR01H061/L0000 215/122/53 H0610/TR01H068/L0000 215/129/53
H0610/TR01H071/L0000 215/132/53 GCH054/TR01H067/L0201 197/128/299
GCH054/TR01H067/L0212 197/128/310 GCH054/TR01H067/L0222 197/128/319
GCH054/TR01H067/L0000 197/128/53 GCH094/TR01H082/L0201 211/142/299
GCH094/TR01H082/L0011 211/142/223 GCH094/TR01H084/L0011 211/144/223
GCH065/TR01H084/L0011 206/144/223 GCH065/TR01H082/L0011 206/142/223
GCH094/TR01H109/L0011 211/164/223 GCH065/TR01H109/L0011 206/164/223
GCH094/TR01H113/L0011 211/168/223 GCH065/TR01H113/L0011
206/168/223
[0573] The Fc region comprised in the multispecific antigen-binding
molecule which is comprised in the anticancer agent or the
pharmaceutical composition of the present invention is not
particularly limited as long as it is an Fc region having reduced
Fc.gamma. receptor-binding activity, but examples of a preferred Fc
region of the present invention include a combination of the
Fc-region portion of E22Hh and the Fc-region portion of E22Hk, a
combination of the Fc-region portion of E2702GsKsc and the
Fc-region portion of E2704sEpsc, and a combination of the Fc-region
portion of E2702sKsc and the Fc-region portion of E2704sEpsc.
[0574] Examples of a preferred multispecific antigen-binding
molecule comprised in an anticancer agent or a pharmaceutical
composition of the present invention include bispecific antibodies
comprising an antibody variable region having glypican 3-binding
activity and an antibody variable region having
CD3.epsilon.-binding activity. More preferably, the cytotoxic
activity is the same or greater than that of the GPC3_ERY22_rCE115
bispecific antibody. Examples of such bispecific antibodies include
bispecific antibodies comprising H and L chains described in Table
17, and bispecific antibodies that bind to an epitope overlapping
with an epitope bound by the above antibodies, and which contain an
Fc region with reduced Fc.gamma. receptor-binding activity.
[0575] Whether an antibody recognizes an epitope that overlaps with
an epitope recognized by another antibody can be confirmed by the
competition between the two antibodies against the epitope.
Competition between the antibodies can be evaluated by competitive
binding assays using means such as enzyme-linked immunosorbent
assay (ELISA), fluorescence energy transfer method (FRET), and
fluorometric microvolume assay technology (FMAT (Registered
trademark)). The amount of an antibody bound to an antigen
indirectly correlates with the binding ability of a candidate
competitor antibody (a test antibody) that competitively binds to
the overlapping epitope. In other words, as the amount or affinity
of a test antibody against the overlapping epitope increases, the
amount of the antibody bound to the antigen decreases, and the
amount of the antigen-bound test antibody increases. Specifically,
the appropriately labeled antibody and antibody to be evaluated are
simultaneously added to the antigen, and the antibody bound as a
result are detected using the label. The amount of the
antigen-bound antibody can be easily determined by labeling the
antibody beforehand. This label is not particularly limited, and
the labeling method is selected according to the assay technique
used. Specifically, the labeling method includes fluorescent
labeling, radiolabeling, enzymatic labeling, and such.
[0576] For example, the fluorescently labeled antibody and the
unlabeled antibody or test antibody are simultaneously added to
beads immobilized with GPC3 or CD3.epsilon., and the labeled
antibody is detected by fluorometric microvolume assay
technology.
[0577] Herein, the "antibody that binds to the overlapping epitope"
refers to a test antibody that can reduce the amount of the bound
labeled antibody by at least 50% at a concentration that is usually
100 times higher, preferably 80 times higher, more preferably 50
times higher, even more preferably 30 times higher, and still more
preferably 10 times higher than the concentration at which the
non-labeled antibody reduces 50% of the amount of the labeled
antibody bound (IC.sub.50).
[0578] Multispecific antigen-binding molecules, which have the
antigen-binding sites of antibodies that bind to epitopes
overlapping with epitopes bound by the above-mentioned antibodies,
can yield excellent cytotoxic activity.
[0579] The multispecific antigen-binding molecules of the present
invention are produced by the same technique as the method for
producing recombinant antibodies mentioned above.
Combination Therapies and Pharmaceutical Compositions
[0580] In a non-limiting embodiment of the present invention, the
combination therapy of the present invention provides methods for
damaging cells, for suppressing cell proliferation, for activating
immunity towards cancer cells or cancer cell-comprising tumor
tissues, for treating cancer, or for preventing cancer, each of the
methods comprising administering effective amounts of the
above-described bispecific antibody and another anticancer agent.
In several embodiments, the combination therapy of the present
invention is highly effective for damaging cells, suppressing cell
proliferation, activating immunity towards cancer cells or cancer
cell-comprising tumor tissues, treating cancer, or preventing
cancer, as compared to monotherapy using the above-described
bispecific antibody or the other anticancer agent. In another
embodiment, the combination therapy of the present invention has
synergistic effects or additive effects on damaging cells,
suppressing cell proliferation, activating immunity towards cancer
cells or cancer cell-comprising tumor tissues, treating cancer, or
preventing cancer.
[0581] In several embodiments, the term "effective amount" in the
present invention refers to a dose of the above-described
bispecific antibody and/or another anticancer agent that is
effective for treating or preventing a disease in an individual.
The disease is not particularly limited but is preferably
cancer.
[0582] In several embodiments, "treatment/treating/therapeutic" in
the present invention means that the combination therapy of the
present invention decreases the number of cancer cells in
individuals, suppresses cancer cell proliferation, decreases tumor
size, suppresses infiltration of cancer cells into peripheral
organs, suppresses cancer cell metastasis, or ameliorates various
symptoms caused by cancer. Furthermore, in several embodiments,
"prevention/preventing/prophylactic" in the present invention
refers to inhibiting increase in the number of cancer cells due to
repopulation of cancer cells that have been decreased, inhibiting
repopulation of cancer cells whose proliferation has been
suppressed, and inhibiting the decreased tumor size to become large
again.
[0583] In several embodiments, the combination therapy of the
present invention provides methods for enhancing therapeutic or
prophylactic effects of the other anticancer agent by using the
above-described bispecific antibody, in cancer treatment or
prevention with the other anticancer agent. In another embodiment,
the combination therapy of the present invention provides methods
for enhancing therapeutic or prophylactic effects of the
above-described bispecific antibody by using the other anticancer
agent, in cancer treatment or prevention with the bispecific
antibody. Herein, enhancement of therapeutic or prophylactic
effects refers to, for example, increase in efficacy rate of the
treatment, decrease in the amount of the anticancer agent that is
administered for the treatment, and/or shortening of the period of
the treatment with an anticancer agent, but is not limited thereto.
In another embodiment, the combination therapy of the present
invention provides methods for extending progression-free survival
in individuals, the method comprising administering an effective
amount of the above-described bispecific antibody and another
anticancer agent.
[0584] In several embodiments, the combination therapy of the
present invention comprises administering the above-described
bispecific antibody and another anticancer agent. The bispecific
antibody and the other anticancer agent can be administered by any
appropriate methods known in the art. For example, the bispecific
antibody and the other anticancer agent can be administered in
parallel (i.e., simultaneously) or successively (i.e., at different
time points). In several embodiments, when the bispecific antibody
and the other anticancer agent are administered successively (i.e.,
at different time points), the interval between administration of
the bispecific antibody and the other anticancer agent is not
particularly limited and the interval can be determined by taking
account for factors such as the administration route and dosage
form. The interval is, for example, 0 to 168 hours, preferably 0 to
72 hours, more preferably 0 to 24 hours, and even more preferably 0
to 12 hours, but is not limited thereto.
[0585] In several embodiments, the above-described bispecific
antibody and the other anticancer agent are administered
simultaneously. In several embodiments, the bispecific antibody is
administered at intervals (i.e., intermittently). In several
embodiments, the bispecific antibody is administered before
administration of the other anticancer agent. In several
embodiments, the bispecific antibody is administered after
administration of the other anticancer agent.
[0586] In several embodiments, the other anticancer agent is
administered at intervals (i.e., intermittently). In several
embodiments, the other anticancer agent is administered before
administration of the bispecific antibody. In several embodiments,
the other anticancer agent is administered after administration of
the bispecific antibody.
[0587] In several embodiments, the bispecific antibodies described
herein and anticancer agents which are known or described herein
can be used in the above-described combination therapies using
bispecific antibodies and other anticancer agents.
[0588] In several embodiments, an additional therapy can be
performed in addition to the combination therapies using the
above-described bispecific antibody and another anticancer agent.
In several embodiments, a therapy to add to the combination therapy
of the present invention may comprise additional administration of
the bispecific antibody and/or the other anticancer agent.
[0589] A non-limiting embodiment of the present invention provides
agents for inducing cytotoxicity, agents for suppressing cell
proliferation (agents for inhibiting cell proliferation), agents
for activating immune response towards cancer cells or cancer
cell-comprising tumor tissues, agents for treating cancer, and
agents for preventing cancer (herein below, referred to as
pharmaceutical compositions and such), each comprising the
above-described bispecific antibody, another anticancer agent, or a
combination of the bispecific antibody and the other anticancer
agent. In several embodiments, the pharmaceutical compositions and
such of the present invention can be used in the combination
therapy of the present invention. In several embodiments, the
pharmaceutical compositions and such of the present invention are
highly effective for damaging cells, suppressing cell
proliferation, activating immunity towards cancer cells or cancer
cell-comprising tumor tissues, treating cancer, or preventing
cancer due to combined use of the above-described bispecific
antibody and the other anticancer agent, as compared to monotherapy
using the bispecific antibody or the other anticancer agent. In
another embodiment, the pharmaceutical compositions of the present
invention have synergistic effects or additive effects on damaging
cells, suppressing cell proliferation, activating immunity towards
cancer cells or cancer cell-comprising tumor tissues, treating
cancer, or preventing cancer due to combined use of the
above-described bispecific antibody and the other anticancer
agent.
[0590] In several embodiments, the pharmaceutical compositions and
such according to the present invention "comprising a combination
of a bispecific antibody and another anticancer agent" refers to
pharmaceutical compositions and such in which the above-described
bispecific antibody and the other anticancer agent are combined for
use in simultaneous, separate, or sequential administration in
treatment or prevention of a disease. For example, the
pharmaceutical compositions and such of the present invention can
be provided in the form of a combination preparation containing
both a bispecific antibody and another anticancer agent.
Alternatively, for example, as the pharmaceutical compositions and
such of the present invention, a pharmaceutical agent containing a
bispecific antibody and a pharmaceutical agent containing another
anticancer agent can be separately provided, and these
pharmaceutical agents may be used simultaneously or sequentially.
The disease is not particularly limited but is preferably
cancer.
[0591] In several embodiments, the present invention provides
pharmaceutical compositions and such for use in combination with
another anticancer agent, the compositions comprising the
above-described bispecific antibody as an active ingredient.
[0592] In several embodiments, the present invention provides
pharmaceutical compositions and such for use in combination with
the above-described bispecific antibody, the compositions
comprising another anticancer agent as an active ingredient.
[0593] In several embodiments, the present invention provides
pharmaceutical compositions and such for enhancing therapeutic
effects of another anticancer agent in cancer treatment with said
another anticancer agent, by using the above-described bispecific
antibody in combination with said another anticancer agent.
[0594] In several embodiments, the present invention provides
pharmaceutical compositions and such for enhancing therapeutic
effects of the above-described bispecific antibody in cancer
treatment with the bispecific antibody, by using another anticancer
agent in combination with the bispecific antibody.
[0595] In several embodiments, the present invention provides use
of the above-described bispecific antibody and/or another
anticancer agent for the production of pharmaceutical compositions
and such comprising as active ingredients the bispecific antibody
and/or the other anticancer agent.
[0596] In the present invention, "comprising as active ingredients
the above-described bispecific antibody and/or another anticancer
agent" means "containing the bispecific antibody and/or the other
(another) anticancer agent as major active component(s)", and does
not limit the content of the bispecific antibody and/or the other
anticancer agent.
[0597] In several embodiments, the bispecific antibodies described
herein and other anticancer agents which are known or described
herein can be used in the above-described pharmaceutical
compositions and others.
[0598] In a non-limiting embodiment of the present invention, the
above-described other anticancer agents include, but are not
limited to, nitrogen mustard analogues, alkyl sulfonates, ethylene
imines, nitrosoureas, epoxides, other alkylating agents, folic acid
analogues, purine analogues, pyrimidine analogues, other
antimetabolites, vinca alkaloids or analogues, podophyllotoxin
derivatives, camptothecan analogues, colchicine derivatives,
taxanes, other plant alkaloids or natural substances,
actinomycines, anthracyclines or related substances, other
cytotoxic antibiotics, platinum compounds, methylhydrazines, kinase
inhibitors, angiogenic inhibitors, hormonal agents, inhibitors of
DNA modification enzymes, immunostimulators, proteasome inhibitors,
enzymes, histone deacetylase inhibitors, inhibitors of DNA
modification enzymes, cytokine preparations, retinoids, immune
checkpoint inhibitors, indoleamine 2,3-Dioxygenase (IDO)
inhibitors, co-stimulatory molecule activators, natural killer cell
activators, monoclonal antibodies, other molecular-targeted agents,
and other anticancer agents. In a non-limiting embodiment, other
anticancer agents in the present invention include, but are not
limited to, for example, antibodies described in WO2015/174439 and
WO2015/156268.
[0599] In several embodiments, an "immune checkpoint" of the
present invention refers to a molecule that is expressed on
immunocompetent cells (including T cells) and binds to a ligand to
thereby transduce to the immunocompetent cells signals inhibiting
immune response. Examples of immune checkpoints and ligands thereof
include, but are not limited to, molecules such as PD-1, CTLA-4,
TIM3, LAG3, PD-L1, PD-L2, BTNL2, B7-H3, B7-H4, CD48, CD80, 2B4,
BTLA, CD160, CD60, CD86, and VISTA. In several embodiments, an
"immune checkpoint inhibitor" of the present invention refers to a
pharmaceutical agent that inhibits binding between an immune
checkpoint and its ligand, and thereby inhibits signal transduction
mediated by the immune checkpoint.
[0600] A non-limiting embodiment of the present invention provides
pharmaceutical compositions and such in which the other anticancer
agent is a chemotherapeutic agent, a T cell-activating agonist
agent, an immune checkpoint inhibitor, or an angiogenic
inhibitor.
[0601] In a non-limiting embodiment of the present invention,
chemotherapeutic agents include, but are not limited to,
antimetabolites, plant alkaloids, and platinum compounds. Preferred
examples of an antimetabolite include, but are not limited to,
enocitabine, capecitabine, carmofur, gemcitabine, cytarabine,
tegafur, tegafur uracil, nelarabine, fluorouracil, fludarabine,
pemetrexed, pentostatin, and methotrexate. Particularly preferred
antimetabolites include, for example, capecitabine. Preferred
examples of a plant alkaloid include, but are not limited to,
irinotecan, etoposide, sobuzoxane, docetaxel, nogitecan,
paclitaxel, vinorelbine, vincristine, vindesine, and vinblastine.
Particularly preferred plant alkaloids include, for example,
paclitaxel. Preferred examples of a platinum compound include, but
are not limited to, oxaliplatin, carboplatin, cisplatin, and
nedaplatin. Particularly preferred platinum compounds include, for
example, cisplatin.
[0602] In a non-limiting embodiment of the present invention, T
cell-activating agonist agents include, but are not limited to, TNF
receptor superfamily (TNFRSF) agonist antibodies and co-stimulatory
molecule agonist antibodies against. Target molecules of "TNF
receptor superfamily agonist antibodies" are not particularly
limited as long as they are factors that activate cells expressing
the TNF receptor superfamily (for example, T cells and NK cells),
but are preferably factors belonging to the "TNF superfamily" or
the "TNF receptor superfamily". Known factors belonging to the "TNF
superfamily" or the "TNF receptor superfamily" include ligands
having a trimeric structure and receptors having a trimeric
structure to which the ligands bind, which contribute to activation
of various immune cells (Nat. Rev. Immunol., 2012, 12, 339-51).
Examples of a factor belonging to the TNF superfamily or TNF
receptor superfamily include CD137, CD137L, CD40, CD40L, OX40,
OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153, GITR,
GITRL, TNFRSF25, and TL1A. Preferred factors include, for example,
CD137. Examples of a CD137 agonist antibody include Urelumab (CAS
No. 934823-49-1), PF-05082566, and various known CD137 agonist
antibodies.
[0603] Factors belonging to co-stimulatory molecules include
TMIGD2, HHLA2, ICOS, ICOS ligand, CD28, CD80, CD86, and such.
Examples of an OX40 agonist antibody include MOXR0916, MEDI6469,
MEDI0562, MEDI6383, PF-04518600, GSK-3174998, and various known
OX40 agonist antibodies. Examples of a CD40 agonist antibody
include RG-7876, ADC-1013, SEA-CD40, APX005M, Dacetuzumab, and
various known CD40 agonist antibodies. Examples of a GITR agonist
antibody include AMG228, AMK-1248, MK-4166, BMS-986156, TRX518, and
various known GITR agonist antibodies. Examples of a CD27 agonist
antibody include Varlilumab (CAS No. 1393344-72-3) and various
known CD27 agonist antibodies.
[0604] In a non-limiting embodiment of the present invention,
preferred examples of an immune checkpoint inhibitor include, but
are not limited to, PD1 antibodies, PDL1 antibodies, CTLA-4
antibodies, TIM3 antibodies, and LAG3 antibodies. Examples of a
PD-1 antibody include Pembrolizumab (CAS No. 1374853-91-4),
Nivolumab (CAS No. 946414-94-4), MEDI0680, PDR001, BGB-A317,
REGN2810, SHR-1210, PF-06801591, and various known PD1 antibodies.
Examples of a PD-L1 antibody include Atezolizumab (CAS No.
1380723-44-3), Avelumab (CAS No. 1537032-82-8), Durvalumab (CAS No.
1428935-60-7), MDX-1105, and various known PD-L1 antibodies.
Examples of a CTLA-4 antibody include Ipilimumab (CAS No.
477202-00-9), Tremelimumab (CAS No. 745013-59-6), and various known
CTLA-4 antibodies. Examples of a TIM3 antibody include MBG452 and
various known TIM3 antibodies. Examples of an LAG3 antibody include
BMS-986016, LAG525, and various known LAG3 antibodies.
[0605] In a non-limiting embodiment of the present invention,
preferred examples of an angiogenic inhibitor include, but are not
limited to, VEGFR2 antibodies. Examples of an angiogenic inhibitor
include Bevacizumab, Sorafenib, Everolimus, Temsirolimus, and
various known angiogenic inhibitors.
[0606] In several embodiments, other anticancer agents of the
present invention are not particularly limited and any anticancer
agent can be used as long as, when used in combination with the
bispecific antibody of the present invention, therapeutic effect or
prophylactic effect of the other anticancer agent is enhanced or
therapeutic effect or prophylactic effect of the bispecific
antibody is enhanced.
[0607] In a non-limiting embodiment of the present invention, the
combination therapy of the present invention may comprise the
above-described bispecific antibody and at least one other
therapeutic agent, an immunomodulator, a therapeutic cancer
vaccine, adoptive T cell therapy, Treg depletion, or such, but the
therapy is not limited thereto. Preferred therapeutic cancer
vaccines include, but are not limited to, whole tumor cell
vaccines, tumor antigen vaccines, vector-based vaccines, oncolytic
viral vaccines, and dendritic cell vaccines. Multimodality therapy
may be performed using surgical operation, radiation treatment, or
such in combination, in addition to the above-described
therapies.
[0608] In a non-limiting embodiment of the present invention, the
combination therapy of the present invention can be performed using
the above-described bispecific antibody in combination with
cytokine therapy using a cytokine as an anti-tumor immune
response-enhancing agent. In such therapy, cytokines include, but
are not limited to, IL-2, IL-7, IL-12, IL-15, IL-17, IL-18, IL-21,
IL-23, IL-27, GM-CSF, interferon-.alpha. (IFN.alpha.),
IFN.alpha.-2b, IFN.beta., and IFN.gamma..
[0609] A non-limiting embodiment of the present invention provides
agents for inducing cytotoxicity, agents for suppressing cell
proliferation, agents for inhibiting cell proliferation, agents for
activating immune response, agents for treating cancer, and agents
for preventing cancer, each comprising the above-described
pharmaceutical composition.
[0610] In several embodiments, an "individual" to which the
above-described bispecific antibody and/or another anticancer agent
is administered refers to a human or a non-human animal, for
example, a mammal such as cattle, horse, dog, sheep, or cat. The
individual is preferably a human. The individual includes patients
(including human and non-human mammals). In several embodiments,
the individual is a patient who has cancer cells or cancer
cell-comprising tumor tissues. Cancer cells or cancer
cell-comprising tumor tissues which become targets of the
anticancer agent or the combination therapy of the present
invention are not particularly limited, as long as they express
glypican 3. In the present invention, preferred glypican
3-expressing cells, i.e., glypican 3-positive cells, are cancer
cells. More preferred cancer types include, but are not limited to,
for example, gastric cancer, head and neck cancer (H&N),
esophageal cancer, lung cancer, liver cancer, ovary cancer, breast
cancer, colon cancer, kidney cancer, skin cancer, muscle tumor,
pancreas cancer, prostate cancer, testis cancer, uterine cancer,
cholangiocarcinoma, Merkel cell carcinoma, bladder cancer, thyroid
cancer, schwannoma, adrenal cancer (adrenal gland), anus cancer,
central nervous system tumor, neuroendocrine tissue tumor, penis
cancer, pleura tumor, salivary gland tumor, vulva cancer, thymoma,
and childhood cancer (Wilms tumor, neuroblastoma, sarcoma,
hepatoblastoma, and germ cell tumor). Still more preferred cancer
types include, but are not limited to, gastric cancer, head and
neck cancer (H&N), esophageal cancer, lung cancer, liver
cancer, ovary cancer, breast cancer, colon cancer, kidney cancer,
skin cancer, muscle tumor, pancreas cancer, prostate cancer, testis
cancer, and uterine cancer (Tumori. (2012) 98, 478-484; Tumor Biol.
(2015) 36, 4671-4679; Am J Clin Pathol (2008) 130, 224-230; Adv
Anat Pathol (2014) 21, 450-460; Med Oncol (2012) 29, 663-669;
Clinical Cancer Research (2004) 10, 6612-6621; Appl Immunohistochem
Mol Morphol (2009) 17, 40-46; Eur J Pediatr Surg (2015) 25,
138-144; J Clin Pathol (2011) 64, 587-591; Am J Surg Pathol (2006)
30, 1570-1575; Oncology (2007) 73, 389-394; Diagnostic Pathology
(2010) 64, 1-6; Diagnostic Pathology (2015) 34, 1-6; Am J Clin
Pathol (2008) 129, 899-906; Virchows Arch (2015) 466, 67-76).
[0611] In several embodiments, patients are those who have received
treatment with the above-described bispecific antibody and/or some
kind of anticancer agent(s) prior to the combination therapy using
the bispecific antibody and another anticancer agent. In several
embodiments, patients are those who cannot receive standard therapy
or for whom standard therapy is ineffective. In several
embodiments, cancer which a patient has is early-stage or
end-stage.
[0612] As used herein, "cancer" refers not only to epithelial
malignancy such as ovary cancer or gastric cancer but also to
non-epithelial malignancy including hematopoietic tumors such as
chronic lymphocytic leukemia or Hodgkin's lymphoma. Herein, the
terms "cancer", "carcinoma", "tumor", "neoplasm" and such are not
differentiated from each other and are mutually
interchangeable.
[0613] Meanwhile, in several embodiments, cancer types which become
targets of anticancer agents or pharmaceutical compositions
(combination therapy) of the present invention are preferably those
in which the number of glypican-3 antigens on the cell surface per
cell is 100 or more, more preferably those in which the number of
glypican-3 antigens on the cell surface per cell is 200 or more,
300 or more, 400 or more, 500 or more, 600 or more, 700 or more,
800 or more, 900 or more, 1000 or more, 1200 or more, 1400 or more,
1600 or more, 1800 or more, or 2000 or more, and still more
preferably those in which the number of glypican-3 antigens on the
cell surface per cell is 3000 or more, 4000 or more, 5000 or more,
6000 or more, 7000 or more, 8000 or more, 9000 or more, 10000 or
more, 20000 or more, 30000 or more, 40000 or more, or 50000 or
more.
[0614] The number of glypican-3 antigens on cell surface per cell
can be appropriately determined using methods described herein or
known to those skilled in the art, for example, by calculating
antibody binding capacity (ABC) of GPC3 on the cell surface with
flow cytometry using QIFIKIT (DAKO). The number of glypican-3
antigens on cell surface per cell in a tissue sample isolated from
a target candidate can be determined in order to assess whether the
candidate can be a target to which an anticancer agent or
pharmaceutical composition (combination therapy) of the present
invention is administered. When in the sample the number of
glypican-3 antigens on cell surface per cell meets the criterion
described above, the target from which the sample is derived can be
the target to which the anticancer agent or pharmaceutical
composition (combination therapy) of the present invention is
administered.
[0615] In a non-limiting embodiment of the present invention,
anticancer agents of the present invention can be used to treat
patients who have cancer which is refractory to treatment with an
immune checkpoint inhibitor. For example, patients with glypican
3-positive cancer, in whom administration of an immune checkpoint
inhibitor has failed to achieve a desired drug efficacy, can be
treated with the anticancer agent of the present invention. In
other words, glypican 3-positive cancer that has been already
treated with therapy using an immune checkpoint inhibitor can be
treated with the anticancer agent of the present invention.
[0616] In a non-limiting embodiment of the present invention,
pharmaceutical compositions (combination therapy) of the present
invention can be used to treat patients who have cancer which is
refractory to treatment with an immune checkpoint inhibitor. For
example, patients with GPC-positive cancer, in whom administration
of an immune checkpoint inhibitor has failed to achieve a desired
drug efficacy, can be treated with the pharmaceutical composition
(combination therapy) of the present invention. In other words,
glypican 3-positive cancer that has been already treated with
therapy using an immune checkpoint inhibitor can be treated with
the pharmaceutical composition (combination therapy) of the present
invention. Preferred examples of another anticancer agent comprised
in the pharmaceutical composition include immune checkpoint
inhibitors, but are not limited thereto.
[0617] In a non-limiting embodiment of the present invention,
pharmaceutical compositions (combination therapy) of the present
invention can be used to treat patients who have cancer which is
refractory to treatment with the anticancer agent of the present
invention. For example, patients with GPC-positive cancer, whose
cancer has become resistant to the anticancer agent of the present
invention after administration of the anticancer agent or in whom
administration of the anticancer agent of the present invention has
failed to achieve a desired drug efficacy, can be treated with the
pharmaceutical composition (combination therapy) of the present
invention. In other words, glypican 3-positive cancer that has been
already treated with therapy using the anticancer agent of the
present invention can be treated with the pharmaceutical
composition (combination therapy) of the present invention.
Preferred examples of another anticancer agent comprised in the
pharmaceutical composition include immune checkpoint inhibitors,
but are not limited thereto.
[0618] With respect to glypican 3-positive cancer (cancer confirmed
to express glypican 3), those skilled in the art can appropriate
examine positivity for glypican 3 using methods known to those
skilled in the art such as immunohistochemical staining or flow
cytometry.
[0619] From another viewpoint, the present invention provides
anticancer agents comprising as the active ingredient a
multispecific antigen-binding molecule that comprises: (1) a domain
comprising an antibody variable region having glypican 3-binding
activity, (2) a domain comprising an antibody variable region
having T-cell receptor complex-binding activity, and (3) a domain
comprising an Fc region with reduced binding activity towards an
Fc.gamma. receptor.
[0620] In the present invention "comprising as the active
ingredient a multispecific antigen-binding molecule that comprises
(1) a domain comprising an antibody variable region having glypican
3-binding activity, (2) a domain comprising an antibody variable
region having T-cell receptor complex-binding activity, and (3) a
domain comprising an Fc region with reduced binding activity
towards an Fc.gamma. receptor" means comprising the antigen-binding
molecule as a major active component, without limitation to the
content ratio of the antigen-binding molecule.
[0621] If necessary, multispecific antigen-binding molecules of the
present invention may be encapsulated in microcapsules (e.g., those
made of hydroxymethylcellulose, gelatin, and
poly(methylmetacrylate)), or incorporated as components of a
colloidal drug delivery system (e.g., liposomes, albumin
microspheres, microemulsion, nanoparticles, and nanocapsules) (see,
for example, "Remington's Pharmaceutical Science 16th edition",
Oslo Ed. (1980)). Methods for preparing the pharmaceutical agents
as controlled-release pharmaceutical agents are also well known,
and such methods may be applied to the multispecific
antigen-binding molecules of the present invention (J. Biomed.
Mater. Res. (1981) 15: 267-277; Chemtech. (1982) 12: 98-105; U.S.
Pat. No. 3,773,719; European Patent Application Publication Nos. EP
58,481 and EP 133,988; Biopolymers (1983) 22: 547-556).
[0622] The pharmaceutical compositions or anticancer agents of the
present invention may be administered to patients by oral or
parenteral administration, and parenteral administration is
preferred. Specific examples of the administration method include
administration by injection, transnasal administration,
transpulmonary administration, and transdermal administration.
Examples of administration by injection include intravenous
injection, intramuscular injection, intraperitoneal injection, and
subcutaneous injection. A pharmaceutical composition or anticancer
agent of the present invention can be administered systemically or
locally, for example, through administration by injection. The
method of administration can be selected appropriately according to
the age and symptoms of the patient. The dose can be selected from
the range of 0.0001 mg to 1000 mg per kilogram body weight for a
single administration. Alternatively, for example, the dose may be
selected from the range of 0.001 mg/body to 100000 mg/body per
patient. The dose may be defined, for example, as the amount of the
multispecific antigen-binding molecule of the present invention
comprised as the active ingredient in the pharmaceutical
composition. However, the pharmaceutical compositions or anticancer
agents of the present invention are not limited to these doses.
[0623] The pharmaceutical compositions or anticancer agents of the
present invention can be formulated according to conventional
methods (for example, Remington's Pharmaceutical Science, latest
edition, Mark Publishing Company, Easton, U.S.A), and may also
contain pharmaceutically acceptable carriers and additives.
Examples include, but are not limited to surfactants, excipients,
coloring agents, perfumes, preservatives, stabilizers, buffers,
suspending agents, isotonization agents, binders, disintegrants,
lubricants, fluidity promoting agents, and flavoring agents; and
other commonly used carriers can be suitably used. Specific
examples of the carriers include light anhydrous silicic acid,
lactose, crystalline cellulose, mannitol, starch, carmellose
calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl
methyl cellulose, polyvinylacetal diethylaminoacetate,
polyvinylpyrrolidone, gelatin, medium chain fatty acid
triglyceride, polyoxyethylene hardened castor oil 60, saccharose,
carboxymethyl cellulose, corn starch, inorganic salt, and such.
[0624] A non-limiting embodiment of the present invention also
provides methods for damaging glypican 3 antigen-expressing cells
or tumor tissues containing the antigen-expressing cells, or
methods for suppressing growth of these cells or tumor tissues by
contacting the glypican 3 antigen-expressing cells with a
multispecific antigen-binding molecule of the present invention
that binds to the antigen and another anticancer agent. The
multispecific antigen-binding molecule that binds to the antigen is
as described above for an antigen-binding molecule of the present
invention that binds to the antigen, which is comprised in the
anticancer agents of the present invention. The cells bound by a
multispecific antigen-binding molecule of the present invention
that binds to the antigen are not particularly limited as long as
they are cells expressing the antigen.
[0625] In the present invention, "contact" is carried out, for
example, by adding a multispecific antigen-binding molecule of the
present invention which binds to the antigen and another anticancer
agent to the culture medium of GPC3 antigen-expressing cells
cultured in vitro. In this case, a liquid or a solid obtained by
freeze-drying or such may be suitably used as the form of the added
antigen-binding molecule and/or another anticancer agent. When
added as an aqueous solution, it may be an aqueous solution that
simply contains only the multispecific antigen-binding molecule of
the present invention, or it may be a solution containing also, for
example, the above-mentioned surfactants, excipients, coloring
agents, perfumes, preservatives, stabilizers, buffers, suspending
agents, isotonization agents, binders, disintegrants, lubricants,
fluidity promoting agents, and flavoring agents. The concentration
at which the addition is performed is not particularly limited, but
a suitable final concentration in the culture solution is
preferably in the range of 1 pg/ml to 1 g/ml, more preferably 1
ng/ml to 1 mg/ml, and even more preferably 1 pg/mL to 1 mg/mL.
[0626] Furthermore, in another embodiment, "contact" of the present
invention is also carried out by administering an antigen-binding
molecule of the present invention and another anticancer agent to
non-human animals with cells expressing the GPC3 antigen
transplanted into their bodies, and to animals carrying cells that
intrinsically express the antigen. The method of administration may
be oral or parenteral, and parenteral administration is
particularly preferred. Specific examples of the administration
method include administration by injection, transnasal
administration, transpulmonary administration, and transdermal
administration. Examples of administration by injection include
intravenous injection, intramuscular injection, intraperitoneal
injection, and subcutaneous injection. A pharmaceutical composition
or anticancer agent of the present invention can be administered
systemically or locally, for example, through administration by
injection. The method of administration can be selected
appropriately according to the age and symptoms of the test animal.
When administered as an aqueous solution, an aqueous solution
containing simply only a multispecific antigen-binding molecule of
the present invention may be used, or a solution containing also
the above-mentioned surfactants, excipients, coloring agents,
perfumes, preservatives, stabilizers, buffers, suspending agents,
isotonization agents, binders, disintegrants, lubricants, fluidity
promoting agents, flavoring agents, and such may be used. The dose
can be selected from the range of 0.0001 mg to 1000 mg per kilogram
body weight for a single administration. Alternatively, for
example, the dose may be selected from the range of 0.001 mg/body
to 100000 mg/body per patient. The dose may be defined, for
example, as the amount of the multispecific antigen-binding
molecule of the present invention comprised as the active
ingredient in the pharmaceutical composition. However, the amount
of the multispecific antigen-binding molecule of the present
invention administered is not limited to these doses.
[0627] The following method is suitably used as a method for
evaluating or measuring cytotoxicity induced in cells expressing
the glypican 3 antigen which is bound by a domain carrying an
antibody variable region having glypican 3-binding activity that
constitutes the antigen-binding molecule as a result of contacting
the cells with a multispecific antigen-binding molecule or another
anticancer agent of the present invention. Examples of a method for
evaluating or measuring the cytotoxic activity in vitro include
methods for measuring cytotoxic T cell activity and such. Whether
or not a multispecific antigen-binding molecule of the present
invention has T cellular cytotoxicity can be measured by known
methods (for example, Current protocols in Immunology, Chapter 7.
Immunologic studies in humans, Editor, John E. Coligan et al., John
Wiley & Sons, Inc., (1993) and the like). For activity
measurements, an antigen-binding molecule that binds to an antigen
different from glypican 3, which is an antigen not expressed in the
cells used for the examination, can be used as a control in the
same manner as a multispecific antigen-binding molecule of the
present invention, and the activity can be determined to be present
when the multispecific antigen-binding molecule of the present
invention shows a stronger cytotoxic activity than when the
antigen-binding molecule is used as a control.
[0628] To evaluate or measure cytotoxic activity in vivo, for
example, cells expressing a glypican 3 antigen are intradermally or
subcutaneously transplanted to a non-human test animal, and then a
test antigen-binding molecule and/or another anticancer agent is
intravenously or intraperitoneally administered daily or with an
interval of few days, starting from the day of transplantation or
the following day. Cytotoxic activity can be determined by daily
measurement of tumor size and by observing difference in the change
of tumor size. In a similar manner to the in vitro evaluation, the
cytotoxic activity of an antigen-binding molecule of the present
invention can be determined to be present when administration of a
control antigen-binding molecule shows that the tumor size in the
group subjected to administration of an antigen-binding molecule of
the present invention is significantly smaller than the tumor size
in the group subjected to administration of the control
antigen-binding molecule.
[0629] As a method for evaluating or measuring the suppressive
effect on proliferation of cells expressing a glypican 3 antigen, a
method of measuring the uptake of isotope-labeled thymidine into
cells or the MTT method may be suitably used. As a method for
evaluating or measuring the cell proliferation-suppressing activity
in vivo, the same method described above for evaluating or
measuring cytotoxic activity in vivo may be suitably used.
Kits
[0630] In several embodiments, the present invention provides a kit
comprising:
(1) the above-described bispecific antibody; (2) a container; and
(3) an instruction or a label indicating that the bispecific
antibody and at least one type of anticancer agent are administered
in combination to a test subject for treating cancer in an
individual.
[0631] In another embodiment, the present invention provides a kit
comprising:
(1) another anticancer agent; (2) a container; and (3) an
instruction or a label indicating that said another anticancer
agent and at least one type of the above-described bispecific
antibody are administered in combination to an individual for
treating cancer in the individual.
[0632] In another embodiment, the present invention provides a kit
comprising:
(1) the above-described bispecific antibody; (2) another anticancer
agent; (3) a container; and (4) an instruction or a label
indicating that the bispecific antibody and the other anticancer
agent are administered in combination to an individual for treating
cancer in the individual.
[0633] In several embodiments, the kit further comprises a
pharmaceutically acceptable carrier. The kit can further comprise a
sterile diluent preferably stored in a separate, additional
container. The kit can also comprise an instruction relating to a
combination therapy for treating or preventing cancer.
[0634] In several embodiments, an "instruction" refers to the
written instruction usually contained in a commercially available
box carrying a pharmaceutical, and can include information on
indications, usage, dose, administration, contraindications, and/or
warnings regarding the use of the pharmaceutical.
[0635] The kits may be those which are used exclusively for the
purpose of combined use of the bispecific antibody of the present
invention and another anticancer agent. Alternatively, the kits may
be those which are used for other purposes as long as they are used
for the purpose of combined use of the bispecific antibody of the
present invention and another anticancer agent. For example, as
long as the instruction or label of the kit of the present
invention indicates that the bispecific antibody and other
anticancer agent are administered in combination to an individual,
the instruction or label may indicate other embodiments, for
example, in which the bispecific antibody or other anticancer agent
is used alone.
[0636] The present invention also relates to molecules having
GPC3-binding activity, which contain a domain comprising an
antibody variable region having GPC3-binding activity of the
multispecific antigen-binding molecule comprised in the anticancer
agent or pharmaceutical composition of the present invention.
Furthermore, the present invention relates to a molecule having
GPC3-binding activity, which comprises the antibody variable
regions of H and L chains respectively comprising the three CDRs of
the H and L chains (total of six CDRs) contained in the molecule.
The present invention also relates to molecules having T-cell
receptor complex-binding activity, which contain a domain
comprising an antibody variable region having T-cell receptor
complex-binding activity of the multispecific antigen-binding
molecule comprised in the anticancer agent or pharmaceutical
composition of the present invention. Furthermore, the present
invention relates to a molecule having T-cell receptor
complex-binding activity that comprises the antibody variable
regions of the H and L chains respectively comprising the three
CDRs of the H and L chains (total of six CDRs) contained in the
molecule. Such molecules may be antibodies or polypeptides
comprising antigen-binding fragments of an antibody. The present
invention also relates to antibodies that bind to epitopes
overlapping or competing with these molecules or polypeptides
containing the antigen-binding fragments thereof. Suitable examples
of such polypeptides comprising antigen-binding fragments of an
antibody include scFv, single chain antibody, Fv, single chain Fv 2
(scFv2), Fab, and F(ab').sub.2. Furthermore, these molecules do not
have to be multispecific (bispecific), and may bind only to either
GPC3 or a T cell receptor complex (for example, the CD3.epsilon.
chain).
[0637] These molecules include a molecule comprising a domain that
comprises an antibody variable region having GPC-binding activity
of the multispecific antigen-binding molecule exemplified in detail
in the Reference Examples herein (which comprises the H-chain
variable regions having GPC3-binding activity and the common
L-chain variable region), a molecule comprising a domain that
comprises an antibody variable region having T cell receptor
complex-binding activity of the multispecific antigen-binding
molecule exemplified in the Reference Examples herein (which
comprises the H-chain variable regions having T cell receptor
complex-binding activity and the common L-chain variable region),
and also a molecule having an activity to bind to the same
antigenic protein (GPC3 or T-cell receptor complex), which
comprises the three CDRs of each of the H and L chains (total of
six CDRs) contained in the above molecule.
[0638] These molecules have CDRs that are in common with those of a
multispecific antigen-binding molecule of the present invention;
and therefore, they are expected to bind to an epitope overlapping
with an epitope for the multispecific antigen-binding molecule of
the present invention. Therefore, these molecules can compete with
multispecific antigen-binding molecules of the present invention
when they coexist with the multispecific antigen-binding molecules
of the present invention. Accordingly, these molecules can be used,
for example, as regulatory agents for suppressing activities (such
as antigen-binding activity, cytotoxic activity, and antitumor
activity) of the multispecific antigen-binding molecules of the
present invention. Furthermore, such a molecule can be bound to a
target protein (GPC3 or T cell receptor complex) in advance, and
when a multispecific antigen-binding molecule of the present
invention is added, the molecules that dissociate through
competition can be detected. This way, the molecule is useful as an
agent for detecting binding of a multispecific antigen-binding
molecule of the present invention to a target protein. Here, such
molecules may be labeled appropriately with fluorescent substances
or such. Alternatively, these molecules are useful for screening
novel antibodies that bind to epitopes overlapping with the
epitopes bound by the multispecific antigen-binding molecules of
the present invention. As described above, such a molecule can be
bound to a target protein (GPC3 or T cell receptor complex) in
advance, and when a test antibody is added, if the bound molecules
dissociate, then the test antibody is a candidate for an antibody
against an epitope overlapping with the epitope bound by the
multispecific antigen-binding molecule of the present invention.
This will enable efficient screening of novel multispecific
antigen-binding molecules.
[0639] The combinations presented as examples herein as
combinations of each CDR of the multispecific antigen-binding
molecules of the present invention can be directly used as specific
combinations of CDRs of the H-chain and L-chain variable regions in
these molecules. The antigen affinity of these molecules (KD
values) is preferably a value exemplified herein as the KD value of
a multispecific antigen-binding molecule of the present invention,
but is not limited thereto.
[0640] In the present invention, the indefinite article "a" or "an"
refers to one, or two or more (i.e., at least one) grammatical
object referred to by the indefinite article. For example, "a
component" refers to one component or two or more components.
[0641] Those skilled in the art will naturally appreciate that any
combinations of one or more of the embodiments described herein are
also included in the present invention as long as they are not
technically inconsistent based on common technical knowledge of
those skilled in the art.
[0642] All prior art documents cited herein are incorporated by
reference into this description.
EXAMPLES
[0643] Hereinbelow, the present invention will be specifically
described with reference to the Examples, but it is not to be
construed as being limited thereto.
[Example 1] T Cell-Dependent Cellular Cytotoxicity (TDCC Activity)
of Each Test Antibody when Human Peripheral Blood Mononuclear Cells
are Used as Effector Cells
[0644] TDCC activity of each test antibody was measured by the
method described below. Human peripheral blood mononuclear cells
(hereinafter referred to as human PBMCs) were used as effector
cells and TDCC activity of each test antibody was measured as
follows.
(1) Preparation of Human PBMC Solution
[0645] 50 ml of peripheral blood was collected from a healthy
person in Chugai Pharmaceutical Co. Ltd. using a syringe pre-loaded
with 500 .mu.l of 5000 units/5 ml heparin solution. The peripheral
blood diluted two-fold with PBS was divided into four aliquots and
added to Leucosep tubes for lymphocyte separation (GE Healthcare)
which had been loaded with 15 ml of Ficoll-Paque PLUS and
centrifuged in advance. The separation tubes containing the
aliquoted peripheral blood were centrifuged at a speed of 1000 g
for 10 minutes at room temperature, and then, the mononuclear cell
layer fraction was collected. After cells contained in each layer
fraction were washed once with RPMI-1640 (SIGMA) containing 10% FBS
(hereinafter referred to as 10% FBS/RPMI-1640), the cells were
suspended at a cell density of 2.times.10.sup.6 cells/ml in culture
medium for each target cell. The cell suspensions were used as
effector cells in subsequent experiments.
(2) LDH Release Test (TDCC Activity)
[0646] TDCC activity was assessed by an LDH release method (LDH
Cytotoxicity Detection Kit, TAKARA). First, an antibody solution
was diluted with each target cell culture medium to concentrations
(0.000004, 0.00004, 0.0004, 0.004, 0.04, 0.4, 4, and 40 .mu.g/ml),
which were four times greater than the final concentrations, and
50-.mu.l aliquots of antibody solutions of respective
concentrations were added to each well of a 96-well round-bottomed
plate. Then, 50-.mu.l aliquots of target cells prepared at
2.times.10.sup.5 cells/ml in culture medium for each target cell
were seeded (1.times.10.sup.4 cells/well) and allowed to stand at
room temperature for 15 minutes. 100-.mu.l aliquots of the human
PBMC suspension prepared in culture medium for each target cell
(2.times.10.sup.5 cells/well) as described in (1) were added to
each well of the plate. The plate was allowed to stand in a 5%
carbon dioxide gas incubator at 37.degree. C. for about 24 hours,
followed by centrifugation. 100 .mu.l of the culture supernatant in
each well of the plate was transferred to a 96-well flat-bottomed
plate. A catalyst solution was dissolved in 1 ml of H.sub.2O and
mixed with a dye solution at ratio of 1:45. The mixed solution of
catalyst and dye solutions was aliquoted at 100 .mu.l/well to the
96-well flat-bottomed plate where the culture supernatants had been
transferred. The plate was allowed to stand at room temperature for
15 to 30 minutes. The absorbance at 490 to 492 nm was measured with
a plate reader. The reference wavelength used was 600 to 620 nm,
and the absorbance was subtracted from that at 490 to 492 nm.
Values obtained by subtracting the mean value for wells containing
culture medium alone (blank) were plugged into the following
equation.
Cytotoxicity (TDCC) (%)=((A-B)-C)).times.100/(D-C) Equation:
Cytotoxic Activity was Determined Based on this Equation.
[0647] Here, A represents the absorbance of a mixture of target
cells, effector cells, and antibody; B represents the absorbance of
effector cells; C represents the absorbance of target cells; and D
represents the absorbance of target cells with Triton X-100
added.
[0648] As a result, TDCC was clearly observed for an anti-human
CD3.epsilon. chain and anti-human GPC3 bispecific antibody (the
antibody of sample No. 38 described in Table 17 of Reference
Example 3) (FIG. 1).
[Example 2] Determination of the Amount of GPC3 Expressed on Cell
Surface in Each Cell Line
[0649] Antibody binding capacity (ABC) of GPC3 on cell surface was
calculated for each cell line by flow cytometry using QIFIKIT
(DAKO).
[0650] After washing with CellWASH (BD Bioscience) supplemented
with 0.5% BSA (hereinafter referred to as FACS/PBS),
5.times.10.sup.5 cells of each cell line were prepared in 50 .mu.l
of a solution containing mouse anti-human GPC antibody or control
antibody at a final concentration of 20 .mu.g/ml and allowed to
stand on ice for 30 minutes. The cells were washed with FACS/PBS.
Then, 50 .mu.l of a solution containing FITC-labeled goat
anti-mouse IgG antibody which had been diluted 50-fold with
FACS/PBS was added to the cells. The cells were allowed to stand on
ice for 30 minutes. After washing with FACS/PBS, the cells were
analyzed by flow cytometry. ABC was calculated by the method
described in the instruction manual of QIFI KIT.
[0651] ABC of GPC3 on cell surface was calculated for each cell
line and is shown in Table 6.
TABLE-US-00007 TABLE 6 Cell name Tissue Histology ABC MKN-74
Gastric Adenocarcinoma 2.97E3 FU-97 Gastric Adenocarcinoma 5.52E5
SNU-1 Gastric Adenocarcinoma 1.85E4 STM-03 Gastric Adenocarcinoma
Not applicable (PDX) SCC152 Hypopharynx Squamous carcinoma 3.77E4
KYSE70 Esophageal Squamous carcinoma 5.35E4 PC-10 Lung Squamous
carcinoma 1.21E5 NCI-H446 Lung Small cell carcinoma 6.95E4 huH-1
Liver Carcinoma 5.66E4 HuH-7 Liver Carcinoma 4.85E4 RMG-1 Ovary
Adenocarcinoma 3.00E2 TOV-21G Ovary Clear cell 1.15E4
adenocarcinoma MDA-MB-134VI Breast Carcinoma, ductal 1.07E4
carcinoma HCC1419 Breast Carcinoma, ductal 1.76E4 carcinoma C2BBe1
Colorectal Adenocarcinoma 5.77E4 RCC-HB Kidney Adenocarcinoma,
1.03E4 clear cell WM-115 Skin Melanoma 1.37E3 VMRC-MELG Skin
Melanoma 1.58E3 SJCRH30 Striated muscle Sarcoma 9.26E3 BxPC-3
Pancreas Adenocarcinoma 1.06E3 Shmac 4 Prostate Carcinoma 3.46E3
NTERA-2 Testis Teratocarcinoma 7.60E3 C-33 A Uterine cervix
Carcinoma 2.10E3
[Example 3] Evaluation of In Vivo Drug Efficacy (Anti-Human
CD3.epsilon. Chain and Anti-Human GPC3 Bispecific Antibody) (Drug
Efficacy Evaluation Studies when Antibodies of Sample Nos. 30, 31,
32, 33, and 38, or Antibodies of Sample Nos. 39 and 40 Described in
Table 17 of Reference Example 3 are Used as a Single Agent)
[0652] In vivo drug efficacy was evaluated using tumor-bearing
models of some of the lines in which cytotoxic activity was
observed by the in vitro assay described in Example 1 as well as in
vivo-passaged lines.
[0653] Methods called an NOD scid/T cell-injected model, a
humanized NOG mouse model, and a human CD3.epsilon.6.gamma.
gene-modified mouse model were used in the in vivo drug efficacy
evaluation. Assay using an NOD scid/T cell-injected model was
performed as follows. Some of the lines in which cytotoxic activity
was observed in the in vitro assay and in vivo-passaged cell lines
were transplanted into NOD scid mice. T cells expanded by culturing
human PBMCs in vitro were injected into the NOD scid mice with
confirmed tumor establishment. The mice were treated by
administering bispecific antibody-38 and bispecific antibody-30,
-31, -32, or -33. Assay using a humanized NOG mouse model was
performed as follows. CD34-positive hematopoietic stem cells
derived from umbilical cord blood were transplanted into NOG mice
by tail vein injection. Human T cells are constantly supplied in
the mice, which are called humanized NOG mice. The PC-10 cell line
was transplanted into humanized NOG mice. The humanized NOG mice
with confirmed establishment of PC-10 tumor were treated by
administering antibody-38. Assay using a human CD3.epsilon.6.gamma.
gene-modified mouse model was performed as follows. Cells of
mouse-derived cell line forced to express human GPC3 were
transplanted into a human CD3.epsilon.6.gamma. gene-modified mouse
model, which had been prepared according to Reference Example 10.
The human CD3.epsilon.6.gamma. gene-modified mice with confirmed
tumor establishment were treated by administering antibody-38.
[0654] The drug efficacy evaluation studies for bispecific
antibody-38, and bispecific antibody-30, -31, -32, and -33 in NOD
scid/T cell-injected model were performed as follows. T cell
expansion culture was performed using PBMCs separated from blood
collected from a healthy person and T cell activation/expansion
kit/human. Each of human cancer cell lines (MKN74, FU-97, SNU-1,
SCC152, KYSE70, PC-10, HuH-7, TOV-21G, RMG-1, and SK-pca31a
(SK-HEP-1/hGPC3)) was mixed with Matrigel.TM. Basement Membrane
Matrix, and transplanted subcutaneously into NOD scid mice (CLEA
Japan). An in vivo-passaged line (STM-03) was transplanted as about
2-mm tumor tissue cubes subcutaneously into NOD scid mice (CLEA
Japan). The day of transplantation was defined as Day 0. On the day
before transplantation, anti-asialo GM1 antibody (Wako Pure
Chemical Industries) was administered intraperitoneally at 0.2
mg/mouse. When tumor was clearly established after transplantation,
the mice were separated into groups based on tumor size and body
weight. Then, anti-asialo GM1 antibody was administered
intraperitoneally at 0.2 mg/mouse again. On the following day, T
cells obtained by the aforementioned expansion culture were
transplanted intraperitoneally at 1.5.times.10.sup.7 to
3.times.10.sup.7 cells/mouse. About two to five hours after T cell
transplantation, antibody-38 was administered at 1 mg/kg (MKN74,
FU-97, SNU-1, SCC152, KYSE70, PC-10, HuH-7, TOV-21G, RMG-1, and
STM-03) through the tail vein. The administration was performed
only once. Bispecific antibody-30, -31, -32, and -33 were
administered at 5 mg/kg (SK-pca31a) through the tail vein. The
administration was performed only once.
[0655] Bispecific antibody-39 and -40 were administered at 1 mg/kg
(PC-10) through the tail vein. The administration was performed
only once. Antibody-39 and -40 used in the experiment have
different constant region sequences, but they share the same amino
acid sequences of the CD3-binding variable region, GPC3-binding
variable region, and common L chain variable region (CD3-binding
variable region: SEQ ID NO: 433, GPC-binding variable region: SEQ
ID NO: 434, common L chain variable region: SEQ ID NO: 435). The
antibodies were produced by a method known to those skilled in the
art.
[0656] As a result, bispecific antibody-38 produced an evident
anti-tumor effect as compared to the vehicle-administered group
(FIG. 2).
[0657] As a result, bispecific antibody-39 and -40 produced an
evident anti-tumor effect as compared to the vehicle-administered
group (FIG. 3).
[0658] As a result, bispecific antibody-30, -31, -32, and -33
produced an evident anti-tumor effect as compared to the
vehicle-administered group (FIG. 4).
[0659] The drug efficacy evaluation study for antibody-38 in the
humanized NOG mouse model was performed as follows. 2.5 Gy of X ray
was irradiated to NOG mice (In-Vivo Science Inc., Y). On the
following day, 1.times.10.sup.5 CD34-positive hematopoietic stem
cells which are derived from umbilical cord blood were transplanted
to the NOG mice by tail vein injection. After 16 weeks, human PC-10
cancer cells were mixed with Matrigel.TM. Basement Membrane Matrix,
and transplanted subcutaneously into the humanized NOG mice. The
day of transplantation was defined as Day 0. When tumor was clearly
established, the mice were separated into groups based on tumor
size and body weight. Antibody-38 was administered at 0.008, 0.04,
0.2, or 1 mg/kg through the tail vein. Antibody-38 was administered
only once.
[0660] As a result, an evident, dose-dependent anti-tumor effect
was produced in the antibody-38-administered group as compared to
the vehicle-administered group (FIG. 5).
[0661] The drug efficacy evaluation study for antibody-38 in assay
using a human CD3.epsilon..delta..gamma. gene-modified mouse model
was performed as follows. LLC1/hGPC3 cancer cells, which are
mouse-derived cancer cell line with over-expression of human GPC3,
were transplanted subcutaneously into human
CD3.epsilon..delta..gamma. gene-modified mice (Chugai
Pharmaceutical Co. Ltd., Y). The day of transplantation was defined
as Day 0. On Day 11, the mice were separated into groups based on
tumor size and body weight. Antibody-38 was administered at 5 mg/kg
through the tail vein. Antibody-38 was administered twice (Day 11
and Day 14). An anti-human GPC3 antibody (WO2006/006693, clone
name: GC33), anti-mouse CTLA-4 antibody (BioXCell, Catalog
#BE0032), anti-mouse PD-1 antibody (BioXCell, Catalog #BE0146), and
anti-mouse PD-L1 antibody (BioXCell, Catalog #BE0101), which were
pharmaceutical agents used as controls, were administered at 25
mg/kg through the tail vein. The anti-human GPC3 antibody,
anti-mouse CTLA-4 antibody, anti-mouse PD-1 antibody, and
anti-mouse PD-L1 antibody were administered twice (Day 11 and Day
14).
[0662] As a result, antibody-38 produced an evident anti-tumor
effect as compared to the solvent-administered group, or the
anti-human GPC3 antibody, anti-mouse CTLA-4 antibody, anti-mouse
PD-1 antibody, and anti-mouse PD-L1 antibody (FIG. 6).
[Example 4] Evaluation of In Vivo Drug Efficacy (Drug Efficacy
Evaluation Study for Combination of Antibody-38 and Other
Agents)
[0663] In vivo drug efficacy evaluation study for combination of
antibody-38 and other agents was carried out using tumor-bearing
models. In vivo drug efficacy by the combination was evaluated
using the NOD scid/T cell-injected model described in Example 3,
the human CD3.epsilon..delta..gamma. gene-modified mouse model
described in Example 3, or a human CD3.epsilon. gene-modified mouse
model. Combination study using the NOD scid/T cell-injected model
was performed as follows. MKN45 or NCI-H446 cell line was
transplanted into NOD scid mice. T cells expanded by culturing
human PBMCs in vitro were injected into the NOD scid mice with
confirmed evident tumor establishment. The mice were treated by
administering antibody-38 in combination with capecitabine,
cisplatin, or paclitaxel. Combination study using the human
CD3.epsilon.6.gamma. gene-modified mouse model was performed as
follows. LLC1/hGPC3 cancer cells or Hepa1-6/hGPC3 cells, which are
mouse-derived cell lines forced to express human GPC3, were
transplanted into human CD3.epsilon..delta..gamma. gene-modified
mice. The human CD3.epsilon..delta..gamma. gene-modified mice with
confirmed evident tumor establishment were treated by administering
antibody-38 in combination with an anti-mouse TIM-3 antibody
(BioXCell, Catalog #BE0115), anti-mouse LAG-3 antibody (BioXCell,
Catalog #BE0174), anti-mouse CD137 antibody (BioXCell, Catalog
#BE0169), or anti-mouse VEGFR2 antibody (BioXCell, Catalog
#BP0060). Combination study using a human CD3.epsilon.
gene-modified mouse model was performed as follows. Hepa1-6/hGPC3
cancer cells, which are a mouse-derived cancer line forced to
express human GPC3, were transplanted into human CD3.epsilon.
gene-modified mice. The human CD3.epsilon. gene-modified mice with
confirmed evident tumor establishment were treated by administering
antibody-38 in combination with an anti-mouse PD-1 antibody
(BioXCell, Catalog #BE0146) or anti-mouse PD-L1 (BioXCell, Catalog
#BE0101) antibody.
[0664] The drug efficacy evaluation study for antibody-38 and other
agents using the NOD scid/T cell-injected model was performed as
follows. T cell expansion culture was performed using PBMCs
separated from blood collected from a healthy person and T cell
activation/expansion kit/human. Each of human cancer cell lines
(MKN45 and NCI-H446) was mixed with Matrigel.TM. Basement Membrane
Matrix, and transplanted subcutaneously into NOD scid mice (CLEA
Japan). The day of transplantation was defined as Day 0. On the day
before transplantation, anti-asialo GM1 antibody was administered
intraperitoneally at 0.2 mg/mouse. When evident tumor establishment
was confirmed after transplantation, the mice were separated into
groups based on tumor size and body weight. Then, anti-asialo GM1
antibody was administered intraperitoneally at 0.2 mg/mouse again.
On the following day, T cells obtained by the aforementioned
expansion culture were transplanted intraperitoneally at
3.times.10.sup.7 cells/mouse. About two to five hours after T cell
transplantation, antibody-38 was administered. In combination with
antibody-38, capecitabine, cisplatin, or paclitaxel was
administered according to the dose and dosing regimen shown in
Table 7.
TABLE-US-00008 TABLE 7 Pharmaceutical agent for use Pharmaceutical
Pharmaceutical in combination agent for use agent for use Dose and
in combination Antibody-38 Antibody-38 Cell line in combination
administration route Dosing regimen Dose Dosing regimen MKN45
capecitabine 431 mg/kg Repetive 5 mg/kg, Single Oral administration
Tail vein administration administration for five days
administration Day 14 from Day 17 cisplatin 7.5 mg/kg Single 1
mg/kg, Single Tail vein administration Tail vein administration
administration Day 13 administration Day 14 paclitaxel 20 mg/kg
Single 1 mg/kg, Single Tail vein administration Tail vein
administration administration Day 13 administration Day 14 NCI-H446
cisplatin 7.5 mg/kg Single 1 mg/kg, Single Tail vein administration
Tail vein administration administration Day 14 administration Day
15 paclitaxel 20 mg/kg Days 14 and 21 1 mg/kg, Single Tail vein
Tail vein administration administration administration Day 15
[0665] As a result, the anti-tumor effect was enhanced in the
combination groups to which antibody-38 and the pharmaceutical
agent for use in combination were administered as compared to
groups where antibody-38 or the pharmaceutical agent for use in
combination was used alone (FIGS. 7 and 8).
[0666] RNA was extracted from tumor samples after administration of
paclitaxel, cisplatin, or capecitabine and antibody-38.
Comprehensive RNA analysis using nCounter, tumor infiltrating
lymphocyte (TIL) analysis, and pathological analysis were performed
as follows.
[0667] The tumor samples were prepared as follows. The tumor
samples when antibody-38 was used in combination with a
chemotherapeutic agent in the humanized NOG mouse model were
prepared by the procedure described below. 2.5 Gy of X ray was
irradiated to NOG mice (In-Vivo Science Inc., Y). On the following
day, 1.times.10.sup.5 CD34-positive hematopoietic stem cells which
are derived from umbilical cord blood were transplanted into the
NOG mice by tail vein injection. After 16 to 20 weeks, each of
human cancer cell lines (MKN45 and NCI-H446) was mixed with
Matrigel.TM. Basement Membrane Matrix, and transplanted
subcutaneously into the humanized NOG mice. The day of
transplantation was defined as Day 0.
[0668] When tumor was established, the mice were separated into
groups based on tumor size and body weight. Antibody-38 and the
pharmaceutical agents for use in combination were administered
according to the dose and dosing regimen described in Table 21. The
mice were euthanized at the timing described in Table 21, and tumor
was excised and preserved for use in tumor infiltrating lymphocyte
(TIL) analysis, pathological analysis, or RNA analysis.
TABLE-US-00009 TABLE 21 Pharmaceutical agent for use Pharmaceutical
Pharmaceutical in combination agent for use Antibody-38 agent for
use Dose and in combination Antibody-38 Dosing Cell line in
combination administration route Dosing regimen Dose regimen Tumor
sampling Analysis MKN45 capecitabine 359 mg/kg Repetive 5 mg/kg
Single 14th day from RNA Oral administration Tail vein
administration the start of administration for five days
administration Day 22 capecitabine from Day 21 administration NCI-
cisplatin 7.5 mg/kg Single 5 mg/kg Single Sixth day after TIL H446
Tail vein administration Tail vein administration antibody-38
administration Day 20 administration Day 21 administration
paclitaxel 20 mg/kg Day 20 5 mg/kg Single Sixth day after TIL, Tail
vein Tail vein administration antibody-38 RNA, administration
administration Day 21 administration Pathology
[0669] RNA analysis was performed as follows. RNA was extracted
(miRNeasy Mini Kit, QIAGEN) from the above-described tumor samples
and the RNA concentrations were determined (NanoDrop, Thermo Fisher
Scientific). 100 ng of RNA was subjected to comprehensive (human)
RNA expression analysis using nCounter PanCancer Pathway Panel and
PanCancer Immune Profiling Panel (NanoStrng). Normalization was
performed using the housekeeping gene included in the Panel. The
analysis software used was nSolver (NanoStrng).
[0670] As a result, the expressions of immune cell markers,
chemokines, cytokines, genes involved in cell death, genes involved
in cell cycle regulation were increased and the expressions of
genes involved in the progress of cell cycle were suppressed when
antibody-38 was used in combination with a chemotherapeutic agent,
paclitaxel or capecitabine, as compared to the group to which
antibody-38 or the chemotherapeutic agent was administered alone
(FIG. 35-1 to 35-6).
[0671] TIL analysis was performed as follows. Tumor tissues which
were dissected from mice transplanted with NCI-H446 described in
Table 21 on the sixth day after antibody-38 administration were
dissociated into cells by enzyme treatment using gentle MACS.TM.
Octo Dissociator. The cells were labeled with CD45, CD3, CD4, CD8,
and GZMB antibodies and examined for their positive rates in each
fraction of TIL using BD LSRFortessa X-20.
[0672] As a result, cell populations expressing T cell markers,
activated T cell markers, and proteins involved in cytotoxic
activity were increased when antibody-38 was used in combination
with a chemotherapeutic agent, paclitaxel or capecitabine, as
compared to the group to which antibody-38 or the chemotherapeutic
agent was administered alone (FIGS. 36-1 and 36-2).
[0673] With respect to histological analysis of tumor tissues,
tumor samples from mice six days after administration of
antibody-38 and paclitaxel were fixed in 10% formaldehyde neutral
buffer solution. Then, HE-stained samples were prepared according
to a conventional method, and histopathological evaluation was
performed using a light microscope.
[0674] As a result, the immune cell infiltration was observed
around the tumor periphery when antibody 38 was administered, and
the infiltration area was expanded when antibody-38 was used in
combination with paclitaxel.
[0675] The drug efficacy evaluation study for antibody-38 and other
agents using the human CD3.epsilon.6.gamma. gene-modified mouse
model was performed as follows. Hepa1-6/hGPC3 and LLC1/hGPC3 cancer
cell lines, which are a mouse cancer cell line with over-expression
of human GPC3, were transplanted subcutaneously into human
CD3.epsilon..delta..gamma. gene-modified mice (Chugai
Pharmaceutical Co. Ltd.). The day of transplantation was defined as
Day 0. When evident tumor establishment was confirmed, the mice
were separated into groups based on tumor size and body weight.
Treatment was performed by administering antibody-38 in combination
with the anti-mouse TIM-3 antibody, anti-mouse LAG-3 antibody,
anti-mouse CD137 antibody, or anti-mouse VEGFR2 antibody. The dose
of each pharmaceutical agent that was used in combination with
antibody-38 is shown in Table 8.
TABLE-US-00010 TABLE 8 Pharmaceutical agent for use Pharmaceutical
Pharmaceutical in combination agent for use Antibody-38 agent for
use Dose and in combination Antibody-38 Dosing Cell line Model in
combination administration route Dosing regimen Dose regimen Hepa1-
Human TIM3 Ab 10 mg/kg Day 13 0.2 mg/kg, Day 13 6/hGPC3
CD3.epsilon..delta..gamma. (Clone Intravenous Single Tail vein
Single gene- RMT 3-23) administration administration administration
administration modified LAG-3 Ab 10 mg/kg Day 13 0.2 mg/kg, Day 13
mouse (Clone Tail vein Single Tail vein Single C9B7W)
administration administration administration administration CD137
Ab 10 mg/kg Day 13 0.2 mg/kg, Day 13 (Clone Tail vein Single Tail
vein Single L0B12.3) administration administration administration
administration VEGFR2 Ab 10 mg/kg Day 13 0.2 mg/kg, Day 13 (Clone
Tail vein Single Tail vein Single DC101) administration
administration administration administration LLC1/hGPC3 Human CD137
Ab 10 mg/kg Days 11 5 mg/kg, Days 11 CD3.epsilon..delta..gamma.
(Clone Intraperitoneal and 15 Tail vein and 15 gene- L0B12.3)
administration administration modified mouse
[0676] As a result, the anti-tumor effect was enhanced in the
combination groups to which antibody-38 and a pharmaceutical agent
for use in combination were administered as compared to the groups
where antibody-38 or the pharmaceutical agent for use in
combination was used alone (FIGS. 9 and 10).
[0677] The drug efficacy evaluation study for antibody-38 with
other agents in the human CD3.epsilon. gene-modified mouse model
was performed as follows. Hepa1-6/hGPC3 cancer cell line, which is
a mouse cancer cell line with over-expression of human GPC3, was
transplanted subcutaneously into human CD3.epsilon. gene-modified
mice (Chugai Pharmaceutical Co. Ltd.). The day of transplantation
was defined as Day 0. On Day 15, the mice were separated into
groups based on tumor size and body weight. Treatment was performed
by administering antibody-38 in combination with the anti-mouse
PD-1 antibody or anti-mouse PD-L1 antibody.
[0678] The dose of each pharmaceutical agent that was used in
combination with antibody-38 is shown in Table 9.
TABLE-US-00011 TABLE 9 Pharmaceutical agent for use Pharmaceutical
Pharmaceutical in combination agent for use Antibody-38 agent for
use Dose and in combination Antibody-38 Dosing Cell line Model in
combination administration route Dosing regimen Dose regimen Hepa1-
Human PD-1 Ab 200 .mu.g/mouse Days 15 5 mg/kg, Days 15 6/hGPC3
CD3.epsilon. (clone Tail vein and 18 Tail vein and 18 gene-
RMP1-14) administration administration modified PD-L1 Ab 200
.mu.g/mouse Days 15 5 mg/kg, Days 15 mouse (clone Tail vein and 18
Tail vein and 18 10F.9G2) administration administration
[0679] As a result, the anti-tumor effect was enhanced in the
combination groups to which antibody-38 and a pharmaceutical agent
for use in combination were administered as compared to groups
where antibody-38 or the pharmaceutical agent for use in
combination was used alone (FIG. 11).
REFERENCE EXAMPLES
[Reference Example 1] Production of GPC3_ERY22_rCE115 and
Measurement of Cytotoxic Activity
(1-1) Production of GPC3_ERY22_rCE115
[0680] A molecule in which one of the Fabs has been replaced with a
CD3 epsilon-binding domain was produced using IgG against a cancer
antigen (GPC3) as the basic structure. In this case, the IgG Fc
used as the basic structure was a silent Fc with attenuated
affinity for FcgR (an Fc .quadrature. (Fc gamma) receptor). An
anti-GPC3 antibody, H0000 (SEQ ID NO: 40)/GL4 (SEQ ID NO: 41), was
used as the GPC3-binding domain. An anti-CD3 antibody,
rCE115H/rCE115L (SEQ ID NO: 42/SEQ ID NO: 43), was used as the
CD3-binding domain.
[0681] G1d produced by removing Gly and Lys at the C terminus of
IgG1 was used as the antibody H-chain constant region, and this was
used in combination with H0000/GL4 and rCE115H/rCE115L. When the
antibody H-chain constant region was named H1, the sequence
corresponding to the H chain of the antibody carrying H0000 in the
variable region was shown as H0000-H1. Here, an amino acid
alteration was shown, for example, as D356K. The first alphabet
(corresponding to D in D356K) is the one-letter code representation
for the amino acid residue before modification, the number that
follows (corresponding to 356 of D356K) is the position of
modification indicated by EU numbering, and the final alphabet
(corresponding to K of D356K) is the one-letter code representation
for the amino acid residue after modification. Gldh (SEQ ID NO: 44)
produced by removing Gly and Lys at the C terminus of IgG1,
ERY22_Hk (SEQ ID NO: 45) produced by introducing the
L234A/L235A/Y349C/T366W mutations into Gldh, and ERY22_Hh (SEQ ID
NO: 46) produced by introducing the
L234A/L235A/D356C/T366S/L368A/Y407V mutations into Gldh were
prepared according to the method of Reference Example 5. The L234A
and L235A mutations were introduced into the respective H chains to
attenuate affinity for FcgR (an Fc Q receptor), and the Y349C/T366W
and D356C/T366S/L368A/Y407V mutations were introduced to
efficiently form heteromers of each H chain when producing
heterodimeric antibodies comprising two types of H chains.
[0682] The heterodimeric antibody, GPC3_ERY22_rCE115, produced by
substitution with the VH and VL domains of Fab against GPC3 was
prepared according to Reference Example 5 (FIG. 12a).
[0683] A series of expression vectors inserted with a
polynucleotide encoding each of GL4-ERY22_Hk (SEQ ID NO: 47),
H0000-ERY22_L (SEQ ID NO: 48), rCE115H-ERY22_Hh (SEQ ID NO: 49),
and rCE115L-k0 (SEQ ID NO: 50) were produced by methods well-known
to those skilled in the art, such as PCR methods using primers
added with an appropriate sequence similar to those in the
above-described method.
[0684] The following combination of expression vectors were
introduced into FreeStyle 293-F cells for transient expression of
each target molecule.
[0685] Target molecule: GPC3_ERY22_rCE115
[0686] Polypeptides encoded by the polynucleotides inserted into
the expression vectors: GL4-ERY22_Hk, H0000-ERY22_L,
rCE115H-ERY22_Hh, rCE115L-k0
(1-2) Purification of GPC3_ERY22_rCE115
[0687] The obtained culture supernatant was added to an anti-FLAG
M2 column (Sigma), and then the column was washed, followed by
elution using 0.1 mg/mL of a FLAG peptide (Sigma). The fractions
containing the molecule of interest were added to a HisTrap HP
column (GE Healthcare), and then the column was washed, followed by
elution using an imidazole concentration gradient. Fractions
containing the molecule of interest were concentrated using an
ultrafiltration membrane, then the fractions were added to a
Superdex 200 column (GE Healthcare), and each of the purified
molecules of interest was obtained by collecting only the monomeric
fractions from the eluted solution.
(1-3) Measurement of the Cytotoxic Activity of GPC3_ERY22_rCE115
Using Human Peripheral Blood Mononuclear Cells
[0688] The in vitro cytotoxic activity of GPC3_ERY22_rCE115 was
assessed.
(1-3-1) Preparation of a Human Peripheral Blood Mononuclear Cell
(PBMC) Solution
[0689] Using a syringe preloaded with 100 .mu.L of 1,000 units/mL
heparin solution (Novo Heparin for injection, 5000 units, Novo
Nordisk), 50 mL of peripheral blood was collected from each healthy
volunteer (adult individual). This peripheral blood was diluted
two-fold in PBS(-), divided into four aliquots, and added into a
Leucosep tube for lymphocyte separation (Cat. No. 227290, Greiner
Bio-One) that had been loaded with 15 mL of Ficoll-Paque PLUS and
subjected to centrifugation in advance. This separation tube was
centrifuged (at 2150 rpm for ten minutes at room temperature), and
then the mononuclear cell fraction was collected. The cells in the
mononuclear cell fraction were washed once with the Dulbecco's
Modified Eagle's Medium containing 10% FBS (manufactured by SIGMA,
hereinafter referred to as 10% FBS/D-MEM), and then prepared to
have a cell density of 4.times.10.sup.6 cells/mL using 10%
FBS/D-MEM. The cell suspension prepared this way was used as the
human PBMC solution in the experiment below.
(1-3-2) Measurement of Cytotoxic Activity
[0690] Cytotoxic activity was assessed by the rate of cell
proliferation inhibition using the xCELLigence Real-Time Cell
Analyzer (Roche Diagnostics). The NCI-H446 human cancer cell line
or the PC-10 human cancer cell line, which expresses human GPC3,
was used as the target cell. NCI-H446 or PC-10 was detached from
the dish, then the cells were plated into E-Plate 96 (Roche
Diagnostics) in aliquots of 100 L/well by adjusting the cells to
1.times.10.sup.4 cells/well, and measurement of live cells was
begun using the xCELLigence Real-Time Cell Analyzer. On the
following day, the plate was removed from the xCELLigence Real-Time
Cell Analyzer, and 50 .mu.L of the respective antibodies prepared
at each concentration (0.004, 0.04, 0.4, 4, or 40 nM) were added to
the plate. After 15 minutes of reaction at room temperature, 50
.mu.L of the human PBMC solution prepared in (1-2) was added
(2.times.10.sup.5 cells/well), and measurement of live cells was
begun by setting the plate into the xCELLigence Real-Time Cell
Analyzer again. The reaction was carried out under the conditions
of 5% carbon dioxide gas at 37.degree. C., and from the Cell Index
value obtained 72 hours after addition of the human PBMC, the cell
proliferation inhibition rate (%) was determined using the equation
below. The Cell Index value used in the calculation was a
normalized value where the Cell Index value immediately before
antibody addition was defined as 1.
Cell proliferation inhibition rate (%)=(A-B).times.100/(A-1)
[0691] A represents the mean value of the Cell Index values in
wells without antibody addition (containing only the target cells
and human PBMCs), and B represents the mean value of the Cell Index
values in each well. The examinations were performed in
triplicate.
[0692] When peripheral blood mononuclear cells (PBMCs) prepared
from human blood were used as the effector cell to measure the
cytotoxicity of GPC3_ERY22_rCE115, a very strong activity was
observed (FIG. 13).
[Reference Example 2] Humanization of the H Chain of the Anti-CD3
Antibody, rCE115, and Sharing of a Common L Chain
[0693] (2-1) Design of hCE115HA, the Humanized rCE115 H-Chain
Variable Region
[0694] The H-chain variable region of the rCE115 anti-CD3 antibody
(SEQ ID NO: 42) was humanized. CDR and FR were determined as
defined by Kabat (Kabat numbering).
[0695] First, a human FR sequence was selected by comparing the
human antibody variable region sequences in a database to the
rCE115 rat variable region sequence. The IMGT Database
(http://www.imgt.org/) and NCBI GenBank
(http://www.ncbi.nlm.nih.gov/genbank/) were used for the database.
A humanized H-chain variable region sequence was designed by
linking the H-chain CDR sequence of the rCE115 variable region with
the selected human FR sequence. This yielded a humanized H-chain
variable region sequence, hCE115HL (SEQ ID NO: 51).
[0696] The amino acid residue at position 93 indicated by Kabat
numbering is Ala in the selected human H-chain FR3 sequence, but is
Arg in the rCE115 variable region sequence. Using the database of
rat and human germline sequences (IMGT Database
(http://www.imgt.org/)), only few sequences were found to contain
Arg at this site. It is reported that the amino acid residue at
position 94 indicated by Kabat numbering contributes to
stabilization of the antibody structure by upper core formation
(Ewert et al. Methods. 2004 October; 34(2):184-99). Based on such
information, a humanized H-chain variable region sequence, in which
the amino acid residues at Kabat positions 93 and 94 in the H-chain
FR3 were substituted with those residues present in the rCE115
variable region sequence, was newly designed. This was the
humanized H-chain variable region sequence, hCE115HA (SEQ ID NO:
52).
(2-2) Design of the Common L Chain, L0000, for the rCE115 Anti-CD3
Antibody and the Anti-GPC3 Antibody
[0697] The FR/CDR shuffling of the L-chain variable region rCE115L
(SEQ ID NO: 43) of the rCE115 anti-CD3 antibody and the L-chain
variable region GL4 (SEQ ID NO: 41) of the anti-GPC3 antibody was
performed.
[0698] The FR sequence of GL4 was selected as the L-chain FR
sequence. L-chain CDR2 was the same for rCE115L and GL4. The
L-chain CDR1 was selected from the CDR sequences of GL4, and the
L-chain CDR3 was selected from the CDR sequences of rCE115L,
respectively. Furthermore, the L-chain CDR3 produced by
substituting the amino acid residue Asp at Kabat position 94 of the
selected L-chain CDR3 with the Val residue present in GL4 was newly
designed.
[0699] A humanized L chain variable region sequence was designed by
linking FR and CDR selected above. This yielded a humanized L-chain
variable region sequence, L0000 (SEQ ID NO: 53).
(2-3) Evaluation of the Affinity for Human GPC3
[0700] The activity to bind human GPC3 when using GL4 (SEQ ID NO:
41) and L0000 (SEQ ID NO: 53) as the L-chain variable regions was
evaluated. This was performed using the molecular form of a
single-arm antibody having a single Fab at the Fc region of a human
IgG1 heterodimerized by the knobs-into-hole technique. H0000 (SEQ
ID NO: 40) was used for the anti-GPC3 antibody H-chain variable
region.
[0701] The affinity and binding rate constants of an anti-GPC3
antibody for an antigen were measured by the multi-cycle kinetics
method of a surface plasmon resonance assay using Biacore.TM.-T200
(GE Healthcare Japan). HBS-EP+ (GE Healthcare Japan) was used for
the running buffer, and an amine coupling kit (GE Healthcare Japan)
was used to covalently bind Protein A/G to the CM5 chip
(carboxymethyl dextran-coated chip). Each anti-GPC3 antibody was
prepared so that approximately 100 RU will be captured by Protein
A/G. Human GPC3 used as the analyte was prepared at 8, 16, 32, 64,
and 128 nM using HBS-EP+. Measurements were carried out by first
allowing Protein A/G to capture the antibody solution, and then
injecting the human GPC3 solution at a flow rate of 30 .mu.L/min
for three minutes to allow reaction to take place. Then, the
solution was switched to HBS-EP+ and the dissociation phase was
measured for 15 minutes. After completion of the dissociation phase
measurement, the sensor chip was regenerated by washing with 10 mM
Gly-HCl at pH 1.5. Measurement at the concentration of 0 was
similarly carried out by allowing Protein A/G to capture the
antibody solution, performing a three-minute HBS-EP+ injection to
allow reaction to take place, and then switching to HBS-EP+ to
measure the dissociation phase for 15 minutes. After completion of
the dissociation phase measurement, the sensor chip was regenerated
by washing with 10 mM Gly-HCl at pH 1.5. A data analysis software
exclusively for Biacore, Biacore T200 Evaluation Software Version
1.0, was used to perform kinetic analyses to calculate the binding
rate constant (ka), dissociation rate constant (kd), and the rate
constant ratio from the obtained sensorgrams. The results are shown
in Table 10.
TABLE-US-00012 TABLE 10 Variable region H-chain L-chain Affinity
for human GPC3 variable region variable region KD (M) ka (1/Ms) kd
(1/s) H0000 GL4 4.2 .times. 10.sup.-9 4.3 .times. 10.sup.5 1.8
.times. 10.sup.-3 H0000 L0000 3.6 .times. 10.sup.-8 3.0 .times.
10.sup.5 1.1 .times. 10.sup.-2
(2-4) Evaluation of the Affinity for Human CD3
[0702] The activity to bind human CD3 when using hCE115HA (SEQ ID
NO: 52) as the H chain variable region and L0000 (SEQ ID NO: 53) as
the L-chain variable region was evaluated. This was performed using
the molecular form of a single-arm antibody having a single Fab at
the Fc region of a human IgG1 heterodimerized by the
knobs-into-hole technique.
[0703] The affinity and binding rate constants of an anti-CD3
antibody for an antigen were measured by the single-cycle kinetics
method of a surface plasmon resonance assay using Biacore.TM.-T200
(GE Healthcare Japan). HBS-EP+(GE Healthcare Japan) was used for
the running buffer, and an amine coupling kit (GE Healthcare Japan)
was used to covalently bind human CD3 to the CM4 chip
(carboxymethyl dextran-coated chip). The anti-CD3 antibody used as
the analyte was prepared at 5 and g/mL using HBS-EP+. Measurements
were carried out by first injecting each of the 5- and 20-.mu.g/mL
anti-CD3 antibody solutions for three minutes continuously at a
flow rate of 20 .mu.L/min to allow reaction to take place. Then,
the solution was switched to HBS-EP+ and the dissociation phase was
measured for 3 minutes. After completion of the dissociation phase
measurement, the sensor chip was regenerated by washing with 10 mM
Gly-HCl at pH 1.5. Measurement at the concentration of 0 was
carried out by performing each of the three-minute HBS-EP+
injections twice successively to allow reaction to take place, and
then switching to HBS-EP+ to measure the dissociation phase for 3
minutes. After completion of the dissociation phase measurement,
the sensor chip was regenerated by washing with 10 mM Gly-HCl at pH
1.5. A data analysis software exclusively for Biacore, Biacore T200
Evaluation Software Version 1.0, was used to perform kinetic
analyses to calculate the binding rate constant (ka), dissociation
rate constant (kd), and the rate constant ratio from the obtained
sensorgrams. The results are shown in Table 11.
TABLE-US-00013 TABLE 11 Variable region H-chain L-chain Affinity
for human CD3 variable region variable region KD (M) ka (1/Ms) kd
(1/s) rCE115H rCE115L 1.0 .times. 10.sup.-7 5.9 .times. 10.sup.4
6.0 .times. 10.sup.-3 hCE115HA L0000 1.2 .times. 10.sup.-7 1.9
.times. 10.sup.5 2.3 .times. 10.sup.-2
(2-5) Preparation of GPC3_ERY27_hCE115
[0704] The IgG4 against a cancer antigen (GPC3) was used as the
basic structure to produce the ERY27 molecule (FIG. 12b), in which
the H-chain variable region of one of the Fabs has been replaced
with a CD3 epsilon-binding domain, and the L chain is common to
both Fabs. In this case, the IgG4 Fc used as the basic structure
was a silent Fc with attenuated affinity for FcgR (an Fc.gamma.
receptor). H0000 (SEQ ID NO: 40) was used as the H-chain variable
region of the GPC3-binding domain, and hCE115HA (SEQ ID NO: 52) was
used as the H-chain variable region of the CD3-binding domain.
L0000 (SEQ ID NO: 53) was used as the L-chain variable region. The
D356K and K439E mutations introduced into the respective H chains
were introduced for efficient heteromer formation of each H chain
when producing heterodimeric antibodies comprising two types of H
chains (WO2006/106905). H435R is a modification that interrupts
binding to Protein A, and was introduced for efficient separation
of the heteromer and homomer (WO/2011/078332).
[0705] A series of expression vectors inserted with a
polynucleotide encoding each of H0000-ERY27_HK (SEQ ID NO: 54),
hCE115HA-ERY27_HE (SEQ ID NO: 55), and L0000-k0 (SEQ ID NO: 56)
were produced by well-known methods.
[0706] The following combination of expression vectors were
introduced into FreeStyle 293-F cells for transient expression of
each target molecule.
[0707] Target molecule: GPC3_ERY27_hCE115
[0708] Polypeptides encoded by the polynucleotides inserted into
the expression vectors: H0000-ERY27_HK, hCE115HA-ERY27_HE, and
L0000-k0
(2-6) Purification of GPC3_ERY27_hCE115
[0709] Each molecule of interest was purified by the method
described in Reference Example 1-2.
(2-7) Measurement of Cytotoxic Activity Using Human Peripheral
Blood Mononuclear Cells
(2-7-1) Preparation of a Human Peripheral Blood Mononuclear Cell
(PBMC) Solution
[0710] The solution was prepared by the method described in
Reference Example 1-3-1.
(2-7-2) Measurement of Cytotoxic Activity
[0711] Cytotoxic activity was measured by the method described in
Reference Example 1-3-2.
[0712] When PBMCs prepared from human blood were used as the
effector cell to measure the cytotoxicity of GPC3_ERY27_hCE115,
reduction of the activity was observed as a result of humanization
of the H chain of rCE115 and sharing of a common L chain (FIG.
13).
[Reference Example 3] Production and Evaluation of Humanized
Bispecific Antibody Variants for Improvement of Various
Properties
[0713] The T-cell-dependent cytotoxic activity of the humanized
anti-human CD3.epsilon. (CD3 epsilon) chain and anti-human GPC3
bispecific antibody obtained in Reference Example 2,
GPC3_ERY27_hCE115 (SEQ ID NOs: 54, 55, and 56), was lower than the
T-cell-dependent cytotoxic activity of GPC3_ERY22_rCE115 (SEQ ID
NOs: 47, 48, 49, and 50). This may be due to attenuation of
affinity for GPC3 and the CD3.epsilon. chain as a result of
humanization and sharing of a common L chain. Regarding GPC3 and
CD3.epsilon.-chain antigens which have independent sequences, there
has been no report so far on humanized bispecific antibodies whose
T-cell dependent cytotoxic activity has been enhanced and whose
affinity for both antigens has been improved by using a common
antibody L chain. Therefore, it has been considered difficult to
obtain humanized antibodies with dual specificity that show a drug
efficacy equivalent to or greater than that of
GPC3_ERY22_rCE115.
[0714] Under such circumstances, the Applicants produced modified
humanized bispecific antibodies with modified affinity for human
GPC3 and human CD3.epsilon. chain by methods known to those skilled
in the art, which involves comprehensively substituting amino acid
residues encoded by the antibody gene to produce antibody variants
against both the human GPC3 and human CD3.epsilon.-chain antigens,
and by performing various evaluations by screening. Furthermore,
similar methods were used to produce modified humanized bispecific
antibodies with modified physicochemical properties. Furthermore,
by combining substitutions of amino acid residues effective for
modifying affinity and physicochemical properties, optimized
bispecific antibodies having a TDCC activity equivalent to or
greater than the T-cell dependent cellular cytotoxicity of
GPC3_ERY22_rCE115 prior to humanization were produced.
[0715] Introduction of point mutations, expression and purification
of antibodies, antigen affinity measurements, and determination of
T-cell dependent cellular cytotoxicity in the optimization of
humanized bispecific antibodies were performed by methods similar
to those in Reference Examples 1 and 2. CDR and FR were determined
according to the Kabat definition (Kabat numbering).
[0716] Depending on the objective, the following were used as the
antibody H-chain constant regions (the numbers indicate EU
numbering): E22Hh (SEQ ID NO: 57) produced by introducing
L234A/L235A/N297A/D356C/T366S/L368A/Y407V/G446 deletion/K447
deletion mutations into human IgG1; E22Hk (SEQ ID NO: 58) produced
by introducing L234A/L235A/N297A/Y349C/T366W/G446 deletion/K447
deletion mutations and a Ser-Ser insertion mutation immediately
before position 118 into human IgG1; Gldh produced by introducing
D356C/T366S/L368A/Y407V/G446 deletion/K447 deletion mutations into
human IgG1; none-Hi-Kn010G3 produced by introducing 118-215
deletion and C220S/Y349C/T366W/H435R mutations into human IgG1;
E2702GsKsc (SEQ ID NO: 60) produced by introducing
L235R/S239K/N297A/E356K/R409K/H435R/L445P/G446 deletion/K447
deletion mutations into human IgG4; E2704sEpsc (SEQ ID NO: 61)
produced by introducing
K196Q/L235R/S239K/N297A/R409K/K439E/L445P/G446 deletion/K447
deletion mutations into human IgG4; and E2702sKsc (SEQ ID NO: 62)
produced by introducing L235R/S239K/N297A/E356K/R409K/L445P/G446
deletion/K447 deletion mutations into human IgG4. Furthermore,
human K (kappa) chain k0 (SEQ ID NO: 63) and E22L (SEQ ID NO: 432)
produced by introducing R108A/T109S mutations into human K chain
were used as the antibody L-chain constant regions.
[0717] The mutation that substitutes Cys for Asp at EU numbering
position 356, the mutation that substitutes Ser for The at EU
numbering position 366, the mutation that substitutes Ala for Leu
at EU numbering position 368, the mutation that substitutes Val for
Tyr at EU numbering position 407, the mutation that substitutes Cys
for Tyr at EU numbering position 349, the mutation that substitutes
Trp for Thr at EU numbering position 366, and the mutation that
inserts Ser-Ser immediately before position 118 are mutations for
efficient formation of heterodimeric molecules for each H chain
when producing heteromeric antibodies. Similarly, the mutation that
substitutes Lys for Glu at EU numbering position 356 and the
mutation that substitutes Glu for Lys at EU numbering position 439
are also mutations for efficient formation of heterodimeric
molecules for each H chain when producing heteromeric antibodies.
They are expected to improve the efficiency of bispecific antibody
production.
[0718] The mutation that substitutes Ala for Leu at EU numbering
position 234, the mutation that substitutes Ala or Arg for Leu at
EU numbering position 235, the mutation that substitutes Lys for
Ser at EU numbering position 239, and the mutation that substitutes
Ala for Asn at EU numbering position 297 are mutations for
attenuating affinity for an Fc.gamma. receptor and a complement
(C1q). They are expected to suppress the binding of Fab to CD3 and
Fc-mediated crosslinking of an Fc.gamma. receptor or a complement,
and avoid cytokine release syndrome that accompanies enhancement of
non-specific effector functions.
[0719] The H chain introduced with deletion mutations at EU
numbering positions 118 to 215 can be combined with a full-length H
chain sequence to produce an antibody that has only one Fab
(monovalent antibody), and it is useful for affinity
evaluation.
[0720] The mutation that substitutes Lys for Arg at EU numbering
position 409 and the mutation that substitutes Arg for His at EU
numbering position 435 are mutations for modifying the antibody
properties to be close to the properties of human IgG1 and human
IgG3, respectively.
(3-1) Modifying the Affinity of a Humanized Anti-CD3 Antibody by
Point Mutations
[0721] First, point mutations were introduced into FR1, FR2, FR3,
CDR1, CDR2, and CDR3 of the humanized anti-human CD3.epsilon. chain
antibody sequence produced in Reference Example 2,
hCE115HA-ERY27_HE (SEQ ID NO: 55), to prepare modified antibodies.
Next, the affinity of these modified antibodies for the soluble
human CD3.epsilon. chain was determined. Combining sites that have
an affinity-enhancing effect yielded modified antibodies having the
affinities shown in Table 12.
TABLE-US-00014 TABLE 12 KD (Human Antibody name CD3)
hCE115HA-E22Hh//-Hi-Kn010G3/L0000-k0 1.43E-07
TR01H083-E22Hh/none-Hi-Kn010G3/L0212-k0 5.86E-11
TR01H040-E22Hh/none-Hi-Kn010G3/L0240-k0 2.17E-09
TR01H002-E22Hh/GLS3108-k0/GL4-E22Hk/H0610-E22L 2.04E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0212-k0 2.17E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0235-k0 2.81E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0238-k0 2.91E-09
TR01H040-E22Hh/none-Hi-Kn010G3/TR01L016-k0 2.52E-09
TR01H083-E22Hh/none-Hi-Kn010G3/L0262-k0 2.45E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0207-k0 2.60E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0241-k0 3.48E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0242-k0 3.58E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0206-k0 2.90E-09
TR01H040-E22Hh/none-Hi-Kn010G3/TR01L019-k0 3.20E-09
TR01H080-E22Hh/none-Hi-Kn010G3/L0000-k0 3.25E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0211-k0 3.22E-09
TR01H002-E22Hh//-Hi-Kn010G3/GLC3108-k0 4.61E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0209-k0 4.25E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0208-k0 4.16E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0224-k0 5.06E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0236-k0 5.64E-09
TR01H083-E22Hh/none-Hi-Kn010G3/L0201-k0 4.42E-09
TR01H084-E2702GsKsc/none-Hi-E2704sE/L0011-k0 4.14E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0210-k0 5.06E-09
TR01H114-E2702GsKsc/none-Hi-E2704sE/L0011-k0 4.22E-09
CE115HA236-E22Hh/GLS3108-k0/GL4-E22Hk/H0610-E22L 6.08E-09
TR01H077-E22Hh/none-Hi-Kn010G3/L0200-k0 6.12E-09
TR01H071-E22Hh/none-Hi-Kn010G3/L0200-k0 6.13E-09
TR01H111-E2702GsKsc/none-Hi-E2704sE/L0011-k0 4.91E-09
TR01H081-E22Hh/none-Hi-Kn010G3/L0262-k0 5.76E-09
TR01H001-E22Hh//-Hi-Kn010G3/GLC3108-k0 8.22E-09
CE115HA179-G1dh//-Hi-Kn010G3/L0000-k0 8.35E-09
TR01H112-E2702GsKsc/none-Hi-E2704sE/L0011-k0 5.12E-09
TR01H113-E2702GsKsc/none-Hi-E2704sE/L0011-k0 5.14E-09
TR01H082-E22Hh/none-Hi-Kn010G3/L0212-k0 4.75E-09
CE115HA236-E22Hh//-Hi-Kn010G3/GLC3108-k0 9.10E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0231-k0 7.75E-09
TR01H037-E22Hh/none-Hi-Kn010G3/L0000-k0 6.93E-09
CE115HA252-E22Hh//-Hi-Kn010G3/L0000-k0 9.48E-09
TR01H083-E22Hh/none-Hi-Kn010G3/L0011-k0 6.70E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0223-k0 8.15E-09
TR01H083-E22Hh/none-Hi-Kn010G3/L0000-k0 6.83E-09
TR01H071-E22Hh/none-Hi-Kn010G3/L0000-k0 8.85E-09
TR01H067-E22Hh/none-Hi-Kn010G3/L0212-k0 5.88E-09
CE115HA178-G1dh//-Hi-Kn010G3/L0000-k0 1.09E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0237-k0 1.02E-08
TR01H083-E22Hh/none-Hi-Kn010G3/L0222-k0 9.42E-09
TR01H084-E22Hh/none-Hi-Kn010G3/L0262-k0 8.51E-09
TR01H071-E22Hh/none-Hi-Kn010G3/L0215-k0 9.51E-09
TR01H040-E22Hh/none-Hi-Kn010G3/L0218-k0 8.20E-09
TR01H081-E22Hh/none-Hi-Kn010G3/L0201-k0 9.46E-09
TR01H071-E22Hh/none-Hi-Kn010G3/L0222-k0 1.04E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0220-k0 9.15E-09
TR01H067-E22Hh/none-Hi-Kn010G3/TR01L016-k0 1.09E-08
TR01H002-E22Hh/GLS3108-k0/GL4-E22Hk/H0000-E22L 4.78E-09
TR01H067-E22Hh/none-Hi-Kn010G3/TR01L019-k0 1.21E-08
TR01H038-E22Hh/none-Hi-Kn010G3/L0000-k0 1.24E-08
TR01H061-E22Hh/none-Hi-Kn010G3/L0200-k0 1.27E-08
TR01H082-E2702GsKsc/none-Hi-E2704sE/L0011-k0 1.01E-08
CE115HA180-G1dh//-Hi-Kn010G3/L0000-k0 1.68E-08
CE115HA251-E22Hh/L0000-k0/GL4-E22Hk/H0610-E22L 1.37E-08
TR01H100-E2702GsKsc/none-Hi-E2704sE/L0011-k0 1.11E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0228-k0 1.60E-08
TR01H081-E22Hh/none-Hi-Kn010G3/L0011-k0 1.35E-08
TR01H061-E22Hh/none-Hi-Kn010G3/L0215-k0 1.54E-08
TR01H110-E2702GsKsc/none-Hi-E2704sE/L0011-k0 1.26E-08
TR01H043-E22Hh/none-Hi-Kn010G3/L0000-k0 1.52E-08
TR01H081-E22Hh/none-Hi-Kn010G3/L0000-k0 1.56E-08
CE115HA251-E22Hh//-Hi-Kn010G3/L0000-k0 2.23E-08
TR01H091-E2702GsKsc/none-Hi-E2704sE/L0011-k0 1.39E-08
CE115HA236-E22Hh/GLS3108-k0/GL4-E22Hk/H0000-E22L 6.95E-09
TR01H084-E22Hh/none-Hi-Kn010G3/L0201-k0 1.65E-08
TR01H072-E22Hh/none-Hi-Kn010G3/L0000-k0 2.03E-08
TR01H099-E2702GsKsc/none-Hi-E2704sE/L0011-k0 1.46E-08
TR01H061-E22Hh/none-Hi-Kn010G3/L0222-k0 1.88E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0239-k0 2.31E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0262-k0 1.81E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0234-k0 2.40E-08
TR01H012-E22Hh/none-Hi-Kn010G3/L0000-k0 7.94E-09
TR01H061-E22Hh/none-Hi-Kn010G3/L0000-k0 1.71E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0243-k0 2.46E-08
TR01H109-E2702GsKsc/none-Hi-E2704sE/L0011-k0 1.64E-08
TR01H047-E22Hh/none-Hi-Kn010G3/L0000-k0 2.04E-08
TR01H082-E22Hh/none-Hi-Kn010G3/L0267-k0 2.29E-08
TR01H082-E22Hh/none-Hi-Kn010G3/L0266-k0 2.29E-08
TR01H084-E22Hh/none-Hi-Kn010G3/L0011-k0 1.98E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0250-k0 2.15E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0204-k0 2.21E-08
TR01H084-E22Hh/none-Hi-Kn010G3/L0000-k0 2.13E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0213-k0 2.01E-08
hCE115HA-E22Hh//-Hi-Kn010G3/L0000-k0 1.43E-07
TR01H040-E22Hh/none-Hi-Kn010G3/L0214-k0 2.02E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0217-k0 2.07E-08
TR01H071-E22Hh/none-Hi-Kn010G3/L0226-k0 2.51E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0200-k0 2.87E-08
TR01H074-E22Hh/none-Hi-Kn010G3/L0000-k0 2.91E-08
TR01H039-E22Hh/none-Hi-Kn010G3/L0000-k0 2.61E-08
CE115HA177-G1dh//-Hi-Kn010G3/L0000-k0 3.55E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0201-k0 2.81E-08
TR01H082-E22Hh/none-Hi-Kn010G3/L0263-k0 3.09E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0000-k0 3.60E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0216-k0 2.53E-08
TR01H051-E22Hh/none-Hi-Kn010G3/L0000-k0 2.91E-08
TR01H003-E22Hh//-Hi-Kn010G3/L0000-k0 4.03E-08
TR01H082-E22Hh/none-Hi-Kn010G3/L0264-k0 3.44E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0232-k0 3.86E-08
TR01H041-E22Hh/none-Hi-Kn010G3/L0000-k0 3.16E-08
CE115HA122-E22Hh//-Hi-Kn010G3/L0000-k0 4.28E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0233-k0 4.01E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0215-k0 3.37E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0203-k0 3.24E-08
TR01H015-E2702GsKsc/GCH019-E2704sEpsc/L0000-k0 2.96E-08
TR01H040-E22Hh/none-Hi-Kn010G3/TR01L008-k0 2.93E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0205-k0 3.42E-08
TR01H015-E22Hh/L0000-k0/GL4-E22Hk/H0610-E22L 3.57E-08
TR01H064-E22Hh/none-Hi-Kn010G3/L0000-k0 3.07E-08
TR01H044-E22Hh/none-Hi-Kn010G3/L0000-k0 3.52E-08
TR01H082-E22Hh/none-Hi-Kn010G3/L0262-k0 3.98E-08
TR01H062-E22Hh/none-Hi-Kn010G3/L0000-k0 3.13E-08
CE115HA251-E22Hh/L0000-k0/GL4-E22Hk/H0000-E22L 1.48E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0011-k0 3.48E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0222-k0 4.65E-08
CE115HA192-E22Hh//-Hi-Kn010G3/L0000-k0 5.05E-08
TR01H040-E22Hh/none-Hi-Kn010G3/TR01L010-k0 3.28E-08
TR01H025-E22Hh/none-Hi-Kn010G3/L0000-k0 3.86E-08
TR01H082-E22Hh/none-Hi-Kn010G3/TR01L023-k0 4.25E-08
TR01H040-E22Hh/none-Hi-Kn010G3/TR01L015-k0 3.95E-08
TR01H055-E22Hh/none-Hi-Kn010G3/L0000-k0 3.88E-08
TR01H082-E22Hh/none-Hi-Kn010G3/L0260-k0 4.53E-08
TR01H040-E22Hh/none-Hi-Kn010G3/TR01L009-k0 3.56E-08
TR01H040-E22Hh/none-Hi-Kn010G3/TR01L011-k0 3.57E-08
TR01H017-E22Hh/none-Hi-Kn010G3/L0000-k0 3.50E-08
CE115HA122-E22Hh/L0000-k0/GL4-E22Hk/H0000-E22L 1.69E-08
TR01H076-E22Hh/none-Hi-Kn010G3/L0000-k0 4.78E-08
TR01H082-E22Hh/none-Hi-Kn010G3/L0258-k0 4.70E-08
TR01H046-E22Hh/none-Hi-Kn010G3/L0000-k0 4.23E-08
rCE115H-G1dh//-Hi-Kn010G3/L0000-k0 5.76E-08
TR01H082-E22Hh/none-Hi-Kn010G3/TR01L024-k0 4.76E-08
TR01H016-E22Hh/none-Hi-Kn010G3/L0000-k0 3.69E-08
TR01H040-E22Hh/none-Hi-Kn010G3/TR01L018-k0 4.51E-08
TR01H084-E22Hh/none-Hi-Kn010G3/L0271-k0 2.76E-08
TR01H084-E22Hh/none-Hi-Kn010G3/L0270-k0 2.76E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0000vk1-k0 4.69E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0219-k0 3.94E-08
TR01H014-E22Hh/none-Hi-Kn010G3/L0000-k0 3.87E-08
TR01H061-E22Hh/none-Hi-Kn010G3/L0226-k0 4.71E-08
TR01H048-E22Hh/none-Hi-Kn010G3/L0000-k0 4.52E-08
TR01H082-E22Hh/none-Hi-Kn010G3/L0259-k0 5.07E-08
TR01H028-E22Hh/none-Hi-Kn010G3/L0000-k0 4.80E-08
TR01H082-E22Hh/none-Hi-Kn010G3/L0201-k0 4.49E-08
TR01H040-E22Hh/none-Hi-Kn010G3/TR01L013-k0 4.15E-08
TR01H033-E22Hh/none-Hi-Kn010G3/L0000-k0 4.88E-08
hCE115HA-G1dh//-Hi-Kn010G3/L0000-k0 6.50E-08
TR01H040-E22Hh/none-Hi-Kn010G3/TR01L012-k0 4.22E-08
TR01H065-E22Hh/none-Hi-Kn010G3/L0000-k0 4.31E-08
TR01H079-E22Hh/none-Hi-Kn010G3/L0000-k0 5.05E-08
TR01H042-E22Hh/none-Hi-Kn010G3/L0000-k0 4.48E-08
TR01H063-E22Hh/none-Hi-Kn010G3/L0000-k0 4.35E-08
TR01H084-E22Hh/none-Hi-Kn010G3/L0272-k0 3.10E-08
CE115HA121-E22Hh//-Hi-Kn010G3/L0000-k0 6.76E-08
TR01H026-E22Hh/none-Hi-Kn010G3/L0000-k0 5.12E-08
TR01H067-E22Hh/none-Hi-Kn010G3/L0262-k0 4.92E-08
TR01H073-E22Hh/none-Hi-Kn010G3/L0000-k0 5.97E-08
TR01H045-E22Hh/none-Hi-Kn010G3/L0000-k0 5.22E-08
TR01H007-E22Hh/none-Hi-Kn010G3/L0000-k0 2.17E-08
TR01H082-E22Hh/none-Hi-Kn010G3/L0203-k0 4.07E-08
TR01H032-E22Hh/none-Hi-Kn010G3/L0000-k0 5.73E-08
TR01H006-E22Hh/none-Hi-Kn010G3/L0000-k0 2.30E-08
TR01H013-E22Hh/none-Hi-Kn010G3/L0000-k0 4.94E-08
TR01H050-E22Hh/none-Hi-Kn010G3/L0000-k0 5.76E-08
TR01H067-E22Hh/none-Hi-Kn010G3/L0200-k0 6.03E-08
TR01H015-E22Hh/none-Hi-Kn010G3/L0000-k0 6.13E-08
hCE115HA-E22Hh/L0000-k0/GL4-E22Hk/H0000-E22L 6.16E-08
hCE115HA-E22stHh/none-Hi-stKn010G3/L0000-k0 5.17E-08
TR01H069-E22Hh/none-Hi-Kn010G3/L0000-k0 7.11E-08
TR01H015-E22Hh/none-Hi-Kn010G3/TR01L003-k0 6.34E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0202-k0 6.19E-08
TR01H067-E22Hh/none-Hi-Kn010G3/L0201-k0 5.93E-08
TR01H020-E22Hh/none-Hi-Kn010G3/L0000-k0 6.48E-08
TR01H082-E22Hh/none-Hi-Kn010G3/L0011-k0 5.95E-08
hCE115HA-E22Hh//-Hi-Kn010G3/L0000-k0 1.43E-07
TR01H082-E22Hh/none-Hi-Kn010G3/TR01L018-k0 4.72E-08
TR01H015-E22Hh/none-Hi-Kn010G3/TR01L005-k0 6.53E-08
TR01H052-E22Hh/none-Hi-Kn010G3/L0000-k0 6.27E-08
TR01H036-E22Hh/none-Hi-Kn010G3/L0000-k0 6.50E-08
TR01H067-E22Hh/none-Hi-Kn010G3/L0203-k0 4.79E-08
TR01H030-E22Hh/none-Hi-Kn010G3/L0000-k0 6.54E-08
TR01H015-E22Hh/none-Hi-Kn010G3/TR01L001-k0 6.56E-08
TR01H100-E22Hh/none-Hi-Kn010G3/L0011-k0 6.25E-08
TR01H029-E22Hh/none-Hi-Kn010G3/L0000-k0 6.70E-08
TR01H019-E22Hh/none-Hi-Kn010G3/L0000-k0 6.85E-08
TR01H082-E22Hh/none-Hi-Kn010G3/L0000-k0 7.37E-08
TR01H018-E22Hh/none-Hi-Kn010G3/L0000-k0 6.93E-08
TR01H027-E22Hh/none-Hi-Kn010G3/L0000-k0 6.95E-08
TR01H049-E22Hh/none-Hi-Kn010G3/L0000-k0 6.79E-08
TR01H066-E22Hh/none-Hi-Kn010G3/L0000-k0 6.02E-08
TR01H091-E22Hh/none-Hi-Kn010G3/L0011-k0 6.67E-08
rCE115H-E22Hh/none-Hi-Kn010G3/L0000-k0 8.00E-08
TR01H015-E22Hh/none-Hi-Kn010G3/TR01L002-k0 7.14E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0226-k0 8.01E-08
TR01H067-E22Hh/none-Hi-Kn010G3/TR01L018-k0 5.26E-08
TR01H093-E22Hh/none-Hi-Kn010G3/L0011-k0 6.80E-08
TR01H067-E22Hh/none-Hi-Kn010G3/L0215-k0 7.41E-08
TR01H015-E22Hh/none-Hi-Kn010G3/TR01L004-k0 7.34E-08
TR01H107-E22Hh/none-Hi-Kn010G3/L0011-k0 6.91E-08
TR01H105-E22Hh/none-Hi-Kn010G3/L0011-k0 6.95E-08
TR01H090-E22Hh/none-Hi-Kn010G3/L0011-k0 6.95E-08
TR01H108-E22Hh/none-Hi-Kn010G3/L0011-k0 6.98E-08
TR01H094-E22Hh/none-Hi-Kn010G3/L0011-k0 7.00E-08
TR01H109-E22Hh/none-Hi-Kn010G3/L0011-k0 7.06E-08
TR01H056-E22Hh/none-Hi-Kn010G3/L0000-k0 7.32E-08
TR01H031-E22Hh/none-Hi-Kn010G3/L0000-k0 7.55E-08
TR01H022-E22Hh/none-Hi-Kn010G3/L0000-k0 7.58E-08
TR01H092-E22Hh/none-Hi-Kn010G3/L0011-k0 7.21E-08
TR01H067-E22Hh/none-Hi-Kn010G3/L0000-k0 7.15E-08
TR01H067-E22Hh/none-Hi-Kn010G3/L0011-k0 7.18E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0248-k0 7.89E-08
TR01H009-E22Hh/none-Hi-Kn010G3/L0000-k0 3.15E-08
TR01H023-E22Hh/none-Hi-Kn010G3/L0000-k0 7.94E-08
TR01H096-E22Hh/none-Hi-Kn010G3/L0011-k0 7.47E-08
TR01H040-E22Hh/none-Hi-Kn010G3/TR01L007-k0 6.82E-08
TR01H054-E22Hh/none-Hi-Kn010G3/L0000-k0 7.79E-08
TR01H021-E22Hh/none-Hi-Kn010G3/L0000-k0 8.05E-08
TR01H103-E22Hh/none-Hi-Kn010G3/L0011-k0 7.72E-08
TR01H099-E22Hh/none-Hi-Kn010G3/L0011-k0 7.74E-08
rCE115H-E22Hh/none-Hi-Kn010G3/L0000vk1-k0 8.52E-08
TR01H101-E22Hh/none-Hi-Kn010G3/L0011-k0 7.87E-08
TR01H053-E22Hh/none-Hi-Kn010G3/L0000-k0 8.23E-08
TR01H035-E22Hh/none-Hi-Kn010G3/L0000-k0 8.49E-08
TR01H067-E22Hh/none-Hi-Kn010G3/TR01L015-k0 8.64E-08
TR01H104-E22Hh/none-Hi-Kn010G3/L0011-k0 8.26E-08
TR01H075-E22Hh/none-Hi-Kn010G3/L0000-k0 9.88E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0227-k0 1.01E-07
TR01H102-E22Hh/none-Hi-Kn010G3/L0011-k0 8.54E-08
TR01H034-E22Hh/none-Hi-Kn010G3/L0000-k0 9.11E-08
TR01H082-E22Hh/none-Hi-Kn010G3/L0222-k0 1.01E-07
rCE115H-E22Hh/rCE115L-k0/GL4-E22Hk/H0000-E22L 9.37E-08
TR01H015-E22Hh/none-Hi-Kn010G3/TR01L006-k0 9.30E-08
TR01H040-E22Hh/none-Hi-Kn010G3/L0246-k0 9.28E-08
TR01H097-E22Hh/none-Hi-Kn010G3/L0011-k0 8.76E-08
TR01H011-E22Hh/none-Hi-Kn010G3/L0000-k0 3.71E-08
TR01H010-E22Hh/none-Hi-Kn010G3/L0000-k0 3.73E-08
TR01H095-E22Hh/none-Hi-Kn010G3/L0011-k0 9.09E-08
TR01H082-E22Hh/none-Hi-Kn010G3/TR01L020-k0 1.06E-07
TR01H098-E22Hh/none-Hi-Kn010G3/L0011-k0 9.14E-08
TR01H082-E22Hh/none-Hi-Kn010G3/TR01L017-k0 1.09E-07
TR01H040-E22Hh/none-Hi-Kn010G3/L0247-k0 1.00E-07
rCE115H-E22Hh/none-Hi-Kn010G3/rCE115L-k0 1.24E-07
TR01H004-E22Hh//-Hi-Kn010G3/L0000-k0 1.35E-07
TR01H067-E22Hh/none-Hi-Kn010G3/L0222-k0 7.63E-08
rCE115H-E22Hh//-Hi-Kn010G3/rCE115L-k0 1.38E-07
TR01H008-E22Hh/none-Hi-Kn010G3/L0000-k0 4.22E-08
TR01H070-E22Hh/none-Hi-Kn010G3/L0000-k0 1.20E-07
TR01H106-E22Hh/none-Hi-Kn010G3/L0011-k0 1.00E-07
TR01H024-E22Hh/none-Hi-Kn010G3/L0000-k0 1.08E-07
CE115HA124-E22Hh//-Hi-Kn010G3/L0000-k0 1.43E-07
TR01H040-E22Hh/none-Hi-Kn010G3/L0249-k0 1.11E-07
TR01H082-E22Hh/none-Hi-Kn010G3/L0271-k0 6.82E-08
TR01H057-E22Hh/none-Hi-Kn010G3/L0000-k0 1.12E-07
TR01H058-E22Hh/none-Hi-Kn010G3/L0000-k0 1.15E-07
TR01H068-E22Hh/none-Hi-Kn010G3/L0000-k0 1.01E-07
TR01H082-E22Hh/none-Hi-Kn010G3/L0270-k0 7.42E-08
TR01H082-E22Hh/none-Hi-Kn010G3/L0272-k0 7.44E-08
hCE115HA-E22Hh/none-Hi-Kn010G3/L0000-k0 1.24E-07
TR01H082-E22Hh/none-Hi-Kn010G3/L0268-k0 1.36E-07
hCE115HAa-E22Hh/none-Hi-Kn010G3/L0000-k0 1.08E-07
TR01H067-E22Hh/none-Hi-Kn010G3/L0226-k0 1.32E-07
TR01H067-E22Hh/none-Hi-Kn010G3/L0248-k0 1.39E-07
(3-2) Modifying the Affinity of a Humanized Anti-GPC3 Antibody
[0722] First, point mutations were introduced into CDR1, CDR2, and
CDR3 of the anti-human GPC3 bispecific antibody sequence produced
in Reference Example 2, H0000-ERY27_HK (SEQ ID NO: 54), to prepare
modified antibodies. Next, the affinity of these modified
antibodies for soluble human GPC3 was determined. Combining sites
that have an affinity-enhancing effect yielded modified antibodies
having the affinities shown in Table 13.
TABLE-US-00015 TABLE 13 KD (Human Antibody name GPC3)
H0610-G1dh/none-Hi-Kn010G3/L0000-k0 3.97E-09
H0610-G1dh/none-Hi-Kn010G3/L0222-k0 1.40E-13
H0610-G1dh/none-Hi-Kn010G3/L0258-k0 3.52E-13
GCH054-G1dh/none-Hi-Kn010G3/L0262-k0 5.25E-13
GCH060-G1dh/none-Hi-Kn010G3/L0222-k0 6.42E-13
H0610-G1dh/none-Hi-Kn010G3/L0246-k0 1.21E-12
GCH057-G1dh/none-Hi-Kn010G3/L0222-k0 1.85E-12
GCH054-G1dh/none-Hi-Kn010G3/L0249-k0 3.61E-12
GCH055-G1dh/none-Hi-Kn010G3/L0222-k0 3.90E-12
GCH094-G1dh/none-Hi-Kn010G3/L0246-k0 4.12E-12
H0610-G1dh/none-Hi-Kn010G3/L0249-k0 6.86E-12
H0610-G1dh/none-Hi-Kn010G3/TR01L017-k0 8.27E-12
H0610-G1dh/none-Hi-Kn010G3/L0265-k0 8.70E-12
H0610-G1dh/none-Hi-Kn010G3/L0261-k0 1.07E-11
GCH065-G1dh/none-Hi-Kn010G3/L0262-k0 1.18E-11
GCH056-G1dh/none-Hi-Kn010G3/L0262-k0 1.19E-11
H0610-G1dh/none-Hi-Kn010G3/L0268-k0 1.69E-11
H0610-G1dh/none-Hi-Kn010G3/TR01L020-k0 2.24E-11
GCH054-G1dh/none-Hi-Kn010G3/L0246-k0 3.15E-11
GCH054-G1dh/none-Hi-Kn010G3/L0222-k0 3.15E-11
GCH073-G1dh/none-Hi-Kn010G3/L0201-k0 3.50E-11
H0610-G1dh/none-Hi-Kn010G3/L0248-k0 5.55E-11
GCH065-G1dh/none-Hi-Kn010G3/L0201-k0 7.74E-11
H0610-G1dh/none-Hi-Kn010G3/L0226-k0 9.30E-11
H0610-G1dh/none-Hi-Kn010G3/L0093-k0 1.06E-10
GCH098-G1dh/none-Hi-Kn010G3/L0201-k0 1.11E-10
H0610-G1dh/none-Hi-Kn010G3/L0267-k0 1.79E-10
H0610-G1dh/none-Hi-Kn010G3/L0228-k0 2.02E-10
H0610-G1dh/none-Hi-Kn010G3/L0262-k0 2.11E-10
H0610-G1dh/none-Hi-Kn010G3/L0266-k0 2.13E-10
H0610-G1dh/none-Hi-Kn010G3/L0264-k0 2.19E-10
H0610-G1dh/none-Hi-Kn010G3/L0224-k0 2.43E-10
H0610-G1dh/none-Hi-Kn010G3/L0167-k0 2.11E-10
CE115HA251-E22Hh/L0000-k0/GL4-E22Hk/H0610-E22L 2.36E-10
TR01H015-E22Hh/L0000-k0/GL4-E22Hk/H0610-E22L 2.63E-10
CE115HA236-E22Hh/GLS3108-k0/GL4-E22Hk/H0610-E22L 2.67E-10
H0610-G1dh/none-Hi-Kn010G3/L0259-k0 3.34E-10
H0610-G1dh/none-Hi-Kn010G3/L0227-k0 4.08E-10
GCH065-G1dh/none-Hi-Kn010G3/L0272-k0 3.93E-10
H0610-G1dh/none-Hi-Kn010G3/L0269-k0 4.59E-10
H0610-G1dh/none-Hi-Kn010G3/L0223-k0 4.75E-10
TR01H002-E22Hh/GLS3108-k0/GL4-E22Hk/H0610-E22L 4.75E-10
GCH054-G1dh/none-Hi-Kn010G3/L0212-k0 5.17E-10
H0610-G1dh/none-Hi-Kn010G3/L0208-k0 5.30E-10
H0610-G1dh/none-Hi-Kn010G3/L0263-k0 5.64E-10
H0610-G1dh/none-Hi-Kn010G3/L0231-k0 5.89E-10
H0610-G1dh/none-Hi-Kn010G3/L0143-k0 5.73E-10
GCH055-G1dh/none-Hi-Kn010G3/L0212-k0 6.14E-10
H0610-G1dh/none-Hi-Kn010G3/L0211-k0 6.47E-10
H0610-G1dh/none-Hi-Kn010G3/L0238-k0 6.37E-10
H0610-G1dh/none-Hi-Kn010G3/L0214-k0 6.57E-10
H0610-G1dh/none-Hi-Kn010G3/L0243-k0 6.49E-10
GCH025-G1dh/none-Hi-Kn010G3/L0204-k0 6.70E-10
GCH054-G1dh/none-Hi-Kn010G3/TR01L016-k0 7.63E-10
H0610-G1dh/none-Hi-Kn010G3/L0168-k0 6.99E-10
GCH094-G1dh/none-Hi-Kn010G3/L0271-k0 6.92E-10
GCH054-G1dh/none-Hi-Kn010G3/TR01L019-k0 8.71E-10
H0610-G1dh/none-Hi-Kn010G3/L0234-k0 7.78E-10
GCH098-G1dh/none-Hi-Kn010G3/L0011-k0 8.02E-10
H0610-G1dh/none-Hi-Kn010G3/L0204-k0 7.27E-10
H0610-G1dh/none-Hi-Kn010G3/L0240-k0 8.48E-10
H0610-G1dh/none-Hi-Kn010G3/L0239-k0 8.74E-10
H0610-G1dh/none-Hi-Kn010G3/L0212-k0 9.94E-10
GCH065-G1dh/none-Hi-Kn010G3/L0011-k0 8.84E-10
H0610-G1dh/none-Hi-Kn010G3/L0200-k0 1.04E-09
H0610-G1dh/none-Hi-Kn010G3/L0124-k0 9.72E-10
GCH073-G1dh/none-Hi-Kn010G3/L0011-k0 9.10E-10
H0610-G1dh/none-Hi-Kn010G3/TR01L016-k0 1.08E-09
GCH054-G1dh/none-Hi-Kn010G3/L0201-k0 1.08E-09
H0610-G1dh/none-Hi-Kn010G3/L0090-k0 1.12E-09
H0610-G1dh/none-Hi-Kn010G3/L0209-k0 1.12E-09
H0610-G1dh/none-Hi-Kn010G3/L0201-k0 1.13E-09
H0610-G1dh/none-Hi-Kn010G3/L0161-k0 9.73E-10
H0610-G1dh/none-Hi-Kn010G3/L0206-k0 8.65E-10
H0610-G1dh/none-Hi-Kn010G3/L0186-k0 1.08E-09
H0610-G1dh/none-Hi-Kn010G3/TR01L019-k0 1.15E-09
H0610-G1dh/none-Hi-Kn010G3/L0085-k0 1.17E-09
GCH055-G1dh/none-Hi-Kn010G3/L0200-k0 1.13E-09
H0610-G1dh/none-Hi-Kn010G3/L0154-k0 1.01E-09
H0610-G1dh/none-Hi-Kn010G3/L0229-k0 1.20E-09
GCH054-G1dh/none-Hi-Kn010G3/L0200-k0 1.18E-09
GCH094-G1dh/none-Hi-Kn010G3/L0201-k0 1.17E-09
H0610-G1dh/none-Hi-Kn010G3/L0000-k0 3.97E-09
H0610-G1dh/none-Hi-Kn010G3/L0205-k0 1.01E-09
GCH099-G1dh/none-Hi-Kn010G3/L0201-k0 1.29E-09
H0610-G1dh/none-Hi-Kn010G3/L0242-k0 1.19E-09
GCH056-G1dh/none-Hi-Kn010G3/L0201-k0 1.16E-09
H0610-G1dh/none-Hi-Kn010G3/L0213-k0 1.25E-09
GCH060-G1dh/none-Hi-Kn010G3/L0200-k0 1.34E-09
GCH065-G1dh/none-Hi-Kn010G3/L0000-k0 1.41E-09
GCH100-G1dh/none-Hi-Kn010G3/L0201-k0 1.37E-09
H0610-G1dh/none-Hi-Kn010G3/L0015-k0 1.31E-09
H0610-G1dh/none-Hi-Kn010G3/L0151-k0 1.25E-09
H0610-G1dh/none-Hi-Kn010G3/L0237-k0 1.31E-09
H0610-G1dh/none-Hi-Kn010G3/L0220-k0 1.36E-09
H0610-G1dh/none-Hi-Kn010G3/L0155-k0 1.28E-09
GCH055-G1dh/none-Hi-Kn010G3/L0215-k0 1.52E-09
H0610-G1dh/none-Hi-Kn010G3/L0202-k0 1.22E-09
GCH056-G1dh/none-Hi-Kn010G3/L0215-k0 1.59E-09
H0610-G1dh/none-Hi-Kn010G3/L0012-k0 1.55E-09
GCH054-G1dh/none-Hi-Kn010G3/L0215-k0 1.62E-09
H0610-G1dh/none-Hi-Kn010G3/L0215-k0 1.64E-09
GCH098-G1dh/none-Hi-Kn010G3/L0000-k0 1.77E-09
H0610-G1dh/none-Hi-Kn010G3/L0125-k0 1.71E-09
GCH057-G1dh/none-Hi-Kn010G3/L0215-k0 1.83E-09
H0610-G1dh/none-Hi-Kn010G3/L0217-k0 1.79E-09
H0610-G1dh/none-Hi-Kn010G3/L0014-k0 1.82E-09
H0610-G1dh/none-Hi-Kn010G3/L0216-k0 1.86E-09
TR01H015-E2702GsKsc/GCH019-E2704sEpscL0000-k0 1.64E-09
H0610-G1dh/none-Hi-Kn010G3/TR01L015-k0 2.16E-09
H0610-G1dh/none-Hi-Kn010G3/TR01L018-k0 2.17E-09
H0610-G1dh/none-Hi-Kn010G3/L0218-k0 1.99E-09
H0610-G1dh/none-Hi-Kn010G3/L0000vk1-k0 2.16E-09
H0610-G1dh/none-Hi-Kn010G3/L0160-k0 2.12E-09
H0610-G1dh/none-Hi-Kn010G3/L0047-k0 2.23E-09
GCH073-G1dh/none-Hi-Kn010G3/L0000-k0 2.00E-09
GCH054-G1dh/none-Hi-Kn010G3/TR01L015-k0 2.45E-09
H0610-G1dh/none-Hi-Kn010G3/L0219-k0 2.28E-09
GCH094-G1dh/none-Hi-Kn010G3/L0272-k0 2.10E-09
H0610-G1dh/none-Hi-Kn010G3/L0149-k0 2.16E-09
GCH054-G1dh/none-Hi-Kn010G3/TR01L018-k0 2.59E-09
GCH054-G1dh/none-Hi-Kn010G3/L0203-k0 2.48E-09
H0610-G1dh/none-Hi-Kn010G3/L0122-k0 2.42E-09
H0610-G1dh/none-Hi-Kn010G3/L0134-k0 2.53E-09
H0610-G1dh/none-Hi-Kn010G3/L0152-k0 2.36E-09
H0610-G1dh/none-Hi-Kn010G3/L0203-k0 2.11E-09
H0610-G1dh/none-Hi-Kn010G3/L0075-k0 2.85E-09
H0610-G1dh/none-Hi-Kn010G3/L0038-k0 2.75E-09
H0610-G1dh/none-Hi-Kn010G3/L0011-k0 2.76E-09
H0610-G1dh/none-Hi-Kn010G3/L0157-k0 2.60E-09
H0610-G1dh/none-Hi-Kn010G3/L0145-k0 2.66E-09
H0610-G1dh/none-Hi-Kn010G3/TR01L010-k0 2.92E-09
H0610-G1dh/none-Hi-Kn010G3/L0009-k0 2.99E-09
GCH099-G1dh/none-Hi-Kn010G3/L0011-k0 2.78E-09
H0610-G1dh/none-Hi-Kn010G3/L0006-k0 3.04E-09
H0610-G1dh/none-Hi-Kn010G3/L0173-k0 2.83E-09
H0610-G1dh/none-Hi-Kn010G3/L0127-k0 3.12E-09
H0610-G1dh/none-Hi-Kn010G3/L0082-k0 3.43E-09
H0610-G1dh/none-Hi-Kn010G3/L0064-k0 3.37E-09
H0610-G1dh/none-Hi-Kn010G3/L0008-k0 3.30E-09
H0610-G1dh/none-Hi-Kn010G3/L0013-k0 3.35E-09
H0610-G1dh/none-Hi-Kn010G3/L0140-k0 3.38E-09
H0610-G1dh/none-Hi-Kn010G3/L0039-k0 3.41E-09
GCH043-G1dh/none-Hi-Kn010G3/L0000-k0 3.74E-09
H0610-G1dh/none-Hi-Kn010G3/TR01L008-k0 3.48E-09
H0610-G1dh/none-Hi-Kn010G3/L0148-k0 3.28E-09
GCH062-G1dh/none-Hi-Kn010G3/L0000-k0 3.73E-09
H0610-G1dh/none-Hi-Kn010G3/L0163-k0 3.38E-09
H0610-G1dh/none-Hi-Kn010G3/L0233-k0 3.55E-09
H0610-G1dh/none-Hi-Kn010G3/L0230-k0 4.00E-09
GCH006-G1dh/none-Hi-Kn010G3/L0000-k0 4.06E-09
H0610-G1dh/none-Hi-Kn010G3/L0032-k0 3.72E-09
H0610-G1dh/none-Hi-Kn010G3/L0181-k0 3.51E-09
H0610-G1dh/none-Hi-Kn010G3/TR01L009-k0 3.81E-09
H0610-G1dh/none-Hi-Kn010G3/L0141-k0 3.86E-09
H0610-G1dh/none-Hi-Kn010G3/L0079-k0 4.23E-09
GCH094-G1dh/none-Hi-Kn010G3/L0270-k0 3.60E-09
GCH066-G1dh/none-Hi-Kn010G3/L0000-k0 4.29E-09
GCH064-G1dh/none-Hi-Kn010G3/L0000-k0 4.14E-09
H0610-G1dh/none-Hi-Kn010G3/L0066-k0 4.20E-09
GCH027-G1dh/none-Hi-Kn010G3/L0000-k0 3.83E-09
H0610-G1dh/none-Hi-Kn010G3/L0003-k0 4.01E-09
H0610-G1dh/none-Hi-Kn010G3/L0042-k0 4.27E-09
H0610-G1dh/none-Hi-Kn010G3/TR01L011-k0 4.02E-09
H0610-G1dh/none-Hi-Kn010G3/L0000-k0 3.97E-09
GCH015-G1dh/none-Hi-Kn010G3/L0000-k0 4.14E-09
H0610-G1dh/none-Hi-Kn010G3/L0175-k0 3.84E-09
GCH100-G1dh/none-Hi-Kn010G3/L0011-k0 3.81E-09
GCH014-G1dh/none-Hi-Kn010G3/L0000-k0 4.20E-09
GCH053-G1dh/none-Hi-Kn010G3/L0000-k0 4.05E-09
hCE115HA-E22Hh/L0000-k0/GL4-E22Hk/H0000-E22L 4.28E-09
GCH094-G1dh/none-Hi-Kn010G3/L0011-k0 3.88E-09
GCH045-G1dh/none-Hi-Kn010G3/L0000-k0 4.63E-09
H0610-G1dh/none-Hi-Kn010G3/TR01L012-k0 4.25E-09
H0610-G1dh/none-Hi-Kn010G3/L0115-k0 4.34E-09
H0610-G1dh/none-Hi-Kn010G3/L0044-k0 4.57E-09
H0610-G1dh/none-Hi-Kn010G3/L0107-k0 4.38E-09
H0610-G1dh/none-Hi-Kn010G3/L0007-k0 4.39E-09
GCH013-G1dh/none-Hi-Kn010G3/L0000-k0 4.44E-09
H0610-G1dh/none-Hi-Kn010G3/L0045-k0 4.66E-09
GCH010-G1dh/none-Hi-Kn010G3/L0000-k0 4.12E-09
GCH040-G1dh/none-Hi-Kn010G3/L0000-k0 4.80E-09
H0610-G1dh/none-Hi-Kn010G3/L0002-k0 4.43E-09
H0610-G1dh/none-Hi-Kn010G3/L0016-k0 4.44E-09
GCH007-G1dh/none-Hi-Kn010G3/L0000-k0 4.93E-09
GCH042-G1dh/none-Hi-Kn010G3/L0000-k0 4.89E-09
rCE115H-E22Hh/rCE115L-k0/GL4-E22Hk/H0000-E22L 4.57E-09
H0610-G1dh/none-Hi-Kn010G3/L0129-k0 4.54E-09
H0610-G1dh/none-Hi-Kn010G3/L0065-k0 4.79E-09
GCH016-G1dh/none-Hi-Kn010G3/L0000-k0 4.59E-09
GCH035-G1dh/none-Hi-Kn010G3/L0000-k0 4.94E-09
GCH039-G1dh/none-Hi-Kn010G3/L0000-k0 4.95E-09
GCH099-G1dh/none-Hi-kn010G3/L0000-k0 4.24E-09
H0610-G1dh/none-Hi-Kn010G3/L0041-k0 4.85E-09
GCH019-G1dh/none-Hi-Kn010G3/L0000-k0 4.36E-09
GCH029-G1dh/none-Hi-Kn010G3/L0000-k0 5.01E-09
GCH056-G1dh/none-Hi-Kn010G3/L0011-k0 4.31E-09
H0610-G1dh/none-Hi-Kn010G3/L0147-k0 4.38E-09
GCH034-G1dh/none-Hi-Kn010G3/L0000-k0 5.09E-09
GCH003-G1dh/none-Hi-Kn010G3/L0000-k0 5.20E-09
H0610-G1dh/none-Hi-Kn010G3/L0139-k0 4.78E-09
H0610-G1dh/none-Hi-Kn010G3/L0089-k0 5.24E-09
H0610-G1dh/none-Hi-Kn010G3/L0113-k0 4.82E-09
H0610-G1dh/none-Hi-Kn010G3/L0180-k0 4.48E-09
GCH005-G1dh/none-Hi-Kn010G3/L0000-k0 5.32E-09
GCH067-G1dh/none-Hi-Kn010G3/L0000-k0 5.24E-09
H0610-G1dh/none-Hi-Kn010G3/L0187-k0 4.92E-09
H0610-G1dh/none-Hi-Kn010G3/L0043-k0 5.14E-09
H0610-G1dh/none-Hi-Kn010G3/L0117-k0 4.92E-09
GCH061-G1dh/none-Hi-Kn010G3/L0000-k0 5.13E-09
GCH022-G1dh/none-Hi-Kn010G3/L0000-k0 4.92E-09
H0610-G1dh/none-Hi-Kn010G3/L0091-k0 5.43E-09
GCH023-G1dh/none-Hi-Kn010G3/L0000-k0 4.94E-09
H0610-G1dh/none-Hi-Kn010G3/L0062-k0 5.28E-09
H0610-G1dh/none-Hi-Kn010G3/L0136-k0 5.04E-09
H0610-G1dh/none-Hi-Kn010G3/TR01L003-k0 5.08E-09
H0610-G1dh/none-Hi-Kn010G3/L0069-k0 5.32E-09
H0610-G1dh/none-Hi-Kn010G3/L0123-k0 5.08E-09
GCH025-G1dh/none-Hi-Kn010G3/L0000-k0 5.05E-09
GCH100-G1dh/none-Hi-Kn010G3/L0000-k0 5.39E-09
H0610-G1dh/none-Hi-Kn010G3/L0046-k0 5.45E-09
H0610-G1dh/none-Hi-Kn010G3/L0144-k0 4.84E-09
GCH026-G1dh/none-Hi-Kn010G3/L0000-k0 5.17E-09
H0610-G1dh/none-Hi-Kn010G3/L0138-k0 5.24E-09
GCH056-G1dh/none-Hi-Kn010G3/L0000-k0 5.03E-09
H0610-G1dh/none-Hi-Kn010G3/L0129-k0 5.28E-09
GCH032-G1dh/none-Hi-Kn010G3/L0000-k0 5.74E-09
H0610-G1dh/none-Hi-Kn010G3/TR01L005-k0 5.37E-09
GCH012-G1dh/none-Hi-Kn010G3/L0000-k0 5.40E-09
GCH055-G1dh/none-Hi-Kn010G3/L0000-k0 5.60E-09
H0610-G1dh/none-Hi-Kn010G3/L0104-k0 5.90E-09
GCH059-G1dh/none-Hi-Kn010G3/L0000-k0 5.70E-09
GCH054-G1dh/none-Hi-Kn010G3/L0000-k0 5.30E-09
GCH008-G1dh/none-Hi-Kn010G3/L0000-k0 5.55E-09
H0610-G1dh/none-Hi-Kn010G3/L0232-k0 5.38E-09
H0610-G1dh/none-Hi-Kn010G3/L0126-k0 5.62E-09
GCH094-G1dh/none-Hi-Kn010G3/L0000-k0 5.89E-09
H0610-G1dh/none-Hi-Kn010G3/L0132-k0 5.65E-09
H0610-G1dh/none-Hi-kn010G3/L0106-k0 5.66E-09
GCH054-G1dh/none-Hi-Kn010G3/L0011-k0 5.25E-09
H0610-G1dh/none-Hi-Kn010G3/L0109-k0 5.70E-09
H0610-G1dh/none-Hi-Kn010G3/L0063-k0 6.03E-09
GCH068-G1dh/none-Hi-Kn010G3/L0000-k0 6.23E-09
GCH057-G1dh/none-Hi-Kn010G3/L0000-k0 5.61E-09
H0610-G1dh/none-Hi-kn010G3/L0137-k0 5.87E-09
(3-3) Modification of pI by Point Mutations
[0723] In commercial production of bispecific antibodies, a high
level of purity is required. When using ion-exchange
chromatography, modifying the molecular isoelectric point (pI) has
been reported to be effective (PLoS One. 2013; 8(2):e57479).
Therefore, point mutations for pI modifications were introduced
into CDR1, CDR2, and CDR3 of the humanized anti-human GPC3 antibody
sequence produced in Reference Example 2, H0000-ERY27_HK (SEQ ID
NO: 54), to prepare modified antibodies. Next, the affinity of
these modified antibodies for soluble human GPC3 was
determined.
[0724] As a result, amino acid modifications that can lower the pI
while maintaining the affinity for human GPC3 were found to be
amino acids at positions 19, 43, 53, and 61 according to Kabat
numbering.
[0725] Combination of sites showing effects of maintaining the
affinity for human GPC3 and lowering the pI yielded antibodies
having the affinities and pI values shown in Table 14.
TABLE-US-00016 Calculated human pI value GPC3 KD Antibody name
(homomeric Antibody name (single-arm Mutation sites based
(homomeric antibody) antibody) (single-arm antibody) antibody) on
H0610-E2704sEpsc H0610-E2704sEpsc/L0000-k0 7.8
H0610-G1dh/none-Hi-Kn010G3/L0000-k0 4.16E-09 --
GCH054-E2704sEpsc/L0011-k0 6.2 GCH054-G1dh/none-Hi-Kn010G3/L0011-k0
5.25E-09 K19T/Q43E/P52aG/K53E/G55P/Q61E GCH065-E2704sEpsc/L0011-k0
6.4 GCH065-G1dh/none-Hi-Kn010G3/L0011-k0 8.84E-10
K19T/Q43E/P52aG/K53P/G55P/Q61E GCH094-E2704sEpsc/L0011-k0 6.2
GCH094-G1dh/none-Hi-Kn010G3/L0011-k0 4.54E-09
K19T/I37V/P40A/Q43E/I48M/P52aG/ K53E/G55P/Q61E
(3-4) Modifying the Extracellular Matrix-Binding Ability by Point
Mutation
[0726] It has been reported that non-specific binding to the
extracellular matrix (ECM) and such may have effects on
pharmacokinetics (MAbs. 2012 November-December; 4(6):753-60).
Therefore, the ECM-binding ability of the modified antibodies
obtained in the Reference Examples was determined by the method
described in Reference Example 8. As a result, the humanized
anti-human CD3.epsilon. chain and anti-human GPC3 bispecific
antibody, GPC3_ERY27_hCE115 (SEQ ID NOs: 54, 55, and 56), were
confirmed to have high ECM-binding abilities. Therefore, any of the
point mutations examined in Reference Examples 3-1, 3-2, and 3-3
for the humanized anti-human CD3.epsilon. chain antibody sequence
hCE115HA-ERY27_HE (SEQ ID NO: 55) was investigated to be a
combination for reducing the ECM-binding ability. As a result,
amino acids at positions 11, 16, 52a, 53, 98, and 100 by Kabat
numbering were found to contribute to the maintenance of affinity
for CD3.epsilon. and to have influence on the reduction of the
ECM-binding ability, and antibodies with a reduced ECM-binding
ability in comparison to that of an antibody variant of the
humanized anti-human CD3.epsilon. chain and anti-human GPC3
bispecific antibody, GPC3_ERY27_hCE115, were obtained (Table
15).
TABLE-US-00017 TABLE 15 ECM binding ratio (stan- Antibody name dard
= 1) GPC3_ERY22_CE115 (rCE115H-E22Hh/ 4.0
rCE115L-k0/GL4-E22Hk/H0000-E22L) GPC3_ERY27
(hCE115HA-E22Hh/L0000-k0/ 50.9 GL4-E22Hk/H0000-E22L)
CE115HA236-E22Hh/GLS3108-k0/GL4-E22Hk/ 429.9 H0610-E22L
CE115HA236-E22Hh/GLS3108-k0/GL4-E22Hk/ 414.8 H0000-E22L
CE115HA251-E22Hh/L0000-k0/GL4-E22Hk/H0000-E22L 346.9
CE115HA251-E22Hh/L0000-k0/GL4-E22Hk/H0610-E22L 334.4
TR01H002-E22Hh/GLS3108-k0/GL4-E22Hk/H0610-E22L 301.1
TR01H002-E22Hh/GLS3108-k0/GL4-E22Hk/H0000-E22L 216.9
TR01H015-E22Hh/L0000-k0/GL4-E22Hk/H0610-E22L 185.7
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0208-k0 50.4
CE115HA122-E22Hh/L0000-k0/GL4-E22Hk/H0000-E22L 47.0
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0211-k0 15.5
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0206-k0 15.4
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0209-k0 7.4
rCE115H-E22Hh/rCE115L-k0/GL4-E22Hk/H0610-E22L 4.6
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0204-k0 4.4
TR01H067-E2702GsKsc/GCH054-E2704sEpsc/L0212-k0 3.3
TR01H113-E2702GsKsc/GCH065-E2704sEpsc/L0011-k0 2.5
TR01H082-E2702GsKsc/GCH065-E2704sEpsc/L0011-k0 1.7
TR01H113-E2702GsKsc/GCH094-E2704sEpsc/L0011-k0 1.6
rCE115H-E22Hh/rCE115L-k0/L0000-E22Hk/H0610-E22L 1.4
TR01H084-E2702GsKsc/GCH065-E2704sEpsc/L0011-k0 1.3
TR01H084-E2702GsKsc/GCH094-E2704sEpsc/L0011-k0 1.2
TR01H082-E2702GsKsc/GCH094-E2704sEpsc/L0201-k0 1.1
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0000-k0 0.8
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0201-k0 0.8
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0203-k0 0.8
TR01H082-E2702GsKsc/GCH094-E2704sEpsc/L0011-k0 0.7
TR01H109-E2702GsKsc/GCH065-E2704sEpsc/L0011-k0 0.7
TR01H067-E2702GsKsc/GCH054-E2704sEpsc/L0222-k0 0.6
TR01H067-E2702GsKsc/GCH054-E2704sEpsc/L0201-k0 0.5
TR01H109-E2702GsKsc/GCH094-E2704sEpsc/L0011-k0 0.4
TR01H113-E2702sKsc/GCH065-E2704sEpsc/L0011-k0 0.3
MRAH-G1d/MRAL-k0(standard) 1
(3-5) Modifying the Binding Ability to the SuRe.TM. Ligand by Point
Mutations
[0727] An example where the binding of an antibody to Protein A
depends on its variable region sequence (VH3) is known (J Biomol
Tech. 2011 July; 22(2):50-2). In the Protein A purification of the
humanized anti-human CD3.epsilon. chain and anti-human GPC3
bispecific antibody, removal of the homomeric anti-CD3 antibody is
important for suppressing non-specific reactions via CD3.
Therefore, it is considered desirable to suppress the binding of
the homomeric anti-CD3 antibody to Protein A. Presumably, the
SuRe.TM. ligand will be used in commercial production, and thus
point mutations for SuRe.TM. ligand binding were introduced into
CDR2 of the humanized anti-CD3 antibody H-chain variants,
TR01H082-E2702GsKsc and TR01H084-E2702GsKsc (SEQ ID NO: 398 and
399), to prepare modified antibodies. The binding ability of these
modified antibodies to the SuRe.TM. ligand was determined by the
method described in Reference Example 9. As a result, amino acids
at positions 19, 57, and 59 by Kabat numbering were found to
contribute to the maintenance of the affinity for CD3.epsilon. and
to have influence on the Sure.TM. ligand-binding ability, and
antibodies with a reduced Sure.TM. ligand-binding ability in
comparison to that of TR01H082-E2702GsKsc/L0011-k0 (SEQ ID NOs: 398
and 410) or TR01H084-E2702GsKsc/L0011-k0 (SEQ ID NOs: 399 and 410)
were obtained (Table 16).
TABLE-US-00018 TABLE 16 SuRe .TM. binding Antibody name (RU)
Mutation sites based on CE115HA000 TR01H084-E2702GsKsc/L0011-k0
5065.8 R16G/A52aD/N53Q/D72A/L78I/G98A/Y100G/A102I
TR01H082-E2702GsKsc/L0011-k0 4469.2 V11L/A52aD/N53Q/G98A/Y100G
TR01H090-E2702GsKsc/L0011-k0 3606.3 V11L/R16G/A52aD/N53Q/G98A/Y100G
TR01H093-E2702GsKsc/L0011-k0 2459.7 V11L/A52aD/N53Q/K64Q/G98A/Y100G
TR01H094-E2702GsKsc/L0011-k0 2351.9 V11L/A52aD/N53Q/K64S/G98A/Y100G
TR01H114-E2702GsKsc/L0011-k0 1485.5
R16G/A52aD/N53Q/T57S/D72A/L78I/G98A/Y100G/A102I
TR01H092-E2702GsKsc/L0011-k0 1159.5 V11L/A52aD/N53Q/K64A/G98A/Y100G
TR01H100-E2702GsKsc/L0011-k0 383.0 V11L/A52aD/N53Q/T57S/G98A/Y100G
TR01H111-E2702GsKsc/L0011-k0 50.7
R16G/R19K/A52aD/N53Q/D72A/L78I/G98A/Y100G/A102I
TR01H110-E2702GsKsc/L0011-k0 29.5 R19K/A52aD/N53Q/G98A/Y100G
TR01H091-E2702GsKsc/L0011-k0 27.5 V11L/R19K/A52aD/N53Q/G98A/Y100G
TR01H091-E2702GsKsc/L0011-k0 15.0 V11L/R19K/A52aD/N53Q/G98A/Y100G
TR01H112-E2702GsKsc/L0011-k0 8.8
R16G/A52aD/N53Q/T57Q/D72A/L78I/G98A/Y100G/A102I
TR01H113-E2702GsKsc/L0011-k0 7.0
R16G/A52aD/N53Q/Y59V/D72A/L78I/G98A/Y100G/A102I
TR01H096-E2702GsKsc/L0011-k0 2.7 V11L/A52aD/N53Q/T57G/G98A/Y100G
TR01H109-E2702GsKsc/L0011-k0 2.2 V11L/A52aD/N53Q/Y59V/G98A/Y100G
TR01H098-E2702GsKsc/L0011-k0 1.6 V11L/A52aD/N53Q/T57P/G98A/Y100G
TR01H107-E2702GsKsc/L0011-k0 1.4 V11L/A52aD/N53Q/Y59Q/G98A/Y100G
TR01H103-E2702GsKsc/L0011-k0 1.4 V11L/A52aD/N53Q/Y59G/G98A/Y100G
TR01H104-E2702GsKsc/L0011-k0 1.0 V11L/A52aD/N53Q/Y59I/G98A/Y100G
TR01H105-E2702GsKsc/L0011-k0 0.8 V11L/A52aD/N53Q/Y59L/G98A/Y100G
TR01H099-E2702GsKsc/L0011-k0 0.6 V11L/A52aD/N53Q/T57Q/G98A/Y100G
TR01H102-E2702GsKsc/L0011-k0 0.5 V11L/A52aD/N53Q/Y59F/G98A/Y100G
TR01H101-E2702GsKsc/L0011-k0 0.5 V11L/A52aD/N53Q/T57V/G98A/Y100G
TR01H108-E2702GsKsc/L0011-k0 0.4 V11L/A52aD/N53Q/Y59T/G98A/Y100G
TR01H097-E2702GsKsc/L0011-k0 0.1 V11L/A52aD/N53Q/T57L/G98A/Y100G
TR01H106-E2702GsKsc/L0011-k0 0.0 V11L/A52aD/N53Q/Y59P/G98A/Y100G
TR01H095-E2702GsKsc/L0011-k0 -0.2
V11L/A52aD/N53Q/T57F/G98A/Y100G
(3-6) Production of Optimized Bispecific Antibodies by Combining
Point Mutations that Lead to Improvement of Various Properties
[0728] Optimized modified antibodies can be produced by combining
the point mutations that lead to improvement of various properties
as described in Reference Examples 3-1 to 3-5. As examples of such
modified antibodies, the antibodies described in Table 17 were
produced, and they were subjected to the T-cell-dependent cellular
cytotoxicity (TDCC) evaluation using methods similar to those of
Reference Example 1. The results are shown in FIGS. 15 to 20. As a
result, optimized humanized anti-human CD3.epsilon. chain and
anti-human GPC3 bispecific antibodies showing a T-cell-dependent
cellular cytotoxicity equivalent to or greater than that of
GPC3_ERY22_rCE115 prior to humanization were obtained.
TABLE-US-00019 TABLE 17 Sample number in TDCC assay, and
abbreviation of antibody name in drug efficacy evaluation Sample
number in TDCC assay Abbreviation of antibody name 1
GPC3_ERY22_CE115 (rCE115H-E22Hh/rCE115L-k0/ GL4-E22Hk/H0000-E22L) 2
GPC3_ERY27 (hCE115HA-E22Hh/L0000-k0/GL4-E22Hk/ H0000-E22L) 3
CE115HA251-E22Hh/L0000-k0/GL4-E22Hk/H0000-E22L 4
CE115HA236-E22Hh/GLS3108-k0/GL4-E22Hk/H0000-E22L 5
TR01H002-E22Hh/GLS3108-k0/GL4-E22Hk/H0000-E22L 6
CE115HA122-E22Hh/L0000-k0/GL4-E22Hk/H0000-E22L 7
rCE115H-E22Hh/rCE115L-k0/L0000-E22Hk/H0610-E22L 8
rCE115H-E22Hh/rCE115L-k0/GL4-E22Hk/H0610-E22L 13
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0000-k0 14
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0201-k0 15
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0203-k0 16
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0204-k0 17
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0206-k0 18
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0208-k0 19
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0209-k0 20
TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0211-k0 21
rCE115H-E2702GsKsc/H0610-E2704sEpsc/L0000-k0 22
TR01H061-E2702GsKsc/H0610-E2704sEpsc/L0000-k0 23
TR01H068-E2702GsKsc/H0610-E2704sEpsc/L0000-k0 24
TR01H071-E2702GsKsc/H0610-E2704sEpsc/L0000-k0 25
TR01H067-E2702GsKsc/GCH054-E2704sEpsc/L0201-k0 26
TR01H067-E2702GsKsc/GCH054-E2704sEpsc/L0212-k0 27
TR01H067-E2702GsKsc/GCH054-E2704sEpsc/L0222-k0 28
TR01H067-E2702GsKsc/GCH054-E2704sEpsc/L0000-k0 29
TR01H082-E2702GsKsc/GCH094-E2704sEpsc/L0201-k0 30
TR01H082-E2702GsKsc/GCH094-E2704sEpsc/L0011-k0 31
TR01H084-E2702GsKsc/GCH094-E2704sEpsc/L0011-k0 32
TR01H084-E2702GsKsc/GCH065-E2704sEpsc/L0011-k0 33
TR01H082-E2702GsKsc/GCH065-E2704sEpsc/L0011-k0 34
TR01H109-E2702GsKsc/GCH094-E2704sEpsc/L0011-k0 35
TR01H109-E2702GsKsc/GCH065-E2704sEpsc/L0011-k0 36
TR01H113-E2702GsKsc/GCH094-E2704sEpsc/L0011-k0 37
TR01H113-E2702GsKsc/GCH065-E2704sEpsc/L0011-k0 38
TR01H113-E2702sKsc/GCH065-E2704sEpsc/L0011-k0
[0729] Reference Examples 3-1 to 3-6 showed that the following
amino acid residues, for example, are important for maintaining the
properties of the optimized anti-human CD3.epsilon. chain and
anti-human GPC3 bispecific antibodies showing a T-cell-dependent
cellular cytotoxicity equivalent to or greater than that of
GPC3_ERY22_rCE115 prior to humanization.
[0730] In anti-human CD3.epsilon. chain antibodies, the examples
are Len at position 11, Gly at position 16, Asp at position 52a,
Gln at position 53, Ala at position 72, Ile at position 78, Ala at
position 98, Gly at position 100, and Ile at position 102. In
anti-human GPC3 antibodies, the examples are Thr at position 19,
Glu at position 43, Gly at position 52a, Pro or Glu at position 53,
Pro at position 55, and Glu at position 61. Furthermore, in common
antibody L chains, the examples are Pro at position 25, Pro at
position 27a, Pro at position 27b, Ile at position 33, Gln at
position 34, Arg or Trp at position 56, and Tyr at position 89.
(All positions are indicated by Kabat numbering).
[Reference Example 4] Evaluation of the In Vivo Efficacy
[0731] Some of the above-described antibodies were evaluated for
their in vivo efficacy using tumor-bearing models.
[0732] Evaluation of the in vivo efficacy was carried out on
representative antibodies from among those shown in Table 17, which
have been confirmed to have cytotoxic activities from the in vitro
assay described in Reference Example 3-6. In the in vivo efficacy
evaluation, any influence caused by differences in the
microenvironment due to tumor aggregate formation on the evaluation
results was taken into consideration. Therefore, two types of human
cancer cell lines having different sensitivities to the antibody
drug efficacy, i.e., PC-10 and NCI-H446, were used for the
evaluation, even though the GPC3 expression levels of these cell
lines were nearly equal. The cell lines were transplanted into the
NOD scid mice, and the NOD scid mice with confirmed tumor
establishment were subjected to injection of T cells grown by in
vitro culturing of human PBMCs. The mice (referred to as a T-cell
injected model) were treated by administration of the optimized
anti-human CD3.epsilon. chain and anti-human GPC3 bispecific
antibodies.
[0733] More specifically, in drug efficacy tests of the optimized
anti-human CD3.epsilon. chain and anti-human GPC3 bispecific
antibodies using the PC-10 T-cell injected model, the tests below
were performed. T cells were expansively cultured using PBMCs
separated from blood collected from healthy volunteers and T cell
activation/expansion kit/human (MACS Miltenyi biotec). The human
cancer cell line PC-10 (1.times.10.sup.7 cells) was mixed with
Matrigel.TM. Basement Membrane Matrix (BD), and transplanted to the
inguinal subcutaneous region of NOD scid mice (CLEA Japan, female,
6W). The day of transplantation was defined as day 0. On the day
before transplantation, the anti-asialo-GM1 antibody (Wako Pure
Chemicals) was administered intraperitoneally to the mice at 0.2
mg/mouse. On days 13 to 15 after the transplantation, the mice were
separated into groups according to their body weight and tumor
size, and the anti-asialo-GM1 antibody was administered again
intraperitoneally to the mice at 0.2 mg/mouse. On the following
day, T cells obtained by the aforementioned expansive culturing
were transplanted intraperitoneally at 3.times.10.sup.7
cells/mouse. Four hours after T-cell transplantation, the optimized
anti-human CD3.epsilon. chain and anti-human GPC3 bispecific
antibodies were administered intravenously through the caudate vein
at 1 mg/kg. The optimized anti-human CD3.epsilon. chain and
anti-human GPC3 bispecific antibodies were administered only
once.
[0734] As a result, anti-tumor activities were more clearly
observed in the optimized anti-human CD3.epsilon. chain and
anti-human GPC3 bispecific antibody-administered group than in the
solvent-administered group (FIG. 21a, b).
[0735] Drug efficacy tests for the optimized anti-human
CD3.epsilon. chain and anti-human GPC3 bispecific antibodies on the
NCI-H446 T-cell injected model were performed by similar methods.
The optimized anti-human CD3.epsilon. chain and anti-human GPC3
bispecific antibodies were administered once intravenously through
the caudate vein at 5 mg/kg against NCI-H446.
[0736] As a result, anti-tumor activities were more clearly
observed in the optimized anti-human CD3.epsilon. chain and
anti-human GPC3 bispecific antibody-administered group than in the
solvent-administered group (FIG. 22a, b).
[Reference Example 5] Production of Antibody Expression Vectors,
and Antibody Expression and Purification
[0737] Amino acid substitutions were introduced by methods known to
those skilled in the art such as using the QuikChange Site-Directed
Mutagenesis Kit (Stratagene), PCR, or the In-fusion Advantage PCR
cloning kit (TAKARA) to construct expression vectors. Nucleotide
sequences of the obtained expression vectors were determined by a
method known to those skilled in the art. The produced plasmids
were transiently introduced into cells of the human embryonic
kidney cancer-derived cell line HEK293H (Invitrogen) or
FreeStyle293 (Invitrogen) to express antibodies. From the obtained
culture supernatants, antibodies were purified using the rProtein A
Sepharose.TM. Fast Flow (GE Healthcare) by a method known to those
skilled in the art. Absorbance at 280 nm of the purified antibody
solutions was measured using a spectrophotometer, and antibody
concentrations were calculated from the determined values using an
absorption coefficient calculated by the PACE method (Protein
Science 1995; 4: 2411-2423).
[Reference Example 6] the ADCC Activity of Each Test Antibody Using
Human Peripheral Blood Mononuclear Cells as the Effector Cell
[0738] The ADCC activity of each test antibody was determined
according to the method below.
[0739] Human peripheral blood mononuclear cells (hereinafter
referred to as human PBMC) were used as the effector cell to
measure the ADCC activity of each test antibody as below.
(1) Preparation of a Human PBMC Solution
[0740] From a healthy volunteer (adult male) of Chugai
Pharmaceutical Co. Ltd., 50 mL of peripheral blood was collected
using a syringe preloaded with 200 .mu.L of a 1000 unit/mL heparin
solution (Novo-Heparin Injection 5000 units, Novo Nordisk). The
peripheral blood was diluted two-fold with PBS(-), divided into
four aliquots, and added into a Leucosep lymphocyte separation tube
(Greiner Bio-one) that had been loaded with 15 mL of Ficoll-Paque
PLUS and subjected to centrifugation in advance. This separation
tube containing aliquots of peripheral blood was centrifuged at
2150 rpm for ten minutes at room temperature, and then the
mononuclear cell fraction was collected. The cells in each fraction
were washed once with Dulbecco's Modified Eagle's Medium (SIGMA)
containing 10% FBS (hereinafter referred to as 10% FBS/D-MEM) and
then suspended in 10% FBS/D-MEM at a cell density of
5.times.10.sup.6 cells/mL. After incubation in an incubator at
37.degree. C. for one hour, the cells were washed once with 10%
FBS/D-MEM, and the cells were suspended in 10% FBS/D-MEM to produce
a cell density of 2.times.10.sup.5 cells/mL. The cell suspension
was subjected to the experiment below as the target cell.
(2) Chromium Release Assay (ADCC Activity)
[0741] The ADCC activity was evaluated from the specific chromium
release rate according to the chromium release method. First,
antibody solutions prepared at each concentration (0, 0.004, 0.04,
0.4, 4, and 40 .mu.g/mL) were added to a 96-well U-bottomed plate
at 50 .mu.L per well. Next, the target cells were seeded at 50
.mu.L per well (1.times.10.sup.4 cells/well), and this was allowed
to stand at room temperature for 15 minutes. The human PBMC
solution prepared in (1) was added at 100 .mu.L per well
(5.times.10.sup.5 cells/well), and the plate was left to stand in a
5% carbon dioxide gas incubator at 37.degree. C. for four hours,
followed by centrifugation. The radioactivity of 100 .mu.L of
culture supernatant in each well of the plate was measured using a
gamma counter. The specific chromium release rate was determined
based on the following equation: Specific chromium release rate
(%)=(A-C).times.100/(B-C)
[0742] In this equation, A represents the mean value of
radioactivity (cpm) of 100 .mu.L of culture supernatant in each
well; B represents the mean value of radioactivity (cpm) of 100
.mu.L of culture supernatant in the well where 100 .mu.L of a 2%
aqueous NP-40 solution (Nonidet P-40, Nacalai Tesque) and 50 .mu.L
of 10% FBS/D-MEM had been added to the target cells; and C
represents the mean value of radioactivity (cpm) of 100 .mu.L of
culture supernatant in the well where 150 .mu.L of 10% FBS/D-MEM
had been added to the target cells. The examinations were performed
in triplicate and the mean values and standard deviations of the
specific chromium release rates (%) in the above-mentioned
examination reflecting the ADCC activity were calculated for each
of the test antibodies.
[Reference Example 7] Assessment of Tm of the Modified Antibodies
by Differential Scanning Fluorimetry
[0743] In this examination, the Tm (thermal denaturation
temperature) value of the modified antibodies was assessed by
differential scanning fluorimetry using Rotor-Gene Q (QIAGEN). It
has been reported that this method has a favorable correlation with
Tm assessment using a differential scanning calorimeter widely
known as a method for evaluating thermal stability of antibodies
(Journal of Pharmaceutical Science 2010; 4: 1707-1720).
[0744] The 5000.times.-concentrated SYPRO.TM. orange (Molecular
Probes) was diluted with PBS (Sigma), and then admixed with the
antibody solutions to prepare measurement samples. Twenty-.mu.L
aliquots of each sample were placed into measurement tubes, and the
temperature was increased from 30.degree. C. to 99.degree. C. at a
temperature elevation rate of 240.degree. C./hr. Changes in
fluorescence accompanying the temperature elevation were detected
at 470 nm (excitation wavelength)/555 nm (fluorescence
wavelength).
[0745] The data were analyzed using the Rotor-Gene Q Series
software (QIAGEN) to calculate the temperature at which
fluorescence transition was observed, and this temperature was
defined as the Tm.
[Reference Example 8] Assessment of the ECM-Binding Ability
[0746] The assessment was carried out according to the method
described in WO2012093704. Specifically, BD Matrigel (BD
Biosciences, #356237) was prepared at 2 mg/mL using TBS (Takara,
#T903), and this was dispensed into a 96-well measurement plate
(Meso Scale Discovery, #L15XB-3(High Bind)) at 5 .mu.L per well and
then allowed to stand overnight in a cool place. Then, 150 .mu.L of
an ECL blocking buffer (PBS containing 0.05% Tween20, 0.5% BSA, and
0.01% sodium azide) was dispensed into each well of the plate, and
this was allowed to stand at room temperature for two hours or
more.
[0747] A goat anti-human IgG(.gamma.) (Invitrogen, #628400) was
ruthenium-labeled with MSD SULFO-TAG NHS Ester (Meso Scale
Discovery, #R91AN-2) by following the attached instructions. This
was diluted in an ECL dilution buffer (PBS containing 0.01%
Tween20, 0.1% BSA, and 0.01% sodium azide) to have a final
concentration of 2 .mu.g/mL. Furthermore, the standard antibody and
the test antibodies were diluted in PBS-T (PBS containing 0.05%
Tween 20 and 0.01% sodium azide) to have a final concentration of 3
.mu.g/mL.
[0748] To a 96-well reaction plate (Thermo scientific, Nunc
#145399), 10 .mu.L of the ECL dilution buffer, 20 .mu.L of the
standard antibody and test antibody (3 .mu.g/mL), and 30 .mu.L of
the ruthenium-labeled antibody (2 .mu.g/mL) were added
sequentially, and this was allowed to react for one hour at room
temperature with stirring in the dark.
[0749] The ECL blocking buffer was removed from the 96-well
measurement plate by tilting, 50 .mu.L of the sample solution from
the 96-well reaction plate was added, and this was allowed to stand
in the dark at room temperature for one hour. This was followed by
removal of the sample solution from the 96-well measurement plate
by tilting, and immediately after addition of 150 .mu.L of
2.times.T buffer (4.times.MSD Read Buffer T (Meso Scale Discovery)
diluted two-fold using the ECL dilution buffer), ECL measurements
were taken. SECTOR Imager 2400 (Meso Scale Discovery) was used for
taking the measurements.
[0750] Analyses were carried out by dividing the fluorescence
intensity of the test antibody by the fluorescence intensity of the
standard antibody to calculate and compare the intensities by
defining the value for the standard antibody to be 1.
[Reference Example 9] Assessment of the SuRe.TM. Ligand-Binding
Ability
[0751] The ability to bind to the SuRe.TM. ligand was assessed by
using Biacore.TM.-T200 (GE Healthcare Japan). HBS-EP+(GE Healthcare
Japan) was used for the running buffer, and an amine coupling kit
(GE Healthcare Japan) was used to covalently bind the Mab Select
SuRe.TM. Ligand (GE Healthcare Japan) to the CM5 chip
(carboxymethyl dextran-coated chip). The antibody used as the
analyte was prepared at 5 .mu.g/mL using HBS-EP+. Measurements were
carried out by first injecting the 5-.mu.g/mL antibody solution at
a flow rate of 10 L/min for 3 minutes, then switching to HBS-EP+,
and measuring the response (RU) after allowing the flow to continue
for 0.5 minutes. After completion of the measurements, the sensor
chip was regenerated by washing with 10 mM Gly-HCl at pH 1.5. For
the control flow cell, a similar experiment was performed without
covalent bonding of the ligand to the chip, and the affinity for
the SuRe.TM. ligand was analyzed by taking the difference between
the responses (RU).
[0752] Sequences corresponding to the SEQ ID NOs mentioned in the
Reference Examples are shown in the Table below.
TABLE-US-00020 TABLE 18 SEQ ID NO: Name 1 GPC3 nucleotide sequence
(NM_001164617.1) 2 GPC3 amino acid sequence (NP_001158089.1) 3
Signal sequence 4 T cell receptor .alpha.-chain peptide
(CAA26636.1) 5 T cell receptor .beta.-chain peptide (C25777) 6 T
cell receptor .gamma.1-chain peptide (A26659) 7 T cell receptor
.gamma.2-chain peptide (AAB63312.1) 8 T cell receptor .delta.-chain
peptide (AAA61033.1) 9 CD3 .gamma.-chain nucleotide (NM_000073.2)
10 CD3 .delta.-chain nucleotide (NM_000732.4) 11 CD3
.epsilon.-chain nucleotide (NM_000733.3) 12 CD3 .gamma.-chain
peptide (NP_000064.1) 13 CD3 .delta.-chain peptide (NP_000723.1) 14
CD3 .epsilon.-chain peptide (NP_000724.1) 15~22 Peptide linker 23
Human C.gamma.1 24 Human C.gamma.2 25 Human C.gamma.3 26 Human
C.gamma.4 27 Fc.gamma.RI nucleotide (NM_000566.3) 28 Fc.gamma.RI
peptide (NP_000557.1) 29 Fc.gamma.RIIA nucleotide (BC020823.1) 30
Fc.gamma.RIIA peptide (AAH20823.1) 31 Fc.gamma.RIIB nucleotide
(BC146678.1) 32 Fc.gamma.RIIB peptide (AAI46679.1) 33
Fc.gamma.RIIIA nucleotide (BC033678.1) 34 Fc.gamma.RIIIA peptide
(AAH33678.1) 35 Fc.gamma.RIIIB nucleotide (BC128562.1) 36
Fc.gamma.RIIIB peptide (AAI28563.1) 37 Fc region (addition of A to
the N terminus of RefSeq accession number AAC82527.1) 38 Fc region
(addition of A to the N terminus of RefSeq accession number
AAB59393.1) 39 Fc region (addition of A to the N terminus of RefSeq
accession number AAB59394.1) 40 H0000, GPC3 H-chain variable region
41 GL4, GPC3 L-chain variable region 42 rCE115H, CE115 H-chain
variable region 43 rCE115L, CE115 L-chain variable region 44 G1dh
45 ERY22_Hk 46 ERY22_Hh 47 GL4-ERY22_Hk 48 H0000-ERY22_L 49
rCE115H-ERY22_Hh 50 rCE115L-k0 51 hCE115HL (Heavy chain of
humanized CE115) 52 hCE115HA (Heavy chain of humanized CE115) 53
L0000 (Light chain of humanized CE115) 54 H0000-ERY27_HK 55
hCE115HA-ERY27_HE 56 L0000-k0 57 E22Hh 58 E22Hk 59 Hi-Kn010G3 60
E2702GsKsc 61 E2704sEpsc 62 E2702sKsc 63 k0 64 CE115HA177 65
CE115HA178 66 CE115HA179 67 CE115HA180 68 hCE115HAa 69 TR01H006 70
TR01H007 71 TR01H008 72 TR01H009 73 TR01H010 74 TR01H011 75
TR01H012 76 TR01H013 77 TR01H014 78 TR01H015 79 TR01H016 80
TR01H017 81 TR01H018 82 TR01H019 83 TR01H020 84 TR01H021 85
TR01H022 86 TR01H023 87 TR01H024 88 TR01H025 89 TR01H026 90
TR01H027 91 TR01H028 92 TR01H029 93 TR01H030 94 TR01H031 95
TR01H032 96 TR01H033 97 TR01H034 98 TR01H035 99 TR01H036 100
TR01H037 101 TR01H038 102 TR01H039 103 TR01H040 104 TR01H041 105
TR01H042 106 TR01H043 107 TR01H044 108 TR01H045 109 TR01H046 110
TR01H047 111 TR01H048 112 TR01H049 113 TR01H050 114 TR01H051 115
TR01H052 116 TR01H053 117 TR01H054 118 TR01H055 119 TR01H056 120
TR01H057 121 TR01H058 122 TR01H061 123 TR01H062 124 TR01H063 125
TR01H064 126 TR01H065 127 TR01H066 128 TR01H067 129 TR01H068 130
TR01H069 131 TR01H070 132 TR01H071 133 TR01H072 134 TR01H073 135
TR01H074 136 TR01H075 137 TR01H076 138 TR01H077 139 TR01H079 140
TR01H080 141 TR01H081 142 TR01H082 143 TR01H083 144 TR01H084 145
TR01H090 146 TR01H091 147 TR01H092 148 TR01H093 149 TR01H094 150
TR01H095 151 TR01H096 152 TR01H097 153 TR01H098 154 TR01H099 155
TR01H100 156 TR01H101 157 TR01H102 158 TR01H103 159 TR01H104 160
TR01H105 161 TR01H106 162 TR01H107 163 TR01H108 164 TR01H109 165
TR01H110 166 TR01H111 167 TR01H112 168 TR01H113 169 TR01H114 170
GCH003 171 GCH005 172 GCH006 173 GCH007 174 GCH008 175 GCH010 176
GCH012 177 GCH013 178 GCH014 179 GCH015 180 GCH016 181 GCH019 182
GCH022 183 GCH023 184 GCH025 185 GCH026 186 GCH027 187 GCH029 188
GCH032 189 GCH034 190 GCH035 191 GCH039 192 GCH040 193 GCH042 194
GCH043 195 GCH045 196 GCH053 197 GCH054 198 GCH055 199 GCH056 200
GCH057 201 GCH059 202 GCH060 203 GCH061 204 GCH062 205 GCH064 206
GCH065 207 GCH066 208 GCH067 209 GCH068 210 GCH073 211 GCH094 212
GCH098 213 GCH099 214 GCH100 215 H0610 216 L0000vk1 217 L0002 218
L0003 219 L0006 220 L0007 221 L0008 222 L0009 223 L0011 224 L0012
225 L0013 226 L0014 227 L0015 228 L0016 229 L0032 230 L0038 231
L0039 232 L0041 233 L0042 234 L0043 235 L0044 236 L0045 237 L0046
238 L0047 239 L0062 240 L0063 241 L0064 242 L0065 243 L0066 244
L0069 245 L0075 246 L0079 247 L0082 248 L0085 249 L0089
250 L0090 251 L0091 252 L0093 253 L0104 254 L0106 255 L0107 256
L0109 257 L0113 258 L0115 259 L0117 260 L0120 261 L0122 262 L0123
263 L0124 264 L0125 265 L0126 266 L0127 267 L0129 268 L0132 269
L0134 270 L0136 271 L0137 272 L0138 273 L0139 274 L0140 275 L0141
276 L0143 277 L0144 278 L0145 279 L0147 280 L0148 281 L0149 282
L0151 283 L0152 284 L0154 285 L0155 286 L0157 287 L0160 288 L0161
289 L0163 290 L0167 291 L0168 292 L0173 293 L0175 294 L0180 295
L0181 296 L0186 297 L0187 298 L0200 299 L0201 300 L0202 301 L0203
302 L0204 303 L0205 304 L0206 305 L0207 306 L0208 307 L0209 308
L0210 309 L0211 310 L0212 311 L0213 312 L0214 313 L0215 314 L0216
315 L0217 316 L0218 317 L0219 318 L0220 319 L0222 320 L0223 321
L0224 322 L0226 323 L0227 324 L0228 325 L0229 326 L0230 327 L0231
328 L0232 329 L0233 330 L0234 331 L0235 332 L0236 333 L0237 334
L0238 335 L0239 336 L0240 337 L0241 338 L0242 339 L0243 340 L0246
341 L0247 342 L0248 343 L0249 344 L0250 345 L0258 346 L0259 347
L0260 348 L0261 349 L0262 350 L0263 351 L0264 352 L0265 353 L0266
354 L0267 355 L0268 356 L0269 357 L0270 358 L0271 359 L0272 360
TR01L001 361 TR01L002 362 TR01L003 363 TR01L004 364 TR01L005 365
TR01L006 366 TR01L007 367 TR01L008 368 TR01L009 369 TR01L010 370
TR01L011 371 TR01L012 372 TR01L013 373 TR01L015 374 TR01L016 375
TR01L017 376 TR01L018 377 TR01L019 378 TR01L020 379 TR01L023 380
TR01L024 381 CE115HA122-E22Hh 382 CE115HA236-E22Hh 383
CE115HA251-E22Hh 384 GCH054-E2704sEpsc 385 GCH065-E2704sEpsc 386
GCH094-E2704sEpsc 387 H0610-E2704sEpsc 388 hCE115HA-E22Hh 389
rCE115H-E22Hh 390 rCE115H-E2702GsKsc 391 TR01H002-E22Hh 392
TR01H015-E22Hh 393 TR01H040-E2702GsKsc 394 TR01H061-E2702GsKsc 395
TR01H067-E2702GsKsc 396 TR01H068-E2702GsKsc 397 TR01H071-E2702GsKsc
398 TR01H082-E2702GsKsc 399 TR01H084-E2702GsKsc 400
TR01H109-E2702GsKsc 401 TR01H113-E2702GsKsc 402 TR01H113-E2702sKsc
403 GL4-E22Hk 404 L0000-E22Hk 405 H0000-E22L 406 H0610-E22L 407
rCE115L-k0 408 GLS3108-k0 409 L0000-k0 410 L0011-k0 411 L0201-k0
412 L0203-k0 413 L0204-k0 414 L0206-k0 415 L0208-k0 416 L0209-k0
417 L0211-k0 418 L0212-k0 419 L0222-k0 420 TR01H001 421 TR01H002
422 TR01H003 423 TR01H004 424 rCE115H 425 CE115HA121 426 CE115HA122
427 CE115HA124 428 CE115HA192 429 CE115HA236 430 CE115HA251 431
CE115HA252 432 E22L
[Reference Example 10] Production of Human CD3 Gene-Substituted
Mice
[0753] (1) Construction of a Mouse Cd3 Gene Region Modification
Vector (FIG. 26A)
[0754] A bacterial artificial chromosome (BAC) clone was used, into
which a genomic region where the mouse CD3.epsilon., CD3.delta.,
and CD3.gamma. genes are positioned had been cloned. A loxP
sequence was inserted at the position approximately 3.5 kb 5'
upstream of the gene region encoding mouse Cd3E in this BAC, and
the genome region further upstream was removed leaving
approximately 3.1 kb. At that time, the loxP sequence was
introduced together with neomycin-resistance (neo) gene cassette
and insertion was conducted by homologous recombination using a
Red/ET system (GeneBridges). In that case, from among the
Escherichia coli clones that grew in a kanamycin-supplemented
medium, clones for which polymerase chain reaction (PCR) method
resulted in correct amplification were selected. Next, loxP
sequence and Rox sequences were placed at 3' downstream of the
Cd3.gamma. gene on the BAC. More specifically, the loxP sequence
and Rox sequences were introduced along with hygromycin-resistance
(Hyg) gene cassette, and insertion was conducted by homologous
recombination using a Red/ET system. In that case, from among the
Escherichia coli clones that grew in a hygromycin-supplemented
medium, clones in which the loxP sequence and Rox sequences were
inserted as expected were selected by PCR method. Next, the genomic
region 3' downstream of the Hyg gene cassette was removed leaving
approximately 3.4 kb.
[0755] (2) Introduction of a Mouse Cd3 Gene Region Modification
Vector into Mouse Embryonic Stem Cells (ES Cells) (FIG. 26A)
[0756] The above-mentioned mouse Cd3 gene region modification
vector was introduced into mouse ES cells (C57BL/6N mouse-derived
cells) via electroporation, and after selective culturing with
G418, drug-resistant clones were obtained. From these clones,
screening for homologous recombinants was performed by a PCR
method. For electroporation, 60 .mu.g of the mouse Cd3 gene region
modification vector was linearized with NotI or the NotI-untreated
circular vector was extracted with phenol/chloroform, precipitated
with ethanol, and then dissolved in PBS.
[0757] ES cells used in screening were cultured on a 96-well plate
and washed twice using 200 .mu.l of PBS solution per well. Then,
the cells were treated at 55.degree. C. for two hours after adding
a cell lysis buffer having the following composition (5 .mu.l of
10.times.LA buffer II (TAKARA LA for Taq), 5 .mu.l of 25 mM
MgCl.sub.2, 5 .mu.l of 5% NP-40, 2 .mu.l of proteinase K (TAKARA,
20 mg/ml), and 33 .mu.l of distilled water), and subsequently
treated at 95.degree. C. for 15 minutes to inactivate proteinase K,
to thereby serve as PCR samples.
[0758] The PCR reaction mixture was made up of 1 .mu.l of the
sample, 2.5 .mu.l of 10.times.LA buffer II, 2.5 .mu.l of 25 mM
MgCl.sub.2, 4 .mu.l of dNTP (2.5 mM), 0.1 .mu.l each of the primers
(50 .mu.M each), 0.25 .mu.l of LA Taq (TAKARA), and 14.55 .mu.l of
distilled water (25 .mu.l in total). The PCR conditions included
preheating at 94.degree. C. for two minutes, 35 cycles of an
amplification cycle of 98.degree. C. for ten seconds and 68.degree.
C. for 4 minutes 30 seconds, and additional heating at 68.degree.
C. for five minutes.
[0759] The following primers were used. The primers were HygF1474
which was positioned within the Hyg gene cassette as a forward
primer, and g4989R which was positioned as a reverse primer at the
mouse genomic region on the 3' downstream side of the 3' homology
arm in the mouse Cd3 gene modification vector (see FIG. 27). In
samples of the ES cells in which homologous recombination occurred,
an approximately 4-kb band was amplified. HygF1474 (forward)
5'-TATCAGAGCTTGGTTGACGG-3' (SEQ ID NO: 436); and g4989R (reverse)
5'-ACTCGTTGTGGCTTAGAAGCAGTAACAATACC-3' (SEQ ID NO: 437).
Furthermore, clones from which amplification signals were obtained
using the above-mentioned primer set were subjected to validation
using a different primer set. More specifically, e27248F was
positioned as a forward primer at the mouse genomic region on the
5' upstream side of the 5' homology arm in the mouse Cd3 gene
modification vector, and Neo0635R was positioned as a reverse
primer within the Neo gene cassette. In samples of ES cells in
which homologous recombination occurred, an approximately 4-kb band
was amplified.
TABLE-US-00021 e27248F (forward) (SEQ ID NO: 438)
5'-ACTGTAATCCTAGTACTTAGGAGGCTGAGG-3'; and Neo0635R (reverse) (SEQ
ID NO: 439) 5'-AATCCATCTTGTTCAATGGCCGATCC-3'.
[0760] (3) Construction of a Human CD3 Gene Region Introduction
Vector (FIG. 26B)
[0761] A BAC clone was used, into which a genomic region where the
human CD3.epsilon., CD3.delta., and CD3.gamma. genes are positioned
had been cloned. A loxP sequence was inserted at 5' upstream of the
gene region encoding human CD3.epsilon. in this BAC. At that time,
the loxP sequence was introduced along with Hyg gene cassette, and
insertion was conducted by homologous recombination using a Red/ET
system (GeneBridges). In that case, from among the Escherichia coli
clones that grew in a hygromycin-supplemented medium, clones for
which PCR method resulted in correct amplification were selected.
Next, at 3' downstream of the human CD3.gamma. gene in the BAC,
puromycin-resistance (Puro) gene flanked on both ends by Frt
sequences was introduced together with Neo gene cassette to
position a Rox sequence further downstream, and insertion was
conducted by homologous recombination using a Red/ET system. In
that case, from among the Escherichia coli clones that grew in a
kanamycin-supplemented medium, clones in which the Frt sequences,
the Puro gene, the Rox sequence, and the Neo gene were inserted as
expected were selected by PCR method.
[0762] (4) Introduction of a Human CD3 Gene Region Introduction
Vector and a Recombinase Expression Vector into Cd3 Gene
Region-Modified Mouse ES Cells
[0763] The human CD3 gene region introduction vector, a Cre
recombinase expression vector, and a Dre recombinase expression
vector were introduced via electroporation into ES cell clones
(1D4, 5H1, 615, and 3A5) in which the loxP sequences and Rox
sequences were correctly inserted at the targeted sites of the
mouse Cd3 gene region in the above-mentioned step; and after
selective culturing with puromycin, the grown ES cell clones were
genotyped.
[0764] First, PCR screening was performed for selection of clones
in which recombination between the loxP sequences and between the
Rox sequences placed at the mouse Cd3 gene region took place by the
action of Cre and Dre, and the genomic region from Cd3.epsilon. to
Cd3.gamma. was deleted. The ES cells used in screening were
cultured on a 96-well plate, washed twice using 200 .mu.l of PBS
per well, and treated at 55.degree. C. for two hours after adding a
cell lysis buffer having the following composition (5 .mu.l of
10.times.LA buffer II (TAKARA LA for Taq), 5 .mu.l of 25 mM
MgCl.sub.2, 5 .mu.l of 5% NP-40, 2 .mu.l of proteinase K (TAKARA,
20 mg/mL), and 33 .mu.l of distilled water), and subsequently
treated at 95.degree. C. for 15 minutes to inactivate proteinase K,
to thereby serve as PCR samples.
[0765] The PCR reaction mixture was made up of 1 .mu.l of the
sample, 2.5 .mu.l of 10.times. LA buffer II, 2.5 .mu.l of 25 mM
MgCl.sub.2, 4 .mu.l of dNTP (2.5 mM), 0.1 .mu.l each of the primers
(50 .mu.M each), 0.25 .mu.l of LA Taq (TAKARA), and 14.55 .mu.l of
distilled water (25 .mu.l in total). The PCR conditions included
preheating at 94.degree. C. for two minutes, 35 cycles of an
amplification cycle of 98.degree. C. for ten seconds and 68.degree.
C. for 4 minutes 30 seconds, and additional heating at 68.degree.
C. for five minutes. The following primers were used. The primers
were e30230F which was positioned as a forward primer at the
genomic region on the 5' upstream side of the mouse Cd3.epsilon.
gene, and g1439R which was positioned as a reverse primer at the
genomic region on the 3' downstream side of the mouse Cd3.gamma.
gene (see FIG. 28A). In samples of the ES cells in which the Cd3
gene region was deleted, an approximately 0.7-kb band was
amplified.
TABLE-US-00022 e30230F (forward) (SEQ ID NO: 440)
5'-TAGCAGCCTTCAGATGAAGAGGTAGGACTC-3'; and g1439R (reverse) (SEQ ID
NO: 441) 5'-TTGATGTGCCACCTCACTGCTGCACTGG-3'.
[0766] PCR screening was performed for selecting clones in which
the human CD3 gene region was introduced from the ES cell clones
deficient in the mouse Cd3 gene region. The PCR samples that were
used for detecting the deletion of the mouse Cd3 gene region were
subjected to the screening. The PCR reaction mixture was made up of
1 .mu.l of the sample, 2.5 .mu.l of 10.times.LA buffer II, 2.5
.mu.l of 25 mM MgCl.sub.2, 4 .mu.l of dNTP (2.5 mM), 0.1 .mu.l each
of the primers (50 .mu.M each), 0.25 .mu.l of LA Taq (TAKARA), and
14.55 .mu.l of distilled water (25 .mu.L in total). The PCR
conditions included preheating at 94.degree. C. for two minutes, 35
cycles of an amplification cycle of 94.degree. C. for 30 seconds,
58.degree. C. for one minute, and 72.degree. C. for five minutes,
and additional heating at 72.degree. C. for five minutes. The
following primers were used. The primers were hCD3e_5arm_F2 which
was positioned as a forward primer at the genomic region on the 5'
upstream side of the human CD3.epsilon. gene, and hCD3e_ex2_R2
which was positioned as a reverse primer within the second exon of
the human CD3.epsilon. gene (see FIG. 28B). In samples of the ES
cells in which the human CD3 gene region was introduced, an
approximately 5.5-kb band was amplified.
TABLE-US-00023 hCD3e_5arm_F2 (forward) (SEQ ID NO: 442)
5'-AACTGACAATGGGACATCAGCTGA-3'; and hCD3e_ex2_R2 (reverse) (SEQ ID
NO: 443) 5'-ATGGGACTGTTACTTTACTAAGAT-3'.
[0767] (5) Production of Mouse Cd3 Gene-Deficient and Human CD3
Gene-Introduced Mice
[0768] The homologous recombinant ES clones were suspended by
trypsin treatment, and washed with the ES cell medium. Female
BALB/c mice which were subjected to superovulation treatment by
administering 5 IU of equine chorionic gonadotropin (eCG) and human
chorionic gonadotropin (hCG) intraperitoneally at 48-hour intervals
were crossed with male mice of the same strain. The day when a plug
was confirmed in a female mouse was regarded as day 0.5. On
gestation day 3.5, blastocyst-stage embryos collected by perfusing
the uterus were used as host embryos, in which 10 to 15 of the ES
cells were injected. The embryos after the injection were
transferred into the uterus of ICR recipient females on Day 2.5
pseudopregnancy, and their offspring were obtained 17 days later.
Screening based on the coat color of the offspring obtained by
injection of the ES cells to the blastocysts, yielded chimeric mice
having a mixture of the recombinant ES cells (black) and the host
blastocyst-derived cells (albino). After sexual maturation, the
male chimeric mice were crossed with C57BL/6N-female mice, and
transmission of the knock-in allele to the next generation was
confirmed by a PCR method using the genomic DNA extracted from the
tissues of the second-generation mice as the template. PCR was
performed by the above-mentioned method used for screening of the
ES cells. As a result, individuals from which the human CD3 gene
region-specific 5.5-kb signal and the mouse Cd3 gene region
deficiency-specific 0.7-kb signal were detected were obtained, and
the human CD3 gene region allele and the mouse Cd3 gene
region-deficient allele were confirmed to be transmitted to these
individuals. Furthermore, breeding of mice having the
above-described genotype yielded mouse individuals whose mouse Cd3
gene region is homozygously deleted and which have the human CD3
gene region, that is, human CD3 gene region-substituted mice were
obtained. Transgenic mice in which human CD3.epsilon. alone had
been introduced (hereinafter, hCD3sTg mice) were produced according
to the report by Wang et al. (Wang et. al. (1994) PNAS.
91:9402-9406), and they were examined as comparisons in the later
experiments.
[0769] (6) Thymus Weights and Spleen Weights of Human CD3
Gene-Substituted Mice
[0770] Spleen and thymus were collected from mice (12 to 14-week
old, male) and the tissue weights were measured. As shown in FIG.
29, the thymus of the human CD3-substituted mice did not show gross
abnormalities. Tissue weight per body weight was calculated for
analysis. The body weights and tissue weights (spleen and thymus)
were measured for four male mice in each group, and represented as
graphs. The tissue weight per body weight ratios were calculated,
the values obtained for each individual are plotted by a black dot,
and the mean value is shown by a column (FIG. 30). Regarding spleen
weight, increasing trend was observed in the Cd3 gene-deficient
mice as compared to mice of other genotypes, but no remarkable
differences were observed. On the other hand, regarding thymus
weight, the Cd3 gene-deficient mice showed decrease down to one
third or so as compared to that of the wild-type. In the human CD3
gene-substituted mice produced by introducing a human CD3 gene into
the Cd3 gene-deficient mice, recovery of thymus weight was
observed, and particularly in the individuals of line no. 1C3,
thymus weight was recovered even to the level equivalent to that of
the wild-type mice. As reported by Wang et al., thymic atrophy was
observed in hCD3sTg mice (Wang et. al. (1994) PNAS.
91:9402-9406).
[0771] (7) Confirmation of Expressions of Human CD3 and Mouse Cd3
in the Respective Lines of Human CD3 Gene-Substituted Mice
[0772] --Confirmation by RT-PCR Method Using Hemocyte RNA--
[0773] Expressions of human CD3.epsilon., human CD3.delta., human
CD3.gamma., mouse Cd3.epsilon., mouse Cd3.delta., and mouse
Cd3.gamma. were analyzed by RT-PCR using hemocyte RNA. Using a
Catrimox-14 RNA Isolation Kit (TaKaRa Bio), total RNA was prepared
from blood collected from the dorsal metatarsal vein or the
abdominal vena cava. A 1 .mu.g portion each of the total RNAs was
used as a template to synthesize cDNAs by performing reverse
transcription reactions with a SuperScript III First Strand cDNA
Synthesis Kit (Invitrogen) using Oligo dT (20) primers. Human
CD3.epsilon., human CD3.delta., human CD3.gamma., mouse
Cd3.epsilon., mouse Cd3.delta., and mouse Cd3.gamma. were detected
by performing PCR using the synthesized cDNAs as templates. Primers
for the protein coding regions were designed to detect the
expression of all of the genes. Human CD3.epsilon. was detected
using the combination of forward primer E0333F
(5'-AAGAAATGGGTGGTATTACACAGACACC-3' (SEQ ID NO: 444)) and reverse
primer E0912R (5'-TGGGCCAGCGGGAGGCAGTGTTCTCCAGAGG-3' (SEQ ID NO:
445)). Human CD36 was detected using the combination of forward
primer D0092F (5'-TAGTTCGGTGACCTGGCTTTATCTACTGG-3' (SEQ ID NO:
446)) and reverse primer D0685R
(5'-ATGGCTGCTTCTAGAAGCCACCAGTCTCAGG-3' (SEQ ID NO: 447)). Human
CD3.gamma. was detected using the combination of forward primer
G0048F (5'-TGCTCCACGCTTTTGCCGGAGGACAG-3' (SEQ ID NO: 448)) and
reverse primer G0666R (5'-TAGGAGGAGAACACCTGGACTACTC-3' (SEQ ID NO:
449)). On the other hand, mouse Cd3.epsilon. was detected using the
combination of forward primer e0065F
(5'-AGCATTCTGAGAGGATGCGGTGGAACAC-3' (SEQ ID NO: 450)) and reverse
primer e0699R (5'-TGCTCGGAGGGCTGGATCTGGGTCCACAG-3' (SEQ ID NO:
451)). Mouse Cd38 was detected using the combination of forward
primer d055F (5'-TCATCCTGTGGCTTGCCTCTATTTGTTGC-3' (SEQ ID NO: 452))
and reverse primer d651R (5'-TTGCTATGGCACTTTGAGAAACCTCCATC-3' (SEQ
ID NO: 453)). Mouse Cd3.gamma. was detected using the combination
of forward primer g080F (5'-AATACTTCTACTGGAGAAGCAAAGAG-3' (SEQ ID
NO: 454)) and reverse primer g316R
(5'-TAGTTGCATTTAGAGGACTTATTATGC-3' (SEQ ID NO: 455)).
[0774] The composition of the PCR reaction solution (25 .mu.l in
total) was made up of 1 .mu.l of the sample, 2.5 .mu.l of 10.times.
Ex buffer, 2 .mu.l of dNTP (2.5 mM), 0.1 .mu.l each of the primers
(50 .mu.M each), 0.25 .mu.l of Ex Taq (TAKARA), and 19.05 .mu.l of
distilled water. The PCR conditions for human CD3.delta., human
CD3.gamma., mouse Cd3.delta., and mouse Cd3.gamma. included
preheating at 94.degree. C. for two minutes, 35 cycles of an
amplification cycle of 94.degree. C. for 30 seconds, 60.degree. C.
for 30 seconds, and 72.degree. C. for two minutes, and additional
heating at 72.degree. C. for five minutes. For human CD3.epsilon.
and mouse Cd3.epsilon., the PCR conditions included preheating at
94.degree. C. for two minutes, 40 cycles of an amplification cycle
of 94.degree. C. for 30 seconds, 60.degree. C. for 30 seconds, and
72.degree. C. for two minutes, and additional heating at 72.degree.
C. for five minutes. PCR primers were designed so that the detected
amplification products of human CD3.epsilon., human CD3.delta., and
human CD3.gamma. will be 580 bp, 594 bp, and 620 bp, respectively,
and those of mouse Cd3.epsilon., mouse Cd3.delta., and mouse
Cd3.gamma. will be 635 bp, 597 bp, and 237 bp, respectively.
[0775] In the Cd3 gene-deficient mice, the respective mouse Cd3
molecule-derived PCR signals were not detected. Only human
CD3.epsilon., human CD3.delta., and human CD3.gamma. were detected,
and none of mouse Cd3.epsilon., mouse Cd3.delta., and mouse
Cd3.gamma. was detected from the samples derived from lines 1C3 and
8I12 of the above-mentioned lines among the human CD3
gene-substituted mouse lines (line nos. 1C3, 3B1, 8112, and 2A4)
produced by introducing the human CD3 gene region to the Cd3
gene-deficient mice (FIG. 31). From the samples derived from
wild-type mice, human CD3.epsilon., human CD3.delta., and human
CD3.gamma. were not detected, and mouse Cd3.epsilon., mouse
Cd3.delta., and mouse Cd3.gamma. were detected (FIG. 31). These
results confirmed that mice expressing human CD3.epsilon.,
CD3.delta., and CD3.gamma. instead of mouse Cd3.epsilon.,
Cd3.delta., and Cd3.gamma. were obtained as designed. Line 4HH3 in
FIG. 31 was analyzed in an individual in which the mouse Cd3 allele
is a wild-type and the human CD3 gene has been introduced, and the
respective human CD3 molecules and the respective mouse Cd3
molecules are both detected. Subsequently, it was cross-bred with
Cd3-deficient mice to establish a mouse Cd3 allele-deficient and
human CD3 gene-expressing line.
[0776] --Analysis by Immunohistological Staining--
[0777] The tissue distribution was examined using the anti-CD3
antibody as the primary antibody. CD3 staining was not observed in
any of the tissues from the Cd3-deficient mice, while CD3-specific
staining equivalent to that of wild-type mice was observed for the
human CD3-substituted mice produced by introducing the human CD3
genes to the Cd3-deficient mice. More specifically, specific
staining was observed in the T cell zones in the thymus (FIG. 32A)
and spleen (FIG. 32B). In all tissues, staining was observed only
in the T cell zone, similarly to the wild-type mice. Furthermore,
staining was not observed in the Cd3 gene-deficient mice,
indicating that staining in the human CD3 gene-substituted mice was
due to the expression of the introduced human CD3 genes.
Furthermore, the detection of CD3.epsilon. in the major organs was
the same as in the wild-type, and ectopic staining was not observed
(Table 19).
TABLE-US-00024 TABLE 19 hCD3, mCD3KOTG mouse, IACUC 14-074;
Findings: - negative; .+-. very slight; + slight; ++ moderate; +++
severe; IHC Staining: - negative; .+-. rare; + occasional; ++
frequent; +++ constant mCD3ko, hCD3TG Line #4HH3 #8I12 #1C3 Animal
No. 008-130 010-163 003-91 003-85 003-86 001-60 168 169 97 Gender
Organs Findings Date of IHC Staining: A, 2014 Jun. 19; B, 2014 Jun.
25 IHC Staining: CD3 A A A A A A A A A Thymus Atrophy + .+-. - -
.+-. - - - - Lymphocyte, cortex ++ +++ +++ +++ +++ +++ +++ +++ +++
Lymphocyte, medulla +++ +++ +++ +++ +++ +++ +++ +++ +++ Other
tissues - - - - - - - - - Mesentery Atrophy + + .+-. - .+-. - - + +
Lymphocyte, paracortex ++ ++ ++ +++ +++ ++ +++ ++ ++ Lymphocyte,
follicle + + + + + + + + + Lymphocyte, medulla + + + + + + + + +
Other tissues - - - - - - - - - Ileum Atrophy of GALT - - - - - - -
- - Lymphocyte, GALT + .+-. .+-. .+-. ++ + + ++ + Lymphocyte,
lamina propria .+-. + + .+-. + + + + + Other tissues - - - - - - -
- - Spleen Atrophy .+-. - - - - .+-. - - - Lymphocyte, PALS +++ +++
+++ +++ +++ +++ +++ +++ +++ Lymphocyte, follicle + + + + + + + + +
Lymphocyte, red pulp ++ ++ ++ ++ ++ ++ ++ ++ ++ Other tissues - - -
- - - - - - Liver Lymphocyte, sinusoid + + + + + + + + + Other
tissues - - - - - - - - - Kidney Lymphocyte, interstitium .+-. -
.+-. .+-. .+-. .+-. .+-. .+-. .+-. Other tissues - - - - - - - - -
Adrenal gland Lymphocyte, interstitium - - - - - .+-. .+-. - .+-.
Other tissues - - - - - - - - - Lung Lymphocyte, alveolar wall .+-.
- .+-. .+-. .+-. .+-. .+-. .+-. .+-. Other tissues - - - - - - - -
- Heart Lymphocyte, interstitium - - .+-. - - - .+-. .+-. .+-.
Other tissues - - - - - - - - - Gastrocnemius muscle - - - - - - -
- - hCD3 .epsilon. TG mCD3KO C57BL/6N Line 3-1-78 #195 None None
Animal No. 195 790 001-63 001-64 001-67 B6-01 B6-02 B6-03 Gender
Organs Positive control Findings Date of IHC Staining: A, 2014 Jun.
19; B, 2014 Jun. 25 IHC Staining: CD3 A A A A A B B B Thymus
Atrophy + - ++ ++ ++ - - - Lymphocyte, cortex +++ +++ - - - +++ +++
+++ Lymphocyte, medulla +++ +++ - - - +++ +++ +++ Other tissues - -
- - - - - - Mesentery NA NA Atrophy ++ ++ ++ - - - Lymphocyte,
paracortex - - - +++ +++ +++ Lymphocyte, follicle - - - + + +
Lymphocyte, medulla - - - + + + Other tissues - - - - - - Ileum NA
NA Atrophy of GALT ++ ++ ++ - - - Lymphocyte, GALT - - - ++ + +
Lymphocyte, lamina propria - - - + + .+-. Other tissues - - - - - -
Spleen Atrophy - - + + + - - - Lymphocyte, PALS +++ +++ - - - +++
+++ +++ Lymphocyte, follicle + + - - - + + + Lymphocyte, red pulp
++ ++ - - - ++ ++ ++ Other tissues - - - - - - - - Liver
Lymphocyte, sinusoid NA NA - - - + + + Other tissues - - - - - -
Kidney NA NA Lymphocyte, interstitium - - - .+-. .+-. .+-. Other
tissues - - - - - - Adrenal gland NA NA Lymphocyte, interstitium -
- - .+-. .+-. .+-. Other tissues - - - - - - Lung NA NA Lymphocyte,
alveolar wall - - - .+-. .+-. .+-. Other tissues - - - - - - Heart
NA NA Lymphocyte, interstitium - - - .+-. .+-. .+-. Other tissues -
- - - - - Gastrocnemius muscle NA NA - - - - - - indicates data
missing or illegible when filed
(8) Evaluation of Abundance Ratio of Mature T Cells in Human CD3
Gene-Substituted Mice
[0778] FACS analyses were performed using spleen cells. Spleens
were collected from mice (12 to 14-week old, male), and cells were
isolated using 70 .mu.m mesh. Erythrocytes were lysed by adding a
hemolytic agent (manufactured by SIGMA). After blocking using an Fc
blocking solution, FITC-labeled anti-mouse Cd3 antibody,
FITC-labeled anti-human CD3 antibody, APC-labeled anti-mouse Cd4
antibody, and PE-labeled anti-mouse Cd8 antibody were used on
2.times.10.sup.6 cells, and the respective positive cell counts
were analyzed by a flow cytometer. It was revealed that the Cd3
gene-deficient mice nearly completely lack in mature T cells, that
is, Cd4 and Cd8 single positive cells, while these cells were
present in the human CD3 gene-substituted mice at a ratio
equivalent to that in the wild-type.
Abundance Ratio of Mature T Cells
TABLE-US-00025 [0779] TABLE 20 Number of Experimental group samples
mCd3 hCD3 mCd4 mCd8 Human CD3s-substituted mouse #1C3 n = 4 ND.
38.8 (.+-.3.1) 19.6 (.+-.0.7) 16.1 (.+-.3.6) Human CD3s-substituted
mouse #4HH3 n = 2 ND. 29.8, 28.9 15.5, 13.9 17.5, 16.4 Human
CD3s-substituted mouse #8I12 n = 4 ND. 31.5 (.+-.5.4) 15.5
(.+-.3.1) 15.3 (.+-.2.7) hCD3E Tg mouse n = 4 19.5 (.+-.3.76) 13.0
(.+-.1.4) 7.4 (.+-.0.6) 7.8 (.+-.0.8) Cd3s-deficient mouse n = 4
ND. ND. 1.8 (.+-.1.3) 2.1 (.+-.0.6) C57BL/6N n = 4 40.4 (.+-.8.42)
ND. 20.3 (.+-.6.7) 12.7 (.+-.2.1)
[0780] The table shows the expression ratios of the respective
marker-positive cells with respect to the spleen cells (unit %).
The mean from four individuals is shown for each the experimental
group, except for human CD3.epsilon.-substituted mice #4HH3, and
the expression ratios of two individuals are shown for line #4HH3.
(The standard deviation is shown in parenthesis.) ND: not
detected.
[Reference Example 11] Evaluation of Immune Function of Human CD3
Gene-Substituted Mice
[0781] (1) Examination of the Ability to Produce Specific
Antibodies in Response to Immunization to Foreign Antigen
[0782] For production of specific antibodies against foreign
antigens, there must exist functional helper T cells that can bind
to antigenic peptides presented together with major
histocompatibility complex (MHC) antigens on the surface of
antigen-presenting cells such as dendritic cells, and the T cells
must have functions of giving instructions to antibody-producing
cells to produce appropriate antibodies. Whether the
above-mentioned human CD3 gene-substituted mice carry helper T
cells having normal functions and produce specific antibodies in
response to immunization to foreign antigens was examined.
Immunization was carried out using chicken ovalbumin (OVA) as the
sensitizing antigen together with Freund's adjuvant. Immunization
to OVA was performed twice with a four-week interval. More
specifically, the first immunization was performed by
subcutaneously applying, 100 .mu.g of OVA per animal with complete
Freund's adjuvant to the dorsal region, and four weeks later,
similar immunization was performed by subcutaneously applying the
antigen with incomplete Freund's adjuvant to the dorsal region. As
human CD3 gene-substituted mice, two lines (line nos. 1C3 and
8I12), each of which is derived from a different modified ES cell
clone, were selected, and compared to human
CD3.epsilon.-overexpressing mice. Furthermore, as controls,
wild-type mice and Cd3 gene-deficient mice were selected and
similar antigen immunizations were performed.
[0783] One week after the second immunization, the animals were
subjected to laparotomy under isoflurane anesthesia, and then
euthanized by collecting whole blood and allowing bleeding from the
abdominal vena cava. Serum was separated from the collected blood,
and the concentrations of OVA-specific IgG1 and OVA-specific IgE
were measured (FIG. 34).
[0784] As a result, neither IgG1 type nor IgE type OVA-specific
antibodies were detected from the serum of mouse Cd3-deficient
mice, whereas OVA-specific IgG1 and IgE were detected in both lines
of the human CD3 gene-substituted mice, and their levels were
equivalent to those of wild-type mice. These results showed that
human CD3 gene-substituted mice have normal ability to produce
antibodies in response to foreign antigen immunization.
INDUSTRIAL APPLICABILITY
[0785] The present invention provides anticancer agents and
pharmaceutical compositions for use in combination with another
anticancer agent, the agents or compositions comprising novel
multispecific antigen-binding molecules that maintain the strong
anti-tumor activity possessed by BiTE and the excellent safety
property of not inducing a cytokine storm or such independently
from cancer antigen, and also have long half-lives in blood.
Anticancer agents and pharmaceutical compositions that comprise an
antigen-binding molecule of the present invention as an active
ingredient can target glypican 3-expressing cells and tumor tissues
containing these cells and induce cytotoxicity. Administration of a
multispecific antigen-binding molecule of the present invention to
patients makes it possible to have a desirable treatment which not
only has a high level of safety but also a reduced physical burden,
and is highly convenient.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210388110A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210388110A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References