NZ795720A - Anti-gpc3 antibody - Google Patents
Anti-gpc3 antibodyInfo
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- NZ795720A NZ795720A NZ795720A NZ79572018A NZ795720A NZ 795720 A NZ795720 A NZ 795720A NZ 795720 A NZ795720 A NZ 795720A NZ 79572018 A NZ79572018 A NZ 79572018A NZ 795720 A NZ795720 A NZ 795720A
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- New Zealand
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Abstract
The present invention addresses the problem of providing: an anti-GPC3 antibody that recognizes an epitope different from those recognized by existing antibodies (e.g., GC33 and GC199), and that can specifically bind to GPC3 localized on a cell membrane even as a single-chain antibody; a CAR including said anti-GPC3 single-chain antibody; an immunocompetent cell expressing said CAR; an anti-GPC3 antibody gene or CAR gene; a vector including said anti-GPC3 antibody gene or CAR gene; a host cell into which said vector has been introduced; a method for specifically detecting GPC3; and a kit for specifically detecting GPC3. This antibody includes the specific heavy-chain CDRs 1-3 and the specific light-chain CDRs 1-3 defined in claim 1, and specifically binds to a human-derived GPC3 polypeptide. This antibody specifically binds to GPC3 localized on a cell membrane. A CAR-immunocompetent cell prepared from a CAR including said single-chain antibody is useful in cancer immunotherapy. ng said anti-GPC3 single-chain antibody; an immunocompetent cell expressing said CAR; an anti-GPC3 antibody gene or CAR gene; a vector including said anti-GPC3 antibody gene or CAR gene; a host cell into which said vector has been introduced; a method for specifically detecting GPC3; and a kit for specifically detecting GPC3. This antibody includes the specific heavy-chain CDRs 1-3 and the specific light-chain CDRs 1-3 defined in claim 1, and specifically binds to a human-derived GPC3 polypeptide. This antibody specifically binds to GPC3 localized on a cell membrane. A CAR-immunocompetent cell prepared from a CAR including said single-chain antibody is useful in cancer immunotherapy.
Description
The present invention ses the problem of providing: an anti-GPC3 antibody that recognizes
an epitope different from those recognized by existing antibodies (e.g., GC33 and GC199), and
that can ically bind to GPC3 localized on a cell membrane even as a single-chain antibody;
a CAR including said PC3 single-chain antibody; an immunocompetent cell expressing said
CAR; an anti-GPC3 antibody gene or CAR gene; a vector including said anti-GPC3 antibody gene
or CAR gene; a host cell into which said vector has been introduced; a method for ically
detecting GPC3; and a kit for specifically detecting GPC3. This antibody includes the specific heavychain
CDRs 1-3 and the specific light-chain CDRs 1-3 defined in claim 1, and specifically binds
to a human-derived GPC3 ptide. This antibody specifically binds to GPC3 localized on a
cell membrane. A CAR-immunocompetent cell prepared from a CAR including said single-chain
antibody is useful in cancer immunotherapy.
NZ 795720
ANTI-GPC3 ANTIBODY
Related applications
The present application is a divisional of New
Zealand patent application 754830, which is the national
phase entry of PCT international application
(published as WO 31586), the
entirety of each of which is incorporated by reference
herein.
cal Field
[0001A]
The present invention relates to: an antibody
specifically binding to GPC3 can-3) (anti-GPC3
antibody); a chimeric antigen receptor (hereinafter, also
referred to as "CAR") comprising PC3 single chain
antibody, a transmembrane region fused with a carboxyl (C)
terminus of the anti-GPC3 single chain antibody, and an
competent cell activation signal transduction region
fused with a C terminus of the transmembrane ; an
immunocompetent cell expressing the CAR; an anti-GPC3
antibody gene or a CAR gene; a vector comprising the anti-
GPC3 antibody gene or the CAR gene; a host cell in which
the vector has been introduced; a method for detecting
GPC3; and a kit for detecting GPC3.
[FOLLOWED BY PAGE 1a]
Background Art
Glypican-3 (GPC3) is an extracellular matrix protein
that is expressed in nic tissues, particularly, the
liver or the kidney, and associated with organogenesis.
The expression of GPC3 is not observed in human adult
tissues except for placenta, but is observed in tissues of
various s such as hepatocellular carcinoma, melanoma,
ovarian clear cell adenocarcinoma, and lung squamous cell
[FOLLOWED BY PAGE 2]
carcinoma. Thus, GPC3 is a protein that is expressed in
embryonic tissues, as in proteins such as α-fetoprotein
(AFP) and carcinoembryonic antigen (CEA), and is therefore
classified into embryonal carcinoma antigens.
Specifically, GPC3 is useful as a target molecule of
cancer treatment, a tumor marker and a diagnostic marker,
because its feature is that the protein is not expressed
in normal tissue cells, but is specifically expressed in
cancer cells.
GPC3 is a member of the proteoglycan family that
functions as extracellular matrix in cell adhesion in
organogenesis or as a receptor of a cell growth factor. A
GPI (glycosylphosphatidylinositol) anchor is added to
serine at position 560 located on the carboxyl (C)-
terminal side of GPC3. The GPI anchor plays a role in
localizing GPC3 on cell surface through covalent binding
to cell membrane lipid. Also, serine at position 495 and
serine at position 509 of GPC3 are modified with a heparan
sulfate chain (HS . The HS chain is known to
regulate a plurality of growth signal transduction
ys such as Wnt signal, FGF , and BMP signal
transduction pathways. A growth signal transduction
pathway involved is known to differ among the types of
cancers. For example, in cellular carcinoma (HCC),
cells grow by the ation of the Wnt signal pathway.
A common feature of the glypican family is the number of
cysteine as abundant as 16 in an extracellular region, and
these cysteine es are considered to contribute to
the stable formation of a mation by forming a
plurality of intramolecular disulfide bonds. The
possibility has been reported that GPC3 on cell membrane
surface is cleaved between ne (R) at position 358
and serine (S) at position 359 S359) by furin
convertase. However, since an amino (N)-terminal subunit
of GPC3 is cross-linked h olecular disulfide
bonds, GPC3, even when cleaved into two subunits, an N-
terminal subunit and a C-terminal subunit, by furin
convertase may probably retain its full-length ure
without dissociating these subunits. The structure of
soluble GPC3 remains a controversial subject. Thus, there
are many unclear points as to the conformation of GPC3
localized on a cell membrane, also including the
structures of isoforms of GPC3.
GPC3 on a cell membrane has a cated structure.
Therefore, for preparing an antibody against GPC3, it has
been considered desirable that the simplest structural
region is an epitope. A representative existing anti-GPC3
antibody includes a monoclonal antibody 1G12 which is
distributed by BioMosaics, Inc. This antibody is an
antibody obtained by immunizing Balb/c mice with an
antigen (C-terminal 70-residue polypeptide of GPC3)
designed so as to circumvent the cated structure or
localization of GPC3, to prepare hybridomas, and screening
the hybridomas using the antigen. Antibodies GC33 and
GC199 developed by a Japanese pharmaceutical cturer
are also monoclonal antibodies established on the basis of
the same concept as above and are dies obtained with
the C-terminal partial fragment of GPC3 as an n
(patent document 1).
Prior Art Document
Patent Document
Patent document 1: Japanese Patent No. 4011100
Summary of the ion
Object to be Solved by the Invention
An object of the present invention is to provide: an
anti-GPC3 antibody that recognizes an epitope different
from that for existing antibodies (e.g., GC33 and GC199)
and can specifically bind, even in the form of single
chain antibody, to GPC3 localized on a cell membrane; CAR
comprising the anti-GPC3 single chain antibody; an
immunocompetent cell expressing the CAR; a gene of the
PC3 antibody or a gene of the CAR; a vector
comprising the anti-GPC3 antibody gene or the CAR gene; a
host cell in which the vector has been introduced; a
method for specifically detecting GPC3; and a kit for
specifically detecting GPC3.
Means to Solve the Object
The present inventors are continuing nt
studies to attain the object. In the course of the
studies, the present inventors have prepared a novel anti-
GPC3 antibody by a phage display method which is an
approach different from tional monoclonal antibody
ation methods involving establishing hybridomas.
Specifically, an immune library of antibody genes was
synthesized using B cells derived from mice zed with
full-length human GPC3, and the genes were reconstituted
into a single chain antibody (scFv) library, which was
then incorporated into a phage display and expressed on
phage surface, followed by biopanning using recombinant
full-length human GPC3 and the GPC3-expressing cell line,
and further, if necessary, a competitor C-terminal
polypeptide of GPC3 serving as an epitope for the existing
antibodies, to prepare an anti-GPC3 antibody. The
prepared anti-GPC3 antibody has also been confirmed to be
useful for cancer therapy using T cells expressing a
chimeric antigen receptor (CAR) (hereinafter, also
referred to as "CAR-T cells"). The present invention has
been completed on the basis of these findings.
Specifically, the present ion is as follows.
An antibody ically g to a human GPC3
(glypican-3)-derived polypeptide consisting of the amino
acid sequence ented by SEQ ID NO: 155 (hereinafter,
also referred to as the "present antibody"), wherein the
antibody
(1-1) comprises a heavy chain complementarity
determining region (CDR) 1 consisting of the amino acid
sequence represented by SEQ ID NO: 1, a heavy chain CDR2
consisting of the amino acid ce represented by SEQ
ID NO: 2, and a heavy chain CDR3 consisting of the amino
acid sequence represented by SEQ ID NO: 3, and
a light chain CDR1 consisting of the amino acid
sequence represented by SEQ ID NO: 4, a light chain CDR2
ting of the amino acid sequence represented by SEQ
ID NO: 5, and a light chain CDR3 consisting of the amino
acid sequence represented by SEQ ID NO: 6; or
(2-1) comprises a heavy chain CDR1 consisting of the
amino acid ce ented by SEQ ID NO: 11, a heavy
chain CDR2 consisting of the amino acid sequence
represented by SEQ ID NO: 12, and a heavy chain CDR3
consisting of the amino acid sequence represented by SEQ
ID NO: 13, and
a light chain CDR1 consisting of the amino acid
sequence represented by SEQ ID NO: 14, a light chain CDR2
ting of the amino acid sequence represented by SEQ
ID NO: 15, and a light chain CDR3 consisting of the amino
acid sequence represented by SEQ ID NO: 16; or
(3-1) comprises a heavy chain CDR1 consisting of the
amino acid sequence represented by SEQ ID NO: 21, a heavy
chain CDR2 consisting of the amino acid sequence
represented by SEQ ID NO: 22, and a heavy chain CDR3
consisting of the amino acid sequence represented by SEQ
ID NO: 23, and
a light chain CDR1 consisting of the amino acid
sequence ented by SEQ ID NO: 24, a light chain CDR2
consisting of the amino acid sequence represented by SEQ
ID NO: 25, and a light chain CDR3 consisting of the amino
acid sequence represented by SEQ ID NO: 26; or
(4-1) comprises a heavy chain CDR1 consisting of the
amino acid sequence represented by SEQ ID NO: 31, a heavy
chain CDR2 consisting of the amino acid sequence
represented by SEQ ID NO: 32, and a heavy chain CDR3
consisting of the amino acid sequence represented by SEQ
ID NO: 33, and
a light chain CDR1 consisting of the amino acid
sequence ented by SEQ ID NO: 34, a light chain CDR2
consisting of the amino acid sequence represented by SEQ
ID NO: 35, and a light chain CDR3 consisting of the amino
acid sequence represented by SEQ ID NO: 36; or
(5-1) comprises a heavy chain CDR1 consisting of the
amino acid sequence represented by SEQ ID NO: 41, a heavy
chain CDR2 consisting of the amino acid sequence
represented by SEQ ID NO: 42, and a heavy chain CDR3
consisting of the amino acid ce ented by SEQ
ID NO: 43, and
a light chain CDR1 consisting of the amino acid
sequence represented by SEQ ID NO: 44, a light chain CDR2
consisting of the amino acid sequence represented by SEQ
ID NO: 45, and a light chain CDR3 consisting of the amino
acid sequence represented by SEQ ID NO: 46; or
(6-1) comprises a heavy chain CDR1 consisting of the
amino acid sequence represented by SEQ ID NO: 51, a heavy
chain CDR2 consisting of the amino acid sequence
represented by SEQ ID NO: 52, and a heavy chain CDR3
consisting of the amino acid ce represented by SEQ
ID NO: 53, and
a light chain CDR1 consisting of the amino acid
sequence represented by SEQ ID NO: 54, a light chain CDR2
consisting of the amino acid sequence ented by SEQ
ID NO: 55, and a light chain CDR3 consisting of the amino
acid sequence represented by SEQ ID NO: 56; or
(7-1) comprises a heavy chain CDR1 consisting of the
amino acid sequence represented by SEQ ID NO: 61, a heavy
chain CDR2 consisting of the amino acid sequence
represented by SEQ ID NO: 62, and a heavy chain CDR3
consisting of the amino acid sequence represented by SEQ
ID NO: 63, and
a light chain CDR1 consisting of the amino acid
sequence represented by SEQ ID NO: 64, a light chain CDR2
consisting of the amino acid sequence represented by SEQ
ID NO: 65, and a light chain CDR3 consisting of the amino
acid sequence represented by SEQ ID NO: 66; or
(8-1) comprises heavy chain CDR1 consisting of the
amino acid sequence represented by SEQ ID NO: 71, a heavy
chain CDR2 consisting of the amino acid ce
represented by SEQ ID NO: 72, and a heavy chain CDR3
consisting of the amino acid sequence represented by SEQ
ID NO: 73, and
a light chain CDR1 consisting of the amino acid
sequence represented by SEQ ID NO: 74, a light chain CDR2
ting of the amino acid sequence represented by SEQ
ID NO: 75, and a light chain CDR3 ting of the amino
acid sequence represented by SEQ ID NO: 76; or
(9-1) comprises a heavy chain CDR1 consisting of the
amino acid sequence represented by SEQ ID NO: 81, a heavy
chain CDR2 consisting of the amino acid sequence
ented by SEQ ID NO: 82, and a heavy chain CDR3
ting of the amino acid sequence represented by SEQ
ID NO: 83, and
a light chain CDR1 consisting of the amino acid
sequence represented by SEQ ID NO: 84, a light chain CDR2
consisting of the amino acid sequence represented by SEQ
ID NO: 85, and a light chain CDR3 consisting of the amino
acid sequence ented by SEQ ID NO: 86; or
(10-1) comprises a heavy chain CDR1 ting of
the amino acid sequence represented by SEQ ID NO: 91, a
heavy chain CDR2 ting of the amino acid sequence
represented by SEQ ID NO: 92, and a heavy chain CDR3
consisting of the amino acid sequence represented by SEQ
ID NO: 93, and
a light chain CDR1 consisting of the amino acid
sequence represented by SEQ ID NO: 94, a light chain CDR2
consisting of the amino acid sequence represented by SEQ
ID NO: 95, and a light chain CDR3 consisting of the amino
acid sequence represented by SEQ ID NO: 96; or
(11-1) comprises a heavy chain CDR1 consisting of
the amino acid sequence represented by SEQ ID NO: 101, a
heavy chain CDR2 consisting of the amino acid sequence
represented by SEQ ID NO: 102, and a heavy chain CDR3
consisting of the amino acid ce represented by SEQ
ID NO: 103, and
a light chain CDR1 consisting of the amino acid
sequence ented by SEQ ID NO: 104, a light chain CDR2
consisting of the amino acid sequence represented by SEQ
ID NO: 105, and a light chain CDR3 consisting of the amino
acid sequence represented by SEQ ID NO: 106.
The antibody according to [1], wherein the antibody
(1-2) comprises a heavy chain variable region
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 7, and a light chain variable
region consisting of an amino acid ce having at
least 80% or higher sequence identity to the amino acid
sequence ented by SEQ ID NO: 8; or
(2-2) comprises a heavy chain variable region
consisting of an amino acid sequence having at least 80%
or higher sequence ty to the amino acid sequence
represented by SEQ ID NO: 17, and a light chain variable
region ting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 18; or
(3-2) comprises a heavy chain variable region
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 27, and a light chain variable
region consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 28; or
(4-2) comprises a heavy chain variable region
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 37, and a light chain variable
region consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 38; or
(5-2) comprises a heavy chain variable region
consisting of an amino acid ce having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 47, and a light chain variable
region ting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence ented by SEQ ID NO: 48; or
(6-2) comprises a heavy chain variable region
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid ce
represented by SEQ ID NO: 57, and a light chain variable
region consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 58; or
(7-2) comprises a heavy chain variable region
consisting of an amino acid ce having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 67, and a light chain variable
region consisting of an amino acid ce having at
least 80% or higher sequence identity to the amino acid
sequence ented by SEQ ID NO: 68; or
(8-2) comprises a heavy chain variable region
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 77, and a light chain variable
region consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 78; or
(9-2) comprises a heavy chain variable region
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
ented by SEQ ID NO: 87, and a light chain variable
region consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 88; or
(10-2) comprises a heavy chain variable region
consisting of an amino acid ce having at least 80%
or higher sequence identity to the amino acid sequence
ented by SEQ ID NO: 97, and a light chain variable
region consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 98; or
(11-2) comprises a heavy chain variable region
consisting of an amino acid sequence having at least 80%
or higher ce identity to the amino acid sequence
represented by SEQ ID NO: 107, and a light chain variable
region consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence ented by SEQ ID NO: 108.
The antibody according to [1] or [2], wherein the
antibody is single chain antibody.
The antibody ing to [3], wherein the single
chain antibody
(1-3) comprises an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 165; or
(2-3) comprises an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 166; or
(3-3) comprises an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 167; or
(4-3) comprises an amino acid sequence having at
least 80% or higher sequence ty to the amino acid
sequence represented by SEQ ID NO: 168; or
(5-3) comprises an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 169; or
(6-3) ses an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
ce represented by SEQ ID NO: 170; or
(7-3) ses an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 171; or
(8-3) comprises an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 172; or
(9-3) comprises an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
ce represented by SEQ ID NO: 173; or
(10-3) comprises an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 174; or
(11-3) comprises an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 175.
The antibody according to [3], n the single
chain antibody
(1-3'-1) comprises an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 178; or
(1-3'-2) comprises an amino acid ce having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 179; or
(1-3'-3) comprises an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 180; or
(2-3'-1) comprises an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 181; or
(2-3'-2) comprises an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 182; or
(2-3'-3) comprises an amino acid sequence having at
least 80% or higher ce ty to the amino acid
sequence represented by SEQ ID NO: 183; or
(2-3'-4) comprises an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence represented by SEQ ID NO: 184.
The antibody according to [1] or [2], wherein the
antibody
(1-4) ses a heavy chain consisting of an amino
acid sequence having at least 80% or higher sequence
identity to the amino acid sequence represented by SEQ ID
NO: 9, and a light chain consisting of an amino acid
sequence having at least 80% or higher sequence identity
to the amino acid sequence ented by SEQ ID NO: 10;
(2-4) comprises a heavy chain consisting of an amino
acid sequence having at least 80% or higher sequence
identity to the amino acid sequence represented by SEQ ID
NO: 19, and a light chain consisting of an amino acid
ce having at least 80% or higher sequence identity
to the amino acid sequence represented by SEQ ID NO: 20;
(3-4) comprises a heavy chain ting of an amino
acid sequence having at least 80% or higher sequence
identity to the amino acid sequence ented by SEQ ID
NO: 29, and a light chain consisting of an amino acid
sequence having at least 80% or higher sequence identity
to the amino acid sequence represented by SEQ ID NO: 30;
(4-4) comprises a heavy chain consisting of an amino
acid sequence having at least 80% or higher sequence
identity to the amino acid sequence represented by SEQ ID
NO: 39, and a light chain consisting of an amino acid
sequence having at least 80% or higher sequence identity
to the amino acid sequence represented by SEQ ID NO: 40;
(5-4) comprises a heavy chain consisting of an amino
acid sequence having at least 80% or higher sequence
ty to the amino acid sequence represented by SEQ ID
NO: 49, and a light chain consisting of an amino acid
sequence having at least 80% or higher ce ty
to the amino acid sequence represented by SEQ ID NO: 50;
(6-4) comprises a heavy chain consisting of an amino
acid sequence having at least 80% or higher sequence
identity to the amino acid sequence represented by SEQ ID
NO: 59, and a light chain consisting of an amino acid
sequence having at least 80% or higher sequence identity
to the amino acid sequence represented by SEQ ID NO: 60;
(7-4) comprises a heavy chain consisting of an amino
acid sequence having at least 80% or higher ce
identity to the amino acid sequence ented by SEQ ID
NO: 69, and a light chain consisting of an amino acid
sequence having at least 80% or higher sequence identity
to the amino acid sequence represented by SEQ ID NO: 70;
(8-4) comprises a heavy chain consisting of an amino
acid sequence having at least 80% or higher ce
identity to the amino acid sequence represented by SEQ ID
NO: 79, and a light chain consisting of an amino acid
sequence having at least 80% or higher sequence identity
to the amino acid sequence represented by SEQ ID NO: 80;
(9-4) comprises a heavy chain consisting of an amino
acid sequence having at least 80% or higher sequence
identity to the amino acid sequence represented by SEQ ID
NO: 89, and a light chain consisting of an amino acid
sequence having at least 80% or higher sequence identity
to the amino acid sequence represented by SEQ ID NO: 90;
(10-4) comprises a heavy chain consisting of an
amino acid sequence having at least 80% or higher sequence
identity to the amino acid sequence ented by SEQ ID
NO: 99, and a light chain consisting of an amino acid
ce having at least 80% or higher sequence identity
to the amino acid sequence represented by SEQ ID NO: 100;
(11-4) comprises a heavy chain consisting of an
amino acid sequence having at least 80% or higher sequence
identity to the amino acid sequence represented by SEQ ID
NO: 109, and a light chain consisting of an amino acid
sequence having at least 80% or higher sequence identity
to the amino acid ce represented by SEQ ID NO: 110.
CAR sing the antibody according to any one of
to [5] (hereinafter, also ed to as the "present
single chain antibody"), a transmembrane region fused with
a carboxyl terminus of the present single chain antibody,
and an immunocompetent cell activation signal transduction
region fused with a carboxyl terminus of the transmembrane
region (hereinafter, also referred to as the "present
CAR").
The CAR according to [7], comprising the amino acid
sequence represented by any of SEQ ID NOs: 185 to 187.
An immunocompetent cell expressing the CAR according
to [7] or [8] nafter, also referred to as the
"present immunocompetent cell").
The competent cell according to [9], further
expressing interleukin 7 (IL-7) and chemokine ligand 19
(CCL19).
An antibody gene encoding the antibody according to
any one of [1] to [6] (hereinafter, also referred to as
the "present antibody gene"), or a CAR gene encoding the
CAR according to [7] or [8] (hereinafter, also referred to
as the "present CAR gene").
An antibody gene ng the antibody according to
any one of [1] to [4] and [6].
A vector comprising a er, and the antibody gene
according to [11] or the CAR gene encoding the CAR
according to [11] operably linked downstream of the
promoter (hereinafter, also referred to as the "present
vector").
A vector sing a promoter, and the dy gene
according to [12] operably linked downstream of the
promoter.
A host cell in which the vector according to [13] or
has been introduced (hereinafter, also referred to as
the "present host cell").
A method for detecting GPC3 (glypican-3), comprising
the step of ing GPC3 using the antibody according to
any one of [1] to [6] (hereinafter, also referred to as
the "present detection method").
A kit for the detection of GPC3 (glypican-3),
comprising the antibody according to any one of [1] to [6],
or a labeled form thereof (hereinafter, also referred to
as the "present kit for detection").
Examples of other embodiments of the present
invention can include the present antibody for use in the
detection of GPC3, and a method for producing the present
antibody, comprising the steps of: immunizing nonhuman
animals (e.g., mice and rats) with ength human GPC3
consisting of the amino acid ce represented by SEQ
ID NO: 157; synthesizing cDNA by reverse ription
reaction from total RNA of B cells derived from the
immunized nonhuman animals, and ying antibody genes
to prepare an antibody gene library; and ucting a
scFv phage library from the antibody gene library, and
infecting E. coli with the library so that cells express
scFv, followed by biopanning using the full-length human
GPC3 and the GPC3-expressing cell line, and further, if
necessary, a competitor C-terminal polypeptide of GPC3
(human-derived GPC3 polypeptide consisting of the amino
acid sequence represented by SEQ ID NO: 156).
Effect of the Invention
The present antibody is an antibody specifically
binding to GPC3 localized on a cell membrane not only in
the form of IgG but in the form of scFv. CAR-T cells
using the present antibody as scFv in CAR have excellent
cytotoxic activity and the ability to produce IFN-γ.
Hence, the present dy is useful for cancer
therapy.
Brief Description of Drawings
e 1] Figure 1 is a diagram showing each round (step)
of biopanning consisting of 5 types of series (A to E
series). A series involves performing 3 rounds of
ning with recombinant GPC3 immobilized on ic
beads as a bait, and performing biopanning in rounds 4 and
with a GPC3-expressing cell line as a bait (round 5 was
carried out only for 1413 #3). In rounds 1 to 4, existing
anti-GPC3 antibodies (GC33 and GC199) were added as
competitive antibodies. B series involves performing
biopanning with GPC3-expressing cells as a bait in the
presence of the competitive antibodies after round 2 of A
series. E series es performing biopanning with
recombinant GPC3 immobilized on magnetic beads as a bait
under conditions of no competitive antibody after round 3
of A series. In C series, 4 rounds in total of biopanning
with a GPC3-expressing cell line as a bait in 2 rounds and
recombinant GPC3 immobilized on magnetic beads as a bait
in 2 rounds were performed in the e of the
competitive antibodies. D series involves ming the
same biopanning as that of A series in the absence of the
competitive antibodies.
e 2] Figure 2 is a diagram showing results of
performing flow cytometry (FCM) using 18 types of anti-
GPC3 scFv clones (TF1413-02d023, , 02d030, 02d039,
02e003, 02e004, , 02e030, 02e040, 03e001, 03e004,
03e005, 03e015, 03e016, 03e019, 03e027, 03e034, and
03e045) and existing anti-GPC3 antibodies (GC33 and ,
and 3 types of cell lines (GPC3 inal fragmentexpressing
cell line, GPC3 inal fragment-expressing
cell line, and GPC3 [full-length]-expressing cell line).
The numeric values in the diagram are indicated by
relative values when the fluorescence intensity of a cell
line expressing no GPC3 (SK-Hep-1 cell line) was defined
as 1 in FCM.
[Figure 3] Figure 3 is a diagram showing results of
performing FCM using IgG antibodies prepared from 11 types
of scFv clones (TF1413-02d028, 02d039, 02e004, 02e014,
02e030, 02e040, 03e001, 03e004, 03e005, 03e015, and
03e034) and existing anti-GPC3 antibodies (GC33 and GC199),
and 3 types of cell lines (GPC3 N-terminal fragmentexpressing
cell line, GPC3 C-terminal fragment-expressing
cell line, and GPC3 [full-length]-expressing cell line).
[Figure 4] Figure 4 is a m showing results of
performing FACS (fluorescence activated cell sorting)
using a GPC3-expressing cell line treated with 3 types of
methods (EDTA, trypsin, and "EDTA + collagenase"), 3 types
of antibody combinations (anti-mouse IgG antibody labeled
with APC [hereinafter, also referred to as "APC use
IgG antibody"], and a combination of the APC anti-mouse
IgG antibody and a scFv clone [TF1413-02d028] antibody).
e 5] Figure 5 is a diagram showing results of
analyzing GPC3 CAR-T cells (T cells expressing CAR of scFv
recognizing GPC3) derived from 5 types of scFv clones
(TF1413-02d028, TF1413-02d039, TF1413-02e014, TF1413-
02e030, and TF1413-03e005) for cytotoxic activity against
a Sk-HEP-1 GPC3 cell line. In each graph, the right peak
depicts CD45-positive cells (GPC3 CAR-T cells), and the
left peak depicts egative cells (residual cancer
cells [Sk-HEP-1 GPC3 ). The ordinate of each graph
depicts the number of cells. The numeric value in each
graph depicts the ratio (%) of the number of CD45-positive
cells to the total number of cells positive cells
and CD45-negative cells). T cells expressing no GPC3 CAR
("Non infection" in the diagram) were used as a control.
e 6] Figure 6 is a graph showing the ratio of CD45-
negative cells in Figure 5 (Figure 6A) and the number of
CD45-negative cells (Figure 6B). In a pair of bar graphs,
the left bar graph depicts "mock" (Sk-HEP-1 mock cell
line), and the right bar graph depicts "GPC3" (Sk-HEP-1
GPC3 cell line).
[Figure 7] Figure 7 is a diagram showing results of
analyzing GPC3 CAR-T cells derived from 5 types of scFv
clones (TF1413-02d028, -02d039, TF1413-02e014,
TF1413-02e030, and TF1413-03e005) for the ability to
produce IFN-γ against a Sk-HEP-1 GPC3 cell line. T cells
expressing no GPC3 CAR ("Non infection" in the diagram)
were used as a control.
Mode of Carrying Out the Invention
The present antibody is an dy comprising the
heavy (H) chain and light (L) chain CDR1 to CDR3 described
above in any of (1-1) to (11-1), and specifically binding
to, as an epitope, at least a portion (usually within the
range of 3 to 30 amino acid es, preferably 4 to 20
amino acid residues, more preferably 5 to 15 amino acid
residues) of a human-derived GPC3 polypeptide consisting
of the amino acid sequence represented by SEQ ID NO: 155
(amino rminal polypeptide consisting of amino acid
residues 32 to 471 [exons 1 to 7] of human-derived fulllength
GPC3 consisting of the amino acid sequence
represented by SEQ ID NO: 157). This antibody
specifically binds not only in the form of IgG but in the
form of scFv to GPC3 zed on a cell membrane, and
usually comprises a H chain variable region comprising the
H chain CDR1 to CDR3 described above in any of (1-1) to
(11-1), and a L chain variable region comprising the L
chain CDR1 to CDR3 described above in any of (1-1) to (11-
1). In this context, the phrase "specifically g"
means that the antibody recognizes and binds to the
polypeptide consisting of the amino acid sequence
represented by SEQ ID NO: 155 through a recognition
mechanism with high antigen-antibody specificity. Thus,
the present dy does not ically bind to a
human-derived GPC3 polypeptide consisting of the amino
acid sequence represented by SEQ ID NO: 156 (carboxyl [C]-
terminal ptide consisting of amino acid residues 472
to 580 [exons 8 and 9] of human-derived full-length GPC3
consisting of the amino acid sequence represented by SEQ
ID NO: 157).
The t antibody is not particularly limited by
its origin, type, class, morphology, etc. The present
antibody includes, for example: a human-derived antibody;
an antibody derived from a nonhuman animal such as a mouse
or a rat; a polyclonal antibody, an oligoclonal antibody
(mixture of several to several tens of antibodies), and a
monoclonal antibody; and a chimeric antibody or a
humanized antibody in which a partial region (e.g.,
constant regions) of an dy has been substituted by a
region d from a different organism species, an
antibody fragment such as a F(ab')2 antibody fragment
obtained by digesting a monoclonal antibody with pepsin, a
Fab' antibody fragment obtained by reducing a F(ab')2
antibody fragment, and Fab obtained by digesting a
monoclonal antibody with papain, and a recombinant
antibody such as scFv ning an antibody heavy (H)
chain variable region and an antibody light (H) chain
variable region linked through amino acid cross-links. In
the case of using the t antibody as CAR, scFv is
preferred.
The present antibody is preferably in a separated
form. In this context, the term "separated" means that
the antibody is t in a state different from the
state where the antibody is originally present in such a
way that the antibody is taken out of an environment
ally involving the antibody or expressed in an
environment different from the environment ally
involving the antibody by an cial ion.
Specifically, the "separated antibody" does not include an
antibody that is derived from a certain individual and is
in a state contained in the body of the individual without
an external ion (artificial operation) or in a
tissue or a body fluid (blood, plasma, serum, etc.)
derived from the body. The present antibody is preferably
an antibody ed by an artificial operation (e.g., the
recombinant antibody described above). Such an "antibody
derived from a cell prepared by an artificial operation or
an antibody produced from the cell" does not include an
antibody that is not subjected to an artificial operation,
for example, an antibody produced from a naturally
occurring B cell.
In the present antibody, a framework region (FR) is
usually linked to the N terminus and/or C terminus of each
of H chain and L chain CDR1 to CDR3 regions. Among such
FRs, examples of the H chain FRs can include H chain FR1
linked to the N terminus of H chain CDR1, H chain FR2
linked to the C terminus of H chain CDR1 (N terminus of H
chain CDR2), H chain FR3 linked to the C us of H
chain CDR2 (N terminus of H chain CDR3), and H chain FR4
linked to the C terminus of H chain CDR3. Among the FRs,
examples of the L chain FRs can include L chain FR1 linked
to the N terminus of L chain CDR1, L chain FR2 linked to
the C terminus of L chain CDR1 (N terminus of L chain
CDR2), L chain FR3 linked to the C terminus of L chain
CDR2 (N terminus of L chain CDR3), and L chain FR4 linked
to the C terminus of L chain CDR3.
Examples of the H chain FR1 can specifically
include: (1-HFR1) a polypeptide consisting of amino acid
residues 1 to 30 of the amino acid sequence represented by
SEQ ID NO: 7, or a polypeptide consisting of an amino acid
sequence having at least 80% or higher sequence identity
to the amino acid sequence of the polypeptide; (2-HFR1) a
polypeptide consisting of amino acid residues 1 to 30 of
the amino acid sequence ented by SEQ ID NO: 17, or a
ptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (3-HFR1) a polypeptide
consisting of amino acid residues 1 to 30 of the amino
acid sequence represented by SEQ ID NO: 27, or a
ptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; 1) a polypeptide
consisting of amino acid residues 1 to 30 of the amino
acid ce ented by SEQ ID NO: 37, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (5-HFR1) a polypeptide
consisting of amino acid residues 1 to 30 of the amino
acid sequence represented by SEQ ID NO: 47, or a
ptide consisting of an amino acid ce having at
least 80% or higher sequence identity to the amino acid
sequence of the ptide; (6-HFR1) a polypeptide
consisting of amino acid residues 1 to 30 of the amino
acid sequence represented by SEQ ID NO: 57, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (7-HFR1) a polypeptide
consisting of amino acid residues 1 to 30 of the amino
acid sequence represented by SEQ ID NO: 67, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (8-HFR1) a ptide
consisting of amino acid residues 1 to 30 of the amino
acid sequence represented by SEQ ID NO: 77, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the ptide; (9-HFR1) a polypeptide
consisting of amino acid residues 1 to 30 of the amino
acid sequence represented by SEQ ID NO: 87, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (10-HFR1) a polypeptide
consisting of amino acid residues 1 to 30 of the amino
acid sequence represented by SEQ ID NO: 97, or a
polypeptide ting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
ce of the polypeptide; and (11-HFR1) a polypeptide
consisting of amino acid residues 1 to 30 of the amino
acid ce represented by SEQ ID NO: 107, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide.
Examples of the H chain FR2 can specifically
include: (1-HFR2) a polypeptide consisting of amino acid
residues 36 to 49 of the amino acid sequence represented
by SEQ ID NO: 7, or a polypeptide consisting of an amino
acid sequence having at least 80% or higher sequence
identity to the amino acid ce of the polypeptide;
(2-HFR2) a polypeptide consisting of amino acid residues
36 to 49 of the amino acid sequence represented by SEQ ID
NO: 17, or a polypeptide consisting of an amino acid
sequence having at least 80% or higher sequence identity
to the amino acid sequence of the polypeptide; (3-HFR2) a
ptide consisting of amino acid residues 36 to 49 of
the amino acid sequence represented by SEQ ID NO: 27, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; 2) a polypeptide
consisting of amino acid residues 36 to 49 of the amino
acid sequence represented by SEQ ID NO: 37, or a
ptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (5-HFR2) a polypeptide
consisting of amino acid residues 36 to 49 of the amino
acid ce represented by SEQ ID NO: 47, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher ce identity to the amino acid
sequence of the polypeptide; (6-HFR2) a polypeptide
consisting of amino acid residues 36 to 49 of the amino
acid sequence represented by SEQ ID NO: 57, or a
ptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (7-HFR2) a polypeptide
consisting of amino acid residues 36 to 49 of the amino
acid sequence represented by SEQ ID NO: 67, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (8-HFR2) a polypeptide
consisting of amino acid residues 36 to 49 of the amino
acid sequence represented by SEQ ID NO: 77, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher ce identity to the amino acid
sequence of the polypeptide; (9-HFR2) a polypeptide
consisting of amino acid es 36 to 49 of the amino
acid sequence represented by SEQ ID NO: 87, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
ce of the polypeptide; (10-HFR2) a polypeptide
consisting of amino acid es 36 to 49 of the amino
acid sequence represented by SEQ ID NO: 97, or a
ptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; and (11-HFR2) a polypeptide
ting of amino acid residues 36 to 49 of the amino
acid sequence represented by SEQ ID NO: 107, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide.
Examples of the H chain FR3 can ically
e: (1-HFR3) a polypeptide consisting of amino acid
residues 67 to 98 of the amino acid sequence represented
by SEQ ID NO: 7, or a polypeptide consisting of an amino
acid sequence having at least 80% or higher ce
identity to the amino acid ce of the polypeptide;
(2-HFR3) a polypeptide consisting of amino acid residues
67 to 98 of the amino acid sequence represented by SEQ ID
NO: 17, or a polypeptide consisting of an amino acid
sequence having at least 80% or higher ce identity
to the amino acid sequence of the polypeptide; (3-HFR3) a
polypeptide consisting of amino acid residues 67 to 98 of
the amino acid sequence represented by SEQ ID NO: 27, or a
polypeptide consisting of an amino acid ce having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (4-HFR3) a polypeptide
consisting of amino acid residues 67 to 99 of the amino
acid sequence represented by SEQ ID NO: 37, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (5-HFR3) a polypeptide
consisting of amino acid residues 67 to 99 of the amino
acid sequence represented by SEQ ID NO: 47, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (6-HFR3) a ptide
consisting of amino acid residues 67 to 98 of the amino
acid sequence represented by SEQ ID NO: 57, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (7-HFR3) a polypeptide
consisting of amino acid residues 67 to 98 of the amino
acid ce represented by SEQ ID NO: 67, or a
polypeptide consisting of an amino acid ce having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; 3) a polypeptide
consisting of amino acid residues 67 to 98 of the amino
acid sequence represented by SEQ ID NO: 77, or a
polypeptide consisting of an amino acid ce having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (9-HFR3) a polypeptide
consisting of amino acid residues 67 to 99 of the amino
acid sequence represented by SEQ ID NO: 87, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; R3) a polypeptide
consisting of amino acid residues 67 to 98 of the amino
acid sequence represented by SEQ ID NO: 97, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; and (11-HFR3) a polypeptide
consisting of amino acid residues 67 to 98 of the amino
acid sequence represented by SEQ ID NO: 107, or a
ptide consisting of an amino acid sequence having at
least 80% or higher ce identity to the amino acid
sequence of the polypeptide.
Examples of the H chain FR4 can specifically
include: (1-HFR4) a polypeptide consisting of amino acid
residues 109 to 118 of the amino acid sequence represented
by SEQ ID NO: 7, or a polypeptide ting of an amino
acid sequence having at least 80% or higher sequence
identity to the amino acid sequence of the polypeptide;
(2-HFR4) a polypeptide consisting of amino acid residues
108 to 117 of the amino acid sequence represented by SEQ
ID NO: 17, or a polypeptide consisting of an amino acid
sequence having at least 80% or higher sequence identity
to the amino acid sequence of the polypeptide; (3-HFR4) a
polypeptide ting of amino acid es 106 to 115
of the amino acid sequence represented by SEQ ID NO: 27,
or a ptide consisting of an amino acid sequence
having at least 80% or higher sequence identity to the
amino acid sequence of the polypeptide; (4-HFR4) a
polypeptide consisting of amino acid residues 111 to 120
of the amino acid sequence represented by SEQ ID NO: 37,
or a polypeptide ting of an amino acid sequence
having at least 80% or higher sequence identity to the
amino acid sequence of the polypeptide; (5-HFR4) a
polypeptide consisting of amino acid residues 108 to 117
of the amino acid sequence represented by SEQ ID NO: 47,
or a polypeptide consisting of an amino acid sequence
having at least 80% or higher sequence identity to the
amino acid sequence of the ptide; (6-HFR4) a
polypeptide consisting of amino acid residues 107 to 116
of the amino acid sequence represented by SEQ ID NO: 57,
or a ptide consisting of an amino acid sequence
having at least 80% or higher sequence identity to the
amino acid sequence of the polypeptide; (7-HFR4) a
polypeptide consisting of amino acid residues 106 to 115
of the amino acid sequence represented by SEQ ID NO: 67,
or a polypeptide consisting of an amino acid sequence
having at least 80% or higher sequence identity to the
amino acid sequence of the polypeptide; (8-HFR4) a
ptide consisting of amino acid residues 106 to 115
of the amino acid sequence represented by SEQ ID NO: 77,
or a polypeptide consisting of an amino acid sequence
having at least 80% or higher sequence identity to the
amino acid ce of the polypeptide; (9-HFR4) a
polypeptide consisting of amino acid residues 111 to 120
of the amino acid sequence represented by SEQ ID NO: 87,
or a polypeptide consisting of an amino acid sequence
having at least 80% or higher sequence identity to the
amino acid sequence of the polypeptide; (10-HFR4) a
polypeptide consisting of amino acid residues 110 to 119
of the amino acid sequence represented by SEQ ID NO: 97,
or a ptide consisting of an amino acid ce
having at least 80% or higher sequence identity to the
amino acid sequence of the polypeptide; and (11-HFR4) a
polypeptide consisting of amino acid residues 109 to 118
of the amino acid sequence represented by SEQ ID NO: 107,
or a polypeptide consisting of an amino acid sequence
having at least 80% or higher ce identity to the
amino acid sequence of the polypeptide.
Examples of the L chain FR1 can specifically
include: (1-LFR1) a polypeptide ting of amino acid
residues 1 to 23 of the amino acid sequence represented by
SEQ ID NO: 8, or a polypeptide consisting of an amino acid
sequence having at least 80% or higher sequence ty
to the amino acid sequence of the polypeptide; (2-LFR1) a
ptide consisting of amino acid residues 1 to 23 of
the amino acid sequence ented by SEQ ID NO: 18, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (3-LFR1) a polypeptide
consisting of amino acid residues 1 to 23 of the amino
acid sequence represented by SEQ ID NO: 28, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (4-LFR1) a polypeptide
consisting of amino acid residues 1 to 23 of the amino
acid sequence represented by SEQ ID NO: 38, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (5-LFR1) a polypeptide
consisting of amino acid residues 1 to 23 of the amino
acid sequence represented by SEQ ID NO: 48, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence ty to the amino acid
sequence of the polypeptide; (6-LFR1) a polypeptide
consisting of amino acid residues 1 to 23 of the amino
acid ce represented by SEQ ID NO: 58, or a
polypeptide consisting of an amino acid ce having at
least 80% or higher sequence identity to the amino acid
ce of the polypeptide; (7-LFR1) a polypeptide
consisting of amino acid residues 1 to 23 of the amino
acid sequence represented by SEQ ID NO: 68, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (8-LFR1) a polypeptide
consisting of amino acid residues 1 to 23 of the amino
acid sequence represented by SEQ ID NO: 78, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (9-LFR1) a polypeptide
consisting of amino acid es 1 to 23 of the amino
acid sequence represented by SEQ ID NO: 88, or a
polypeptide ting of an amino acid ce having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (10-LFR1) a polypeptide
consisting of amino acid residues 1 to 23 of the amino
acid sequence represented by SEQ ID NO: 98, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; and (11-LFR1) a polypeptide
consisting of amino acid residues 1 to 23 of the amino
acid sequence represented by SEQ ID NO: 108, or a
polypeptide ting of an amino acid ce having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide.
Examples of the L chain FR2 can specifically
include: (1-LFR2) a ptide consisting of amino acid
residues 35 to 49 of the amino acid sequence ented
by SEQ ID NO: 8, or a polypeptide consisting of an amino
acid sequence having at least 80% or higher sequence
identity to the amino acid sequence of the polypeptide;
(2-LFR2) a polypeptide consisting of amino acid residues
40 to 54 of the amino acid sequence represented by SEQ ID
NO: 18, or a polypeptide consisting of an amino acid
sequence having at least 80% or higher sequence ty
to the amino acid sequence of the polypeptide; (3-LFR2) a
polypeptide ting of amino acid residues 35 to 49 of
the amino acid sequence represented by SEQ ID NO: 28, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (4-LFR2) a polypeptide
consisting of amino acid residues 35 to 49 of the amino
acid ce represented by SEQ ID NO: 38, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (5-LFR2) a polypeptide
consisting of amino acid residues 41 to 55 of the amino
acid sequence represented by SEQ ID NO: 48, or a
polypeptide ting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (6-LFR2) a polypeptide
consisting of amino acid residues 35 to 49 of the amino
acid ce represented by SEQ ID NO: 58, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (7-LFR2) a polypeptide
consisting of amino acid residues 35 to 49 of the amino
acid sequence represented by SEQ ID NO: 68, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence ty to the amino acid
sequence of the ptide; (8-LFR2) a polypeptide
consisting of amino acid residues 35 to 49 of the amino
acid sequence represented by SEQ ID NO: 78, or a
polypeptide ting of an amino acid ce having at
least 80% or higher ce identity to the amino acid
sequence of the polypeptide; 2) a polypeptide
consisting of amino acid residues 35 to 49 of the amino
acid sequence represented by SEQ ID NO: 88, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher ce identity to the amino acid
sequence of the polypeptide; (10-LFR2) a polypeptide
consisting of amino acid residues 35 to 49 of the amino
acid sequence represented by SEQ ID NO: 98, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; and (11-LFR2) a polypeptide
consisting of amino acid residues 35 to 49 of the amino
acid sequence represented by SEQ ID NO: 108, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence ty to the amino acid
sequence of the polypeptide.
Examples of the L chain FR3 can specifically
include: (1-LFR3) a polypeptide consisting of amino acid
es 57 to 88 of the amino acid sequence represented
by SEQ ID NO: 8, or a polypeptide consisting of an amino
acid sequence having at least 80% or higher sequence
identity to the amino acid sequence of the polypeptide;
(2-LFR3) a polypeptide consisting of amino acid residues
62 to 93 of the amino acid sequence represented by SEQ ID
NO: 18, or a polypeptide consisting of an amino acid
sequence having at least 80% or higher sequence identity
to the amino acid sequence of the ptide; (3-LFR3) a
polypeptide consisting of amino acid residues 57 to 88 of
the amino acid sequence represented by SEQ ID NO: 28, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (4-LFR3) a polypeptide
consisting of amino acid residues 57 to 88 of the amino
acid sequence ented by SEQ ID NO: 38, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence ty to the amino acid
sequence of the polypeptide; (5-LFR3) a polypeptide
consisting of amino acid es 63 to 94 of the amino
acid sequence represented by SEQ ID NO: 48, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (6-LFR3) a polypeptide
consisting of amino acid es 57 to 88 of the amino
acid sequence represented by SEQ ID NO: 58, or a
polypeptide ting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (7-LFR3) a polypeptide
consisting of amino acid residues 57 to 88 of the amino
acid sequence represented by SEQ ID NO: 68, or a
ptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (8-LFR3) a polypeptide
consisting of amino acid residues 57 to 88 of the amino
acid sequence represented by SEQ ID NO: 78, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher ce identity to the amino acid
sequence of the polypeptide; (9-LFR3) a polypeptide
consisting of amino acid residues 57 to 88 of the amino
acid sequence represented by SEQ ID NO: 88, or a
ptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (10-LFR3) a polypeptide
consisting of amino acid residues 57 to 88 of the amino
acid sequence represented by SEQ ID NO: 98, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; and (11-LFR3) a polypeptide
consisting of amino acid residues 57 to 88 of the amino
acid sequence represented by SEQ ID NO: 108, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
ce of the ptide.
Examples of the L chain FR4 can ically
e: (1-LFR4) a polypeptide consisting of amino acid
residues 98 to 108 of the amino acid sequence represented
by SEQ ID NO: 8, or a polypeptide consisting of an amino
acid sequence having at least 80% or higher sequence
identity to the amino acid sequence of the polypeptide;
(2-LFR4) a polypeptide consisting of amino acid residues
103 to 113 of the amino acid sequence represented by SEQ
ID NO: 18, or a polypeptide consisting of an amino acid
sequence having at least 80% or higher sequence identity
to the amino acid ce of the polypeptide; (3-LFR4) a
polypeptide consisting of amino acid residues 97 to 107 of
the amino acid ce represented by SEQ ID NO: 28, or a
ptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; 4) a ptide
consisting of amino acid residues 98 to 108 of the amino
acid sequence represented by SEQ ID NO: 38, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (5-LFR4) a polypeptide
consisting of amino acid residues 104 to 114 of the amino
acid sequence represented by SEQ ID NO: 48, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (6-LFR4) a polypeptide
consisting of amino acid residues 98 to 108 of the amino
acid sequence represented by SEQ ID NO: 58, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (7-LFR4) a polypeptide
consisting of amino acid residues 98 to 108 of the amino
acid ce represented by SEQ ID NO: 68, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher ce identity to the amino acid
sequence of the polypeptide; (8-LFR4) a polypeptide
consisting of amino acid es 98 to 108 of the amino
acid sequence represented by SEQ ID NO: 78, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide; (9-LFR4) a polypeptide
consisting of amino acid residues 98 to 108 of the amino
acid ce represented by SEQ ID NO: 88, or a
ptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the ptide; (10-LFR4) a polypeptide
consisting of amino acid residues 98 to 108 of the amino
acid sequence represented by SEQ ID NO: 98, or a
polypeptide consisting of an amino acid ce having at
least 80% or higher sequence identity to the amino acid
ce of the polypeptide; and (11-LFR4) a polypeptide
consisting of amino acid residues 98 to 108 of the amino
acid sequence represented by SEQ ID NO: 108, or a
polypeptide consisting of an amino acid sequence having at
least 80% or higher sequence identity to the amino acid
sequence of the polypeptide.
The FRs of the present antibody are preferably FRs
of a known human antibody. Examples of such "FRs of a
known human antibody" can include FRs of a human antibody
registered in a sequence se known in the art such as
GenBank, and FRs selected from a common sequence (human
most homologous consensus sequence; Kabat, E. A. et al.,
Sequences of Proteins of Immunological Interest, US Dept.
Health and Human Services, 1991) derived from each
subgroup of a human antibody.
The H chain CDR1 in the present antibody usually
resides at positions H31 to H35 based on Kabat numbering
(see the document "Kabat, E.A. et al., (1991) NIH
Publication No. 91-3242, ces of proteins of
immunological interest"). The H chain CDR2 in the present
antibody usually resides at ons H50 to H52, H52A,
and H53 to H65 based on Kabat numbering. The H chain CDR3
in the present antibody usually resides at positions H95
to H100, H100A, H100B, H101, and H102 based on Kabat
ing. The L chain CDR1 in the present antibody
usually resides at positions L24 to L34 based on Kabat
numbering. The L chain CDR2 in the present antibody
y resides at positions L50 to L56 based on Kabat
numbering. The L chain CDR3 in the t antibody
usually resides at ons L89 to L97 based on Kabat
numbering.
Examples of the antibody comprising the H chain and
L chain CDR1 to CDR3 described above in (1-1) as the
present antibody can include an antibody comprising the H
chain and L chain variable (V) regions described above in
(1-2) and can specifically include: the single chain
antibody described above in (1-3); the single chain
antibody described above in (1-3'-1), the single chain
antibody described above in (1-3'-2), and the single chain
antibody described above in (1-3'-3); and an antibody
comprising the H chain and the L chain described above in
(1-4). Examples of the antibody comprising the H chain
and L chain CDR1 to CDR3 described above in (2-1) can
include an antibody comprising the H chain and L chain V
regions described above in (2-2) and can specifically
include: the single chain antibody bed above in (2-
3); the single chain dy described above in (2-3'-1),
the single chain antibody described above in (2-3'-2), the
single chain antibody described above in (2-3'-3), and the
single chain antibody described above in (2-3'-4); and an
antibody comprising the H chain and the L chain described
above in (2-4). Examples of the antibody comprising the H
chain and L chain CDR1 to CDR3 bed above in (3-1)
can include an antibody comprising the H chain and L chain
V s described above in (3-2) and can ically
include: the single chain antibody described above in (3-
3); and an antibody comprising the H chain and the L chain
described above in (3-4). es of the antibody
comprising the H chain and L chain CDR1 to CDR3 described
above in (4-1) can include an antibody comprising the H
chain and L chain V regions described above in (4-2) and
can specifically include: the single chain antibody
described above in (4-3); and an antibody comprising the H
chain and the L chain described above in (4-4). Examples
of the antibody comprising the H chain and L chain CDR1 to
CDR3 described above in (5-1) can include an antibody
comprising the H chain and L chain V regions described
above in (5-2) and can specifically e: the single
chain antibody described above in (5-3); and an antibody
comprising the H chain and the L chain described above in
(5-4). Examples of the antibody comprising the H chain
and L chain CDR1 to CDR3 described above in (6-1) can
e an dy comprising the H chain and L chain V
regions described above in (6-2) and can specifically
include: the single chain antibody described above in (6-
3); and an antibody comprising the H chain and the L chain
described above in (6-4). Examples of the antibody
comprising the H chain and L chain CDR1 to CDR3 described
above in (7-1) can include an antibody comprising the H
chain and L chain V regions described above in (7-2) and
can specifically include: the single chain antibody
described above in (7-3); and an antibody comprising the H
chain and the L chain bed above in (7-4). Examples
of the antibody comprising the H chain and L chain CDR1 to
CDR3 described above in (8-1) can include an antibody
comprising the H chain and L chain V regions bed
above in (8-2) and can specifically include: the single
chain antibody described above in (8-3); and an antibody
comprising the H chain and the L chain described above in
(8-4). Examples of the antibody comprising the H chain
and L chain CDR1 to CDR3 described above in (9-1) can
include an antibody comprising the H chain and L chain V
regions described above in (9-2) and can specifically
include: the single chain dy described above in (9-
3); and an antibody comprising the H chain and the L chain
described above in (9-4). Examples of the antibody
comprising the H chain and L chain CDR1 to CDR3 described
above in (10-1) can include an antibody comprising the H
chain and L chain V s described above in (10-2) and
can specifically include: the single chain antibody
described above in (10-3); and an antibody comprising the
H chain and the L chain described above in (10-4).
es of the antibody comprising the H chain and L
chain CDR1 to CDR3 described above in (11-1) can include
an antibody comprising the H chain and L chain V regions
described above in (11-2) and can specifically include:
the single chain antibody described above in (11-3); and
an antibody sing the H chain and the L chain
described above in (11-4). The heavy chain variable
region and the light chain variable region in the single
chain antibody are usually bound via a e linker.
The present CAR can se the present single
chain antibody, a transmembrane region fused with the C
terminus of the present single chain antibody, and an
immunocompetent cell activation signal transduction region
fused with the C terminus of the transmembrane region. In
this context, the fusion between the present single chain
antibody and the embrane region, or between the
transmembrane region and the immunocompetent cell
activation signal transduction region may be mediated by a
peptide linker or an IgG4 hinge region.
es of the length of the peptide linker in the
present antibody can include 1 to 100 amino acid es,
preferably 10 to 50 amino acid residues. Examples of the
peptide linker in the t antibody can specifically
include a consecutive e of 3 amino acid sequences
each consisting of 1 to 4 glycine residues and 1 serine
residue.
The transmembrane region can be any peptide that can
penetrate a cell membrane. Examples thereof can include a
transmembrane region derived from CD8, a T cell receptor α
or β chain, CD3ζ, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9,
CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137,
ICOS, CD154, EGFR (epidermal growth factor receptor), or
GITR and can specifically include a human CD8
transmembrane region consisting of amino acid residues 1
to 83 of the amino acid ce represented by SEQ ID NO:
185. atively, the transmembrane region may be
derived from a peptide that can ate cell membrane by
the truncation of C-terminal 1 to 10 amino acid residues,
preferably 6 or 7 amino acid residues. Examples thereof
can include engineered form 1 of the human CD8
transmembrane region consisting of amino acid residues 1
to 77 of the amino acid sequence represented by SEQ ID NO:
186, and engineered form 2 of the human CD8 transmembrane
region consisting of amino acid es 1 to 76 of the
amino acid sequence represented by SEQ ID NO: 187.
The immunocompetent cell activation signal
transduction region can be any region capable of
transducing a signal into immunocompetent cells upon
binding of the present single chain antibody to human GPC3.
The immunocompetent cell activation signal transduction
region preferably comprises at least one or more members
selected from polypeptides of the intracellular regions of
CD28, 4-1BB (CD137), GITR, CD27,OX40, HVEM, CD3ζ, and Fc
receptor-associated γ chain, and more preferably comprises
three polypeptides of the intracellular regions of CD28,
4-1BB, and CD3ζ. Examples of such a ptide of the
intracellular region of CD28 can ically include a
polypeptide of the intracellular region of human CD28
consisting of amino acid residues 85 to 124 of the amino
acid sequence represented by SEQ ID NO: 185. Examples of
the "polypeptide of the intracellular region of 4-1BB" can
ically include a polypeptide of the intracellular
region of human 4-1BB consisting of amino acid residues
125 to 170 of the amino acid sequence represented by SEQ
ID NO: 185. Examples of the polypeptide of the
intracellular region of CD3ζ can specifically include a
polypeptide of the intracellular region of human CD3ζ
consisting of amino acid residues 172 to 283 of the amino
acid sequence ented by SEQ ID NO: 185.
ne (Arg) at position 84 of the amino acid
sequence represented by SEQ ID NO: 185, arginine at
position 78 of the amino acid sequence represented by SEQ
ID NO: 186, and arginine at position 77 of the amino acid
sequence represented by SEQ ID NO: 187 are a common
sequence n the polypeptide of the transmembrane
region derived from human CD8 and the polypeptide of the
intracellular region of human CD28. Leucine (Leu) at
on 171 of the amino acid sequence represented by SEQ
ID NO: 185, leucine at position 165 of the amino acid
sequence represented by SEQ ID NO: 186, and leucine at
position 164 of the amino acid sequence represented by SEQ
ID NO: 187 are a common sequence n the polypeptide
of the intracellular region of human 4-1BB and the
polypeptide of the intracellular region of human CD3ζ.
In the present specification, the "immunocompetent
cell" means a cell responsible for immune functions in a
living body. Examples of the immunocompetent cell can
include: a lymphoid cell such as a T cell, a natural
killer cell (NK cell), and a B cell; an n-presenting
cell such as a monocyte, a macrophage, and a dendritic
cell; and a granulocyte such as a neutrophil, an
eosinophil, a basophil, and a mast cell. Specific
examples thereof can ably e a T cell derived
from a mammal such as a human, a dog, a cat, a pig, or a
mouse, preferably a human-derived T cell. The T cell can
be obtained by isolation or purification from an
immunocompetent cell infiltrating a body fluid such as
blood or bone marrow fluid, a tissue of the spleen, the
thymus, lymph node or the like, or a cancer tissue of
primary tumor, atic tumor, cancerous ascites or the
like. Alternatively, a T cell prepared from an ES cell or
an iPS cell may be utilized. Examples of such a T cell
can e an alpha-beta T cell, a gamma-delta T cell, a
CD8+ T cell, a CD4+ T cell, a tumor-infiltrating T cell, a
memory T cell, a naive T cell, and a NKT cell. The origin
of the competent cell may be the same as or
different from an administration subject. When the
administration subject is a human, an autologous cell
collected from a patient as the administration subject may
be used as the competent cell, or any of other cells
collected from a person other than the administration
subject may be used as the immunocompetent cell.
Specifically, the donor and the recipient may be the same
or different and is preferably the same.
Examples of the administration subject can
preferably include a mammal and a mammalian cell.
Examples of the mammal can more preferably include a human,
a mouse, a dog, a rat, a guinea pig, a rabbit, a bird,
sheep, a pig, cattle, a horse, a cat, a monkey, and a
chimpanzee, particularly preferably a human.
The present CAR is preferably used for ex vivo
expression on the cell surface of the immunocompetent cell
ted from a cancer patient in cancer ent. In
the case of using a T cell as the immunocompetent cell,
examples of the peptide consisting of the transmembrane
region and the immunocompetent cell activation signal
uction region fused with the C terminus of the
transmembrane region in the present CAR can specifically
include a peptide consisting of the amino acid sequence
represented by any of SEQ ID NOs: 185 to 187. Examples of
the present CAR can ically include CAR comprising
single chain antibody selected from the group consisting
of the single chain antibody described above in (1-3), the
single chain antibody bed above in (2-3), the single
chain antibody described above in (1-3'-1), the single
chain antibody described above in (1-3'-2), the single
chain antibody described above in 3), the single
chain antibody described above in (2-3'-1), the single
chain antibody described above in (2-3'-2), the single
chain antibody described above in (2-3'-3), and the single
chain antibody described above in (2-3'-4), and a peptide
consisting of the amino acid sequence represented by any
of SEQ ID NOs: 185 to 187, fused with the C terminus of
the single chain antibody.
Specifically, examples of the present CAR can
include
CAR comprising the single chain antibody described above
in (1-3), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 185,
CAR comprising the single chain antibody described above
in (1-3), and a peptide ting of the amino acid
sequence represented by SEQ ID NO: 186,
CAR sing the single chain antibody described above
in (1-3), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 187,
CAR comprising the single chain dy described above
in (1-3'-1), and a e consisting of the amino acid
sequence represented by SEQ ID NO: 185,
CAR sing the single chain antibody described above
in (1-3'-1), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 186,
CAR comprising the single chain antibody described above
in (1-3'-1), and a peptide consisting of the amino acid
ce represented by SEQ ID NO: 187,
CAR comprising the single chain antibody described above
in 2), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 185,
CAR comprising the single chain antibody described above
in (1-3'-2), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 186,
CAR comprising the single chain antibody described above
in (1-3'-2), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 187,
CAR comprising the single chain antibody described above
in (1-3'-3), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 185,
CAR comprising the single chain antibody described above
in (1-3'-3), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 186,
CAR comprising the single chain antibody described above
in 3), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 187,
CAR comprising the single chain antibody described above
in (2-3), and a peptide consisting of the amino acid
ce represented by SEQ ID NO: 185,
CAR comprising the single chain antibody described above
in (2-3), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 186,
CAR comprising the single chain antibody described above
in (2-3), and a peptide ting of the amino acid
sequence represented by SEQ ID NO: 187,
CAR comprising the single chain antibody bed above
in (2-3'-1), and a peptide ting of the amino acid
sequence represented by SEQ ID NO: 185,
CAR comprising the single chain antibody described above
in (2-3'-1), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 186,
CAR comprising the single chain antibody described above
in (2-3'-1), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 187,
CAR comprising the single chain antibody bed above
in (2-3'-2), and a peptide consisting of the amino acid
sequence ented by SEQ ID NO: 185,
CAR comprising the single chain antibody described above
in (2-3'-2), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 186,
CAR comprising the single chain antibody described above
in (2-3'-2), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 187,
CAR comprising the single chain antibody described above
in (2-3'-3), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 185,
CAR comprising the single chain dy described above
in (2-3'-3), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 186,
CAR comprising the single chain dy described above
in (2-3'-3), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 187,
CAR comprising the single chain antibody described above
in 4), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 185,
CAR comprising the single chain antibody described above
in (2-3'-4), and a peptide consisting of the amino acid
sequence represented by SEQ ID NO: 186, and
CAR sing the single chain antibody bed above
in (2-3'-4), and a peptide ting of the amino acid
ce represented by SEQ ID NO: 187.
The present immunocompetent cell can be any
immunocompetent cell expressing CAR. Since CAR usually
does not occur naturally, the immunocompetent cell
expresses foreign CAR, not endogenous CAR. The present
immunocompetent cell preferably further expresses IL-7
and/or CCL19. When the immunocompetent cell is a cell
found to not express IL-7 and/or CCL19, for example, a T
cell, or when the immunocompetent cell is a cell, other
than a T cell, low expressing IL-7 and/or CCL19, the
present immunocompetent cell preferably expresses foreign
IL-7 and/or CCL19.
The present immunocompetent cell can be prepared by
introducing the present vector comprising the t CAR
gene, and a vector comprising IL-7 and/or CCL19 gene to an
immunocompetent cell. The introduction method can be any
method for introducing DNA to mammalian cells. Examples
thereof can include a method such as electroporation
(Cytotechnology, 3, 133 (1990)), calcium ate method
(Japanese unexamined Patent ation Publication No. 2-
227075), lipofection (Proc. Natl. Acad. Sci. U.S.A., 84,
7413 (1987)), and viral infection method. Examples of
such a viral infection method can include a method which
involves ecting a packaging cell such as a GP2-293
cell (manufactured by Takara Bio Inc.), a Plat-GP cell
(manufactured by Cosmo Bio Co., Ltd.), a PG13 cell (ATCC
CRL-10686), or a PA317 cell (ATCC CRL-9078) with a CAR
expression vector (International Publication No. WO
2016/056228) and a packaging plasmid to prepare a
recombinant virus, and ing a T cell with the
recombinant virus.
The present immunocompetent cell may be produced by
incorporating a nucleotide encoding the t CAR and a
nucleotide encoding IL-7 and/or CCL19 into the genome of a
cell by use of a gene editing que known in the art
such that the nucleotides are expressible under the
control of an appropriate promoter. Examples of the gene
editing que known in the art include a technique
using clease such as zinc finger nuclease, TALEN
(transcription activator-like effector nuclease), or
CRISPR (clustered regularly paced short palindromic
)-Cas system.
The present immunocompetent cell can be used in
ation with an additional anticancer agent. Examples
of the additional anticancer agent can include: an
alkylating drug such as cyclophosphamide, bendamustine,
ifosfamide, and dacarbazine; an antimetabolite such as
tatin, fludarabine, cladribine, rexate, 5-
uracil, 6-mercaptopurine, and enocitabine; a
molecular targeting drug such as rituximab, cetuximab, and
trastuzumab; a kinase inhibitor such as imatinib,
gefitinib, erlotinib, afatinib, dasatinib, sunitinib, and
trametinib; a proteasome inhibitor such as omib; a
calcineurin inhibitory drug such as cyclosporin and
tacrolimus; an anticancer antibiotic such as idarubicin
and doxorubicin mitomycin C; a vegetable alkaloid such as
irinotecan and etoposide; a platinum-containing drug such
as cisplatin, oxaliplatin, and carboplatin; a hormone
therapeutic such as tamoxifen and bicalutamide; and an
immunosuppressive drug such as interferon, nivolumab, and
pembrolizumab.
Examples of the method for "using the present
immunocompetent cell in combination with the additional
anticancer agent" can include a method using treatment
with the additional anticancer agent followed by use of
the present immunocompetent cell, a method using the
present immunocompetent cell and the additional anticancer
agent at the same time, and a method using treatment with
the present immunocompetent cell followed by use of the
additional anticancer agent. Use of the present
competent cell in combination with the additional
anticancer agent can further improve a eutic effect
on a cancer and can also reduce their respective adverse
reactions by decreasing their respective numbers of
administration or doses.
The present antibody gene is not ularly
limited as long as the dy gene (nucleotide) encodes
the present antibody. Examples thereof can include
(1-1D) an antibody gene comprising: a H chain CDR1 gene
consisting of nucleotide residues 91 to 105 of a H chain V
region gene consisting of the nucleotide ce
ented by SEQ ID NO: 111 (gene encoding the H chain
CDR1 described above in (1-1)), or a degenerate codon
engineered form of the H chain CDR1 gene; a H chain CDR2
gene consisting of nucleotide residues 148 to 198 of the H
chain V region gene consisting of the nucleotide sequence
represented by SEQ ID NO: 111 (gene encoding the H chain
CDR2 described above in , or a degenerate codon
engineered form of the H chain CDR2 gene; and a H chain
CDR3 gene consisting of nucleotide residues 295 to 324 of
the H chain V region gene consisting of the nucleotide
sequence represented by SEQ ID NO: 111 (gene encoding the
H chain CDR3 described above in (1-1)), or a degenerate
codon engineered form of the H chain CDR3 gene; and
a L chain CDR1 gene consisting of nucleotide residues 70
to 102 of a L chain V region gene ting of the
nucleotide sequence represented by SEQ ID NO: 112 (gene
encoding the L chain CDR1 described above in , or a
degenerate codon engineered form of the L chain CDR1 gene;
a L chain CDR2 gene consisting of nucleotide residues 148
to 168 of the L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 112 (gene
ng the L chain CDR2 described above in (1-1)), or a
degenerate codon ered form of the L chain CDR2 gene;
and a L chain CDR3 gene consisting of tide residues
265 to 291 of the L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 112 (gene
encoding the L chain CDR3 described above in (1-1)), or a
degenerate codon engineered form of the L chain CDR3 gene,
(2-1D) an antibody gene comprising: a H chain CDR1 gene
consisting of nucleotide residues 91 to 105 of a H chain V
region gene consisting of the nucleotide sequence
represented by SEQ ID NO: 115 (gene encoding the H chain
CDR1 described above in (2-1)), or a degenerate codon
engineered form of the H chain CDR1 gene; a H chain CDR2
gene consisting of nucleotide residues 148 to 198 of the H
chain V region gene consisting of the nucleotide sequence
represented by SEQ ID NO: 115 (gene encoding the H chain
CDR2 described above in (2-1)), or a degenerate codon
engineered form of the H chain CDR2 gene; and a H chain
CDR3 gene ting of nucleotide residues 295 to 321 of
the H chain V region gene consisting of the nucleotide
sequence represented by SEQ ID NO: 115 (gene encoding the
H chain CDR3 described above in (2-1)), or a degenerate
codon engineered form of the H chain CDR3 gene; and
a L chain CDR1 gene consisting of nucleotide residues 70
to 117 of a L chain V region gene consisting of the
tide sequence represented by SEQ ID NO: 116 (gene
ng the L chain CDR1 described above in (2-1)), or a
degenerate codon engineered form of the L chain CDR1 gene;
a L chain CDR2 gene consisting of nucleotide es 163
to 183 of the L chain V region gene consisting of the
nucleotide ce represented by SEQ ID NO: 116 (gene
encoding the L chain CDR2 described above in (2-1)), or a
degenerate codon engineered form of the L chain CDR2 gene;
and a L chain CDR3 gene consisting of nucleotide residues
280 to 306 of the L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 116 (gene
ng the L chain CDR3 described above in (2-1)), or a
rate codon engineered form of the L chain CDR3 gene,
(3-1D) an antibody gene comprising: a H chain CDR1 gene
consisting of nucleotide residues 91 to 105 of a H chain V
region gene ting of the nucleotide sequence
represented by SEQ ID NO: 119 (gene encoding the H chain
CDR1 described above in (3-1)), or a degenerate codon
engineered form of the H chain CDR1 gene; a H chain CDR2
gene consisting of nucleotide residues 148 to 198 of the H
chain V region gene consisting of the nucleotide sequence
represented by SEQ ID NO: 119 (gene encoding the H chain
CDR2 described above in (3-1)), or a degenerate codon
engineered form of the H chain CDR2 gene; and a H chain
CDR3 gene consisting of nucleotide residues 295 to 315 of
the H chain V region gene consisting of the nucleotide
sequence represented by SEQ ID NO: 119 (gene encoding the
H chain CDR3 described above in (3-1)), or a degenerate
codon engineered form of the H chain CDR3 gene; and
a L chain CDR1 gene consisting of nucleotide residues 70
to 102 of a L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 120 (gene
encoding the L chain CDR1 described above in , or a
degenerate codon engineered form of the L chain CDR1 gene;
a L chain CDR2 gene consisting of nucleotide residues 148
to 168 of the L chain V region gene consisting of the
nucleotide sequence ented by SEQ ID NO: 120 (gene
encoding the L chain CDR2 described above in (3-1)), or a
degenerate codon ered form of the L chain CDR2 gene;
and a L chain CDR3 gene consisting of nucleotide residues
265 to 288 of the L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 120 (gene
encoding the L chain CDR3 described above in (3-1)), or a
degenerate codon engineered form of the L chain CDR3 gene,
(4-1D) an antibody gene sing: a H chain CDR1 gene
consisting of tide residues 91 to 105 of a H chain V
region gene consisting of the tide sequence
represented by SEQ ID NO: 123 (gene ng the H chain
CDR1 described above in (4-1)), or a degenerate codon
engineered form of the H chain CDR1 gene; a H chain CDR2
gene consisting of nucleotide residues 148 to 198 of the H
chain V region gene consisting of the nucleotide sequence
represented by SEQ ID NO: 123 (gene encoding the H chain
CDR2 described above in (4-1)), or a degenerate codon
engineered form of the H chain CDR2 gene; and a H chain
CDR3 gene consisting of nucleotide residues 298 to 330 of
the H chain V region gene consisting of the nucleotide
ce represented by SEQ ID NO: 123 (gene encoding the
H chain CDR3 described above in , or a degenerate
codon engineered form of the H chain CDR3 gene; and
a L chain CDR1 gene consisting of nucleotide residues 70
to 102 of a L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 124 (gene
encoding the L chain CDR1 described above in (4-1)), or a
degenerate codon engineered form of the L chain CDR1 gene;
a L chain CDR2 gene consisting of nucleotide residues 148
to 168 of the L chain V region gene consisting of the
tide sequence represented by SEQ ID NO: 124 (gene
encoding the L chain CDR2 described above in (4-1)), or a
degenerate codon ered form of the L chain CDR2 gene;
and a L chain CDR3 gene consisting of nucleotide residues
265 to 291 of the L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 124 (gene
encoding the L chain CDR3 described above in (4-1)), or a
degenerate codon engineered form of the L chain CDR3 gene,
(5-1D) an antibody gene comprising: a H chain CDR1 gene
consisting of tide residues 91 to 105 of a H chain V
region gene consisting of the nucleotide sequence
represented by SEQ ID NO: 127 (gene encoding the H chain
CDR1 described above in , or a degenerate codon
engineered form of the H chain CDR1 gene; a H chain CDR2
gene consisting of nucleotide residues 148 to 198 of the H
chain V region gene consisting of the nucleotide ce
represented by SEQ ID NO: 127 (gene encoding the H chain
CDR2 described above in (5-1)), or a degenerate codon
engineered form of the H chain CDR2 gene; and a H chain
CDR3 gene consisting of nucleotide residues 298 to 321 of
the H chain V region gene ting of the nucleotide
sequence ented by SEQ ID NO: 127 (gene encoding the
H chain CDR3 bed above in (5-1)), or a degenerate
codon engineered form of the H chain CDR3 gene; and
a L chain CDR1 gene consisting of nucleotide residues 70
to 120 of a L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 128 (gene
encoding the L chain CDR1 described above in (5-1)), or a
degenerate codon engineered form of the L chain CDR1 gene;
a L chain CDR2 gene consisting of nucleotide residues 166
to 186 of the L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 128 (gene
encoding the L chain CDR2 described above in (5-1)), or a
degenerate codon engineered form of the L chain CDR2 gene;
and a L chain CDR3 gene consisting of nucleotide residues
283 to 309 of the L chain V region gene consisting of the
tide sequence represented by SEQ ID NO: 128 (gene
encoding the L chain CDR3 described above in (5-1)), or a
degenerate codon engineered form of the L chain CDR3 gene,
(6-1D) an antibody gene comprising: a H chain CDR1 gene
consisting of nucleotide residues 91 to 105 of a H chain V
region gene consisting of the nucleotide sequence
represented by SEQ ID NO: 131 (gene encoding the H chain
CDR1 described above in (6-1)), or a degenerate codon
engineered form of the H chain CDR1 gene; a H chain CDR2
gene consisting of tide residues 148 to 198 of the H
chain V region gene ting of the tide sequence
represented by SEQ ID NO: 131 (gene encoding the H chain
CDR2 described above in (6-1)), or a degenerate codon
engineered form of the H chain CDR2 gene; and a H chain
CDR3 gene consisting of nucleotide residues 295 to 318 of
the H chain V region gene consisting of the nucleotide
sequence ented by SEQ ID NO: 131 (gene ng the
H chain CDR3 described above in (6-1)), or a degenerate
codon engineered form of the H chain CDR3 gene; and
a L chain CDR1 gene consisting of nucleotide residues 70
to 102 of a L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 132 (gene
encoding the L chain CDR1 described above in (6-1)), or a
degenerate codon engineered form of the L chain CDR1 gene;
a L chain CDR2 gene consisting of nucleotide residues 148
to 168 of the L chain V region gene consisting of the
nucleotide sequence ented by SEQ ID NO: 132 (gene
encoding the L chain CDR2 described above in (6-1)), or a
degenerate codon engineered form of the L chain CDR2 gene;
and a L chain CDR3 gene consisting of nucleotide es
265 to 291 of the L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 132 (gene
encoding the L chain CDR3 described above in (6-1)), or a
degenerate codon engineered form of the L chain CDR3 gene,
(7-1D) an antibody gene comprising: a H chain CDR1 gene
consisting of nucleotide residues 91 to 105 of a H chain V
region gene consisting of the nucleotide sequence
represented by SEQ ID NO: 135 (gene ng the H chain
CDR1 described above in (7-1)), or a degenerate codon
engineered form of the H chain CDR1 gene; a H chain CDR2
gene consisting of nucleotide residues 148 to 198 of the H
chain V region gene consisting of the nucleotide ce
represented by SEQ ID NO: 135 (gene encoding the H chain
CDR2 described above in (7-1)), or a degenerate codon
engineered form of the H chain CDR2 gene; and a H chain
CDR3 gene consisting of nucleotide residues 295 to 315 of
the H chain V region gene ting of the tide
sequence represented by SEQ ID NO: 135 (gene encoding the
H chain CDR3 described above in (7-1)), or a degenerate
codon engineered form of the H chain CDR3 gene; and
a L chain CDR1 gene consisting of nucleotide residues 70
to 102 of a L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 136 (gene
encoding the L chain CDR1 described above in (7-1)), or a
degenerate codon ered form of the L chain CDR1 gene;
a L chain CDR2 gene ting of tide residues 148
to 168 of the L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 136 (gene
encoding the L chain CDR2 described above in (7-1)), or a
degenerate codon ered form of the L chain CDR2 gene;
and a L chain CDR3 gene consisting of nucleotide residues
265 to 291 of the L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 136 (gene
encoding the L chain CDR3 described above in (7-1)), or a
degenerate codon engineered form of the L chain CDR3 gene,
(8-1D) an antibody gene comprising: a H chain CDR1 gene
consisting of nucleotide residues 91 to 105 of a H chain V
region gene consisting of the tide sequence
represented by SEQ ID NO: 139 (gene encoding the H chain
CDR1 described above in (8-1)), or a degenerate codon
engineered form of the H chain CDR1 gene; a H chain CDR2
gene consisting of nucleotide residues 148 to 198 of the H
chain V region gene consisting of the nucleotide sequence
represented by SEQ ID NO: 139 (gene encoding the H chain
CDR2 described above in (8-1)), or a rate codon
engineered form of the H chain CDR2 gene; and a H chain
CDR3 gene consisting of nucleotide residues 295 to 315 of
the H chain V region gene consisting of the nucleotide
ce represented by SEQ ID NO: 139 (gene encoding the
H chain CDR3 described above in (8-1)), or a degenerate
codon engineered form of the H chain CDR3 gene; and
a L chain CDR1 gene consisting of tide residues 70
to 102 of a L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 140 (gene
encoding the L chain CDR1 described above in (8-1)), or a
degenerate codon engineered form of the L chain CDR1 gene;
a L chain CDR2 gene consisting of nucleotide residues 148
to 168 of the L chain V region gene ting of the
nucleotide sequence represented by SEQ ID NO: 140 (gene
encoding the L chain CDR2 described above in , or a
degenerate codon engineered form of the L chain CDR2 gene;
and a L chain CDR3 gene consisting of nucleotide es
265 to 291 of the L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 140 (gene
encoding the L chain CDR3 described above in (8-1)), or a
degenerate codon engineered form of the L chain CDR3 gene,
(9-1D) an dy gene comprising: a H chain CDR1 gene
consisting of nucleotide residues 91 to 105 of a H chain V
region gene consisting of the nucleotide sequence
represented by SEQ ID NO: 143 (gene encoding the H chain
CDR1 described above in (9-1)), or a degenerate codon
engineered form of the H chain CDR1 gene; a H chain CDR2
gene consisting of nucleotide residues 148 to 198 of the H
chain V region gene consisting of the nucleotide sequence
represented by SEQ ID NO: 143 (gene encoding the H chain
CDR2 described above in (9-1)), or a degenerate codon
engineered form of the H chain CDR2 gene; and a H chain
CDR3 gene consisting of nucleotide residues 298 to 330 of
the H chain V region gene consisting of the nucleotide
sequence represented by SEQ ID NO: 143 (gene encoding the
H chain CDR3 described above in (9-1)), or a degenerate
codon engineered form of the H chain CDR3 gene; and
a L chain CDR1 gene consisting of nucleotide residues 70
to 102 of a L chain V region gene ting of the
nucleotide sequence represented by SEQ ID NO: 144 (gene
encoding the L chain CDR1 described above in (9-1)), or a
rate codon ered form of the L chain CDR1 gene;
a L chain CDR2 gene consisting of nucleotide residues 148
to 168 of the L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 144 (gene
encoding the L chain CDR2 described above in , or a
degenerate codon engineered form of the L chain CDR2 gene;
and a L chain CDR3 gene consisting of nucleotide residues
265 to 291 of the L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 144 (gene
encoding the L chain CDR3 described above in (9-1)), or a
degenerate codon engineered form of the L chain CDR3 gene,
(10-1D) an antibody gene comprising: a H chain CDR1 gene
ting of nucleotide residues 91 to 105 of a H chain V
region gene consisting of the nucleotide sequence
represented by SEQ ID NO: 147 (gene encoding the H chain
CDR1 described above in (10-1)), or a degenerate codon
engineered form of the H chain CDR1 gene; a H chain CDR2
gene consisting of nucleotide es 148 to 198 of the H
chain V region gene consisting of the nucleotide sequence
represented by SEQ ID NO: 147 (gene encoding the H chain
CDR2 described above in (10-1)), or a degenerate codon
engineered form of the H chain CDR2 gene; and a H chain
CDR3 gene consisting of nucleotide residues 295 to 327 of
the H chain V region gene consisting of the nucleotide
sequence represented by SEQ ID NO: 147 (gene encoding the
H chain CDR3 bed above in (10-1)), or a degenerate
codon engineered form of the H chain CDR3 gene; and
a L chain CDR1 gene consisting of nucleotide es 70
to 102 of a L chain V region gene consisting of the
nucleotide sequence ented by SEQ ID NO: 148 (gene
encoding the L chain CDR1 described above in (10-1)), or a
degenerate codon engineered form of the L chain CDR1 gene;
a L chain CDR2 gene consisting of nucleotide residues 148
to 168 of the L chain V region gene consisting of the
nucleotide sequence ented by SEQ ID NO: 148 (gene
encoding the L chain CDR2 described above in ), or a
degenerate codon engineered form of the L chain CDR2 gene;
and a L chain CDR3 gene consisting of nucleotide residues
265 to 291 of the L chain V region gene consisting of the
nucleotide sequence represented by SEQ ID NO: 148 (gene
encoding the L chain CDR3 described above in (10-1)), or a
degenerate codon ered form of the L chain CDR3 gene,
(11-1D) an antibody gene comprising: a H chain CDR1 gene
consisting of nucleotide residues 91 to 105 of a H chain V
region gene consisting of the nucleotide sequence
represented by SEQ ID NO: 151 (gene encoding the H chain
CDR1 described above in ), or a degenerate codon
engineered form of the H chain CDR1 gene; a H chain CDR2
gene consisting of nucleotide residues 148 to 198 of the H
chain V region gene consisting of the nucleotide ce
represented by SEQ ID NO: 151 (gene encoding the H chain
CDR2 described above in (11-1)), or a degenerate codon
engineered form of the H chain CDR2 gene; and a H chain
CDR3 gene consisting of tide residues 295 to 324 of
the H chain V region consisting of the nucleotide sequence
ented by SEQ ID NO: 151 (gene encoding the H chain
CDR3 described above in (11-1)), or a degenerate codon
engineered form of the H chain CDR3 gene.
Further examples of the present antibody gene can
include
(1-2D) an antibody gene comprising a H chain variable
region gene consisting of a nucleotide ce having at
least 80% or higher sequence identity to the nucleotide
sequence represented by SEQ ID NO: 111 (gene encoding a H
chain variable region consisting of an amino acid sequence
having at least 80% or higher sequence identity to the
amino acid sequence represented by SEQ ID NO: 7), and a L
chain variable region gene consisting of a nucleotide
sequence having at least 80% or higher sequence identity
to the nucleotide ce represented by SEQ ID NO: 112
(gene encoding a L chain variable region consisting of an
amino acid sequence having at least 80% or higher sequence
identity to the amino acid sequence represented by SEQ ID
NO: 8),
(2-2D) an antibody gene comprising a H chain variable
region gene consisting of a nucleotide sequence having at
least 80% or higher ce identity to the nucleotide
sequence represented by SEQ ID NO: 115 (gene encoding a H
chain variable region consisting of an amino acid ce
having at least 80% or higher sequence ty to the
amino acid sequence represented by SEQ ID NO: 17), and a L
chain variable region gene consisting of a nucleotide
sequence having at least 80% or higher sequence identity
to the nucleotide sequence represented by SEQ ID NO: 116
(gene encoding a L chain variable region consisting of an
amino acid sequence having at least 80% or higher sequence
identity to the amino acid sequence represented by SEQ ID
NO: 18),
(3-2D) an antibody gene comprising a H chain variable
region gene consisting of a nucleotide sequence having at
least 80% or higher sequence identity to the nucleotide
sequence represented by SEQ ID NO: 119 (gene encoding a H
chain variable region consisting of an amino acid sequence
having at least 80% or higher sequence identity to the
amino acid sequence represented by SEQ ID NO: 27), and a L
chain variable region gene consisting of a nucleotide
sequence having at least 80% or higher sequence identity
to the nucleotide sequence represented by SEQ ID NO: 120
(gene encoding a L chain variable region consisting of an
amino acid ce having at least 80% or higher sequence
ty to the amino acid sequence represented by SEQ ID
NO: 28),
(4-2D) an antibody gene comprising a H chain variable
region gene consisting of a nucleotide sequence having at
least 80% or higher sequence identity to the nucleotide
sequence represented by SEQ ID NO: 123 (gene encoding a H
chain variable region ting of an amino acid sequence
having at least 80% or higher sequence ty to the
amino acid sequence represented by SEQ ID NO: 37), and a L
chain variable region gene consisting of a nucleotide
sequence having at least 80% or higher sequence identity
to the nucleotide sequence represented by SEQ ID NO: 124
(gene encoding a L chain le region consisting of an
amino acid sequence having at least 80% or higher ce
identity to the amino acid sequence represented by SEQ ID
NO: 38),
(5-2D) an antibody gene comprising a H chain variable
region gene consisting of a nucleotide sequence having at
least 80% or higher sequence identity to the nucleotide
sequence represented by SEQ ID NO: 127 (gene encoding a H
chain le region consisting of an amino acid sequence
having at least 80% or higher sequence ty to the
amino acid sequence ented by SEQ ID NO: 47), and a L
chain variable region gene consisting of a nucleotide
sequence having at least 80% or higher sequence identity
to the nucleotide sequence represented by SEQ ID NO: 128
(gene encoding a L chain variable region consisting of an
amino acid sequence having at least 80% or higher sequence
identity to the amino acid sequence represented by SEQ ID
NO: 48),
(6-2D) an antibody gene comprising a H chain variable
region gene consisting of a nucleotide sequence having at
least 80% or higher sequence identity to the nucleotide
sequence represented by SEQ ID NO: 131 (gene encoding a H
chain variable region consisting of an amino acid sequence
having at least 80% or higher sequence identity to the
amino acid sequence ented by SEQ ID NO: 57), and a L
chain variable region gene consisting of a tide
sequence having at least 80% or higher sequence identity
to the nucleotide sequence ented by SEQ ID NO: 132
(gene encoding a L chain variable region consisting of an
amino acid sequence having at least 80% or higher sequence
identity to the amino acid sequence represented by SEQ ID
NO: 58),
(7-2D) an antibody gene comprising a H chain variable
region gene consisting of a tide sequence having at
least 80% or higher sequence identity to the nucleotide
sequence represented by SEQ ID NO: 135 (gene encoding a H
chain variable region consisting of an amino acid ce
having at least 80% or higher ce identity to the
amino acid sequence represented by SEQ ID NO: 67), and a L
chain variable region gene consisting of a nucleotide
sequence having at least 80% or higher sequence identity
to the nucleotide sequence represented by SEQ ID NO: 136
(gene encoding a L chain variable region consisting of an
amino acid sequence having at least 80% or higher sequence
identity to the amino acid sequence represented by SEQ ID
NO: 68),
(8-2D) an antibody gene comprising a H chain variable
region gene consisting of a nucleotide sequence having at
least 80% or higher sequence identity to the nucleotide
sequence represented by SEQ ID NO: 139 (gene encoding a H
chain variable region consisting of an amino acid ce
having at least 80% or higher sequence identity to the
amino acid sequence represented by SEQ ID NO: 77), and a L
chain variable region gene consisting of a nucleotide
sequence having at least 80% or higher sequence identity
to the nucleotide sequence represented by SEQ ID NO: 140
(gene encoding a L chain variable region consisting of an
amino acid sequence having at least 80% or higher sequence
ty to the amino acid sequence represented by SEQ ID
NO: 78),
(9-2D) an antibody gene sing a H chain variable
region gene consisting of a nucleotide sequence having at
least 80% or higher sequence identity to the nucleotide
sequence represented by SEQ ID NO: 143 (gene encoding a H
chain variable region consisting of an amino acid sequence
having at least 80% or higher sequence identity to the
amino acid sequence ented by SEQ ID NO: 87), and a L
chain variable region gene consisting of a nucleotide
sequence having at least 80% or higher sequence ty
to the nucleotide sequence represented by SEQ ID NO: 144
(gene encoding a L chain variable region consisting of an
amino acid ce having at least 80% or higher sequence
identity to the amino acid sequence represented by SEQ ID
NO: 88),
(10-2D) an antibody gene comprising a H chain variable
region gene consisting of a nucleotide sequence having at
least 80% or higher sequence ty to the nucleotide
sequence represented by SEQ ID NO: 147 (gene encoding a H
chain variable region consisting of an amino acid sequence
having at least 80% or higher sequence identity to the
amino acid ce represented by SEQ ID NO: 97), and a L
chain variable region gene consisting of a nucleotide
sequence having at least 80% or higher sequence identity
to the nucleotide sequence represented by SEQ ID NO: 148
(gene encoding a L chain variable region consisting of an
amino acid sequence having at least 80% or higher sequence
ty to the amino acid ce represented by SEQ ID
NO: 98), and
(11-2D) an antibody gene comprising a H chain le
region gene consisting of a nucleotide sequence having at
least 80% or higher sequence identity to the nucleotide
sequence ented by SEQ ID NO: 151 (gene encoding a H
chain variable region consisting of an amino acid sequence
having at least 80% or higher sequence identity to the
amino acid sequence ented by SEQ ID NO: 107), and a
L chain variable region gene consisting of a nucleotide
sequence having at least 80% or higher sequence identity
to the nucleotide sequence represented by SEQ ID NO: 152
(gene encoding a L chain variable region consisting of an
amino acid sequence having at least 80% or higher sequence
identity to the amino acid sequence represented by SEQ ID
NO: 108).
Particularly, examples of the present antibody gene
can specifically include
(1-4D) an antibody gene comprising a H chain gene
consisting of a tide sequence having at least 80% or
higher sequence identity to the nucleotide sequence
ented by SEQ ID NO: 113 (gene encoding a H chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 9), and a L chain gene
consisting of a nucleotide ce having at least 80% or
higher sequence ty to the nucleotide sequence
represented by SEQ ID NO: 114 (gene encoding a L chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 10),
(2-4D) an antibody gene comprising a H chain gene
consisting of a nucleotide sequence having at least 80% or
higher ce identity to the nucleotide sequence
represented by SEQ ID NO: 117 (gene ng a H chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 19), and a L chain gene
consisting of a nucleotide sequence having at least 80% or
higher sequence identity to the nucleotide sequence
represented by SEQ ID NO: 118 (gene encoding a L chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid ce
represented by SEQ ID NO: 20),
(3-4D) an antibody gene comprising a H chain gene
consisting of a nucleotide sequence having at least 80% or
higher sequence identity to the nucleotide sequence
represented by SEQ ID NO: 121 (gene encoding a H chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 29), and a L chain gene
consisting of a nucleotide sequence having at least 80% or
higher sequence identity to the nucleotide sequence
ented by SEQ ID NO: 122 (gene ng a L chain
consisting of an amino acid sequence having at least 80%
or higher sequence ty to the amino acid sequence
represented by SEQ ID NO: 30),
(4-4D) an antibody gene comprising a H chain gene
consisting of a nucleotide ce having at least 80% or
higher sequence identity to the nucleotide sequence
represented by SEQ ID NO: 125 (gene encoding a H chain
consisting of an amino acid ce having at least 80%
or higher ce identity to the amino acid sequence
ented by SEQ ID NO: 39), and a L chain gene
consisting of a nucleotide sequence having at least 80% or
higher sequence identity to the nucleotide sequence
represented by SEQ ID NO: 126 (gene encoding a L chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 40),
(5-4D) an antibody gene comprising a H chain gene
consisting of a nucleotide sequence having at least 80% or
higher sequence identity to the nucleotide sequence
represented by SEQ ID NO: 129 (gene encoding a H chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 49), and a L chain gene
consisting of a nucleotide sequence having at least 80% or
higher sequence identity to the nucleotide ce
represented by SEQ ID NO: 130 (gene encoding a L chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 50),
(6-4D) an antibody gene comprising a H chain gene
consisting of a nucleotide sequence having at least 80% or
higher sequence identity to the nucleotide sequence
represented by SEQ ID NO: 133 (gene encoding a H chain
consisting of an amino acid ce having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 59), and a L chain gene
consisting of a nucleotide ce having at least 80% or
higher sequence identity to the nucleotide sequence
ented by SEQ ID NO: 134 (gene encoding a L chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 60),
(7-4D) an antibody gene comprising a H chain gene
consisting of a nucleotide sequence having at least 80% or
higher sequence identity to the tide sequence
ented by SEQ ID NO: 137 (gene ng a H chain
consisting of an amino acid sequence having at least 80%
or higher sequence ty to the amino acid sequence
represented by SEQ ID NO: 69), and a L chain gene
consisting of a nucleotide sequence having at least 80% or
higher sequence identity to the nucleotide sequence
represented by SEQ ID NO: 138 (gene encoding a L chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 70),
(8-4D) an antibody gene comprising a H chain gene
consisting of a nucleotide sequence having at least 80% or
higher sequence identity to the nucleotide sequence
represented by SEQ ID NO: 141 (gene encoding a H chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 79), and a L chain gene
consisting of a nucleotide sequence having at least 80% or
higher sequence identity to the tide sequence
represented by SEQ ID NO: 142 (gene encoding a L chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 80),
(9-4D) an antibody gene comprising a H chain gene
consisting of a nucleotide sequence having at least 80% or
higher sequence ty to the nucleotide sequence
represented by SEQ ID NO: 145 (gene encoding a H chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid ce
represented by SEQ ID NO: 89), and a L chain gene
ting of a nucleotide sequence having at least 80% or
higher sequence identity to the nucleotide sequence
represented by SEQ ID NO: 146 (gene encoding a L chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 90),
(10-4D) an antibody gene comprising a H chain gene
consisting of a tide sequence having at least 80% or
higher sequence ty to the nucleotide sequence
represented by SEQ ID NO: 149 (gene encoding a H chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 99), and a L chain gene
consisting of a nucleotide sequence having at least 80% or
higher sequence identity to the nucleotide ce
represented by SEQ ID NO: 150 (gene encoding a L chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 100), and
(11-4D) an antibody gene comprising a H chain gene
consisting of a nucleotide sequence having at least 80% or
higher sequence identity to the nucleotide sequence
represented by SEQ ID NO: 153 (gene encoding a H chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 109), and a L chain gene
ting of a nucleotide sequence having at least 80% or
higher sequence identity to the nucleotide sequence
represented by SEQ ID NO: 154 (gene encoding a L chain
consisting of an amino acid sequence having at least 80%
or higher sequence identity to the amino acid sequence
represented by SEQ ID NO: 110).
The t CAR gene is not particularly limited as
long as the gene (nucleotide) encodes the present CAR.
es f can specifically include
(1-3D) a CAR gene comprising a gene encoding the single
chain antibody described above in (1-3), or a degenerate
codon engineered form of the gene,
(2-3D) a CAR gene comprising a gene encoding the single
chain antibody described above in (2-3), or a rate
codon engineered form of the gene,
(3-3D) a CAR gene comprising a gene encoding the single
chain antibody described above in (3-3), or a rate
codon engineered form of the gene,
(4-3D) a CAR gene comprising a gene encoding the single
chain antibody described above in (4-3), or a degenerate
codon engineered form of the gene,
(5-3D) a CAR gene comprising a gene encoding the single
chain antibody described above in (5-3), or a degenerate
codon ered form of the gene,
(6-3D) a CAR gene comprising a gene encoding the single
chain dy described above in (6-3), or a degenerate
codon engineered form of the gene,
(7-3D) a CAR gene comprising a gene encoding the single
chain antibody described above in (7-3), or a degenerate
codon engineered form of the gene,
(8-3D) a CAR gene sing a gene encoding the single
chain antibody described above in (8-3), or a degenerate
codon engineered form of the gene,
(9-3D) a CAR gene comprising a gene ng the single
chain antibody described above in (9-3), or a degenerate
codon engineered form of the gene,
) a CAR gene comprising a gene encoding the single
chain antibody described above in (10-3), or a degenerate
codon engineered form of the gene,
(11-3D) a CAR gene comprising a gene encoding the single
chain antibody described above in , or a degenerate
codon engineered form of the gene,
(1-3'-1D) a CAR gene comprising a gene encoding the single
chain antibody described above in (1-3'-1), or a
degenerate codon engineered form of the gene,
(1-3'-2D) a CAR gene comprising a gene encoding the single
chain antibody described above in (1-3'-2), or a
degenerate codon engineered form of the gene,
3D) a CAR gene comprising a gene encoding the single
chain antibody described above in (1-3'-3), or a
degenerate codon engineered form of the gene,
(2-3'-1D) a CAR gene comprising a gene encoding the single
chain antibody described above in (2-3'-1), or a
degenerate codon ered form of the gene,
(2-3'-2D) a CAR gene sing a gene encoding the single
chain antibody described above in (2-3'-2), or a
degenerate codon engineered form of the gene,
(2-3'-3D) a CAR gene sing a gene encoding the single
chain antibody bed above in (2-3'-3), or a
degenerate codon engineered form of the gene, and
(2-3'-4D) a CAR gene comprising a gene encoding the single
chain antibody described above in (2-3'-4), or a
degenerate codon engineered form of the gene.
In the present specification, the phrase "at least
80% or higher identity" means that the identity is 80% or
higher, preferably 85% or higher, more preferably 88% or
higher, further preferably 90% or higher, still further
preferably 93% or higher, particularly preferably 95% or
higher, particularly more preferably 98% or higher, most
ably 100%.
In the present specification, the term "identity"
means the degree of similarity between polypeptide or
cleotide sequences (this degree is determined by
matching a query sequence to another sequence, preferably
of the same type (nucleic acid or protein sequence)).
Examples of a preferred er program method for
calculating and determining the "identity" include, but
are not limited to, GCG BLAST (Basic Local Alignment
Search Tool) (Altschul et al., J. Mol. Biol. 1990, 215:
403-410; Altschul et al., Nucleic Acids Res. 1997, 25:
402; and Devereux et al., c Acid Res. 1984,
12: 387), BLASTN 2.0 (Gish W.,
http://blast.wustl.edu,1996-2002), FASTA (Pearson and
Lipman, Proc. Natl. Acad. Sci. USA 1988, 85: 2444-2448),
and GCG GelMerge which determines and aligns a pair of the
longest overlapping contigs (Wibur and Lipman, SIAM J.
Appl. Math. 1984, 44: 557-567; and Needleman and Wunsch, J.
Mol. Biol. 1970, 48: 443-453).
In the present specification, the "amino acid
sequence having at least 80% or higher sequence identity
to the amino acid sequence represented by SEQ ID NO: X" is,
in other words, an "amino acid sequence derived from the
amino acid ce represented by SEQ ID NO: X by the
deletion, substitution, ion, and/or addition of 0, 1
or several amino acid residues" and has functions
equivalent to those of the amino acid sequence represented
by SEQ ID NO: X. In this context, the "amino acid
sequence derived by the deletion, tution, ion,
and/or addition of 1 or several amino acid residues" means
an amino acid sequence in which amino acid residues have
been deleted, substituted, inserted, and/or added, for
example, within the range of 1 to 30 residues, preferably
within the range of 1 to 20 residues, more preferably
within the range of 1 to 15 residues, further ably
within the range of 1 to 10 residues, further preferably
within the range of 1 to 5 residues, further preferably
within the range of 1 to 3 residues, further preferably
within the range of 1 or 2 residues. The mutation
treatment of these amino acid residues can be performed by
an arbitrary method known to those skilled in the art such
as al synthesis, a gene ering approach, or
mutagenesis.
The promoter in the present vector can be any region
that starts the transcription of mRNA encoded by the
present antibody gene located downstream of the promoter.
The promoter usually comprises a transcription start site
(TSS).
The type of the promoter or the vector in the
present vector can be appropriately selected according to
the type of a host cell (or a host organism) to which the
present vector is introduced.
The host cell can express the present antibody by
the transcription of the present antibody gene, or can
express the present CAR by the transcription of mRNA of
the present CAR gene. In the case of ucing a
"vector comprising the t antibody gene" as the
present vector, a yeast, a mammalian cell, an insect cell,
or a plant cell given below can be used as the host cell.
In the case of ucing a "vector comprising the
present CAR gene" as the t vector, the
immunocompetent cell described above can be used as the
host cell.
In the case of using a yeast (e.g., Saccharomyces
cerevisiae and Schizosaccharomyces pombe) as the host cell,
examples of the present vector can include a vector such
as YEP13 7115), YEp24 (ATCC37051), and YCp50
(ATCC37419), and a vector derived from the vector.
es of the promoter can include glycolysis gene (e.g.,
hexose kinase gene) promoter, PHO5 promoter, PGK promoter,
GAP promoter, ADH promoter, gal1 promoter, gal10 promoter,
heat shock protein promoter, MFα1 promoter, and CUP1
promoter.
In the case of using a mammalian cell (e.g., a
human-derived a cell, a monkey-derived COS cell, a
Chinese hamster ovary-derived CHO cell, and a human- or
mouse-derived T cell) as the host cell and using a vector
sing the dy gene as the present vector,
examples of the present vector can include a vector such
as pcDNAI, pcDM8 (manufactured by Funakoshi Co., Ltd.),
pAGE107 (Japanese unexamined Patent Application
Publication No. 3-22979; and Cytotechnology, 3,
133,(1990)), pAS3-3 (Japanese unexamined Patent
Application ation No. 2-227075), pCDM8 (Nature, 329,
840, (1987)), pcDNAI/Amp (manufactured by Invitrogen
Corp.), pREP4 (manufactured by Invitrogen Corp.), pAGE103
(J. Biochemistry, 101, 1307 ), and pAGE210, and a
vector derived from the vector. On the other hand, in the
case of using a mammalian cell (e.g., the human-derived
immunocompetent cell described above) as the host cell and
using a vector comprising the CAR gene as the present
vector, examples of the present vector can include a
retrovirus vector such as a pMSGV vector (Tamada k et al.,
Clin Cancer Res 18: 6436-6445 (2002)) and a pMSCV vector
(manufactured by Takara Bio Inc.), and a vector derived
from the vector.
Examples of the promoter in the present vector can
include cytomegalovirus (CMV) IE (immediate early) gene
promoter, SV40 early promoter, retrovirus promoter,
metallothionein promoter, heat shock er, SRα
promoter, NFAT promoter, and HIF promoter.
In the case of using an insect cell (e.g., a Sf9
cell and a Sf21 cell which are Spodoptera frugiperda
ovarian cells, and a High5 cell which is a Trichoplusia ni
ovarian cell) as the host cell, examples of the t
vector can include a transfer vector for use in
recombinant baculovirus preparation methods, specifically,
a vector such as pVL1392, pVL1393, and pBlueBacIII (all
manufactured by ogen Corp.), and a vector d
from the . Examples of the promoter can include
polyhedrin er and p10 promoter.
In the case of using a plant cell (e.g., o,
potato, tomato, carrot, soybean, rapeseed, alfalfa, rice,
wheat, and barley cells) as the host cell, examples of the
expression vector can include a vector such as Ti plasmid
and tobacco mosaic virus vector, and a vector derived from
the vector. Examples of the promoter can include
cauliflower mosaic virus (CaMV) 35S promoter and rice
actin 1 promoter.
The present vector preferably further comprises the
nucleotide sequences of an enhancer region and a ribosome
binding site (RBS) for further enhancing gene sion
efficiency, and r comprises a drug resistance gene
(e.g., spectinomycin ance gene, chloramphenicol
resistance gene, tetracycline resistance gene, kanamycin
resistance gene, ampicillin ance gene, puromycin
resistance gene, hygromycin resistance gene, blasticidin
resistance gene, and geneticin resistance gene)
appropriate for the type of the host cell for screening
for the present host cell. The enhancer region is usually
arranged upstream of the promoter, and RBS is usually
arranged between the er and the present gene. The
nucleotide sequence of the present antibody gene to be
incorporated into the present vector may be subjected to
the optimization of a codon ce according to the host
cell for expression. The present vector can be prepared
by a method known in the art using a gene recombination
technique.
The t host cell can be obtained by introducing
the present vector to the host cell (transfecting the host
cell therewith) by a method appropriate for the type of
the host cell.
In the case of using the yeast described above as
the host cell, the method for introducing the present
vector to the yeast can be any method for introducing DNA
to the yeast. Examples f can include a method such
as electroporation (Methods l., 194, 182 (1990)),
spheroplast method (Proc. Natl. Acad. Sci. U.S.A, 84, 1929
(1978)), and lithium acetate method (J. Bacteriology, 153,
163 (1983)).
In the case of using the mammalian cell bed
above as the host cell, the method for introducing the
present vector to the mammalian cell can be any method for
introducing DNA to the mammalian cell. Examples thereof
can include a method such as electroporation
(Cytotechnology, 3, 133 (1990)), calcium phosphate method
(Japanese unexamined Patent Application Publication No. 2-
227075), lipofection (Proc. Natl. Acad. Sci. U.S.A., 84,
7413 (1987)), and viral ion method, as mentioned
above. Examples of such a viral infection method can
include a method which involves transfecting a packaging
cell such as a GP2-293 cell (manufactured by Takara Bio
Inc.), a Plat-GP cell actured by Cosmo Bio Co.,
Ltd.), a PG13 cell (ATCC CRL-10686), or a PA317 cell (ATCC
CRL-9078) with a CAR expression vector (International
Publication No.
prepare a recombinant virus, and infecting a T cell with
the recombinant virus, as mentioned above.
In the case of using the insect cell described above
as the host cell, examples of the method for introducing
the t vector to the insect cell can include a method
which involves cotransfecting the insect cell with the
present vector (transfer vector) and baculovirus-derived
genomic DNA to prepare a inant baculovirus,
ing to a method described in nt Protocols in
Molecular Biology", "Baculovirus Expression Vectors, A
Laboratory Manual, W.H. Freeman and Company, New York
(1992)", "Bio/Technology, 6, 47 (1988)", etc. Examples of
such a cotransfection method can include a method such as
calcium ate method (Japanese unexamined Patent
Application Publication No. 2-227075) and lipofection
(Proc. Natl. Acad. Sci. , 84, 7413 (1987).
In the case of using the plant cell described above
as the host cell, examples of the method for introducing
the present vector to the plant cell can include a method
such as a method using Agrobacterium (Japanese ined
Patent Application Publication Nos. 59-140885 and 60-
70080), electroporation (Japanese unexamined Patent
Application Publication No. 60-251887), and a method using
a particle gun (gene gun) (Japanese Patent Nos. 2606856
and 2517813).
The present antibody can be obtained by culturing
the present host cell obtained by the method mentioned
above in a culture solution appropriate for the host cell.
A transgenic animal, such as a mouse, cattle, a goat,
sheep, a chicken, or a pig, in which the present antibody
gene (the present vector) has been incorporated is
prepared by use of a transgenic animal preparation
technique, and an antibody d from the present
antibody gene can also be ed in a large amount from
the blood, milk, or the like of the transgenic animal.
Nonhuman animals (e.g., mice and rats) are immunized
with a substance comprising a human-derived GPC3
ptide consisting of the amino acid sequence
represented by SEQ ID NO: 155 (GPC3 polypeptide antigen).
A phage library of scFv genes is prepared by a phage
display method. The present scFv can be ed by a
ning method using the GPC3 polypeptide antigen
and/or a cell line expressing the GPC3 polypeptide antigen
(preferably a cell line expressing no endogenous GPC3),
and further, preferably, a competitor C-terminal
polypeptide of GPC3 consisting of the amino acid sequence
represented by SEQ ID NO: 159. From the nonhuman animals
thus immunized with the antigen, antibody-producing
hybridomas are prepared by use of a cell fusion technique.
A culture supernatant containing the present antibody can
also be obtained through screening by ELISA using a plate
in which the antigen has been immobilized on a solid phase.
The present antibody can be ted and ed from
the culture supernatant by use of an antibody purification
que known in the art.
The present detection method can be any method
comprising the step of detecting GPC3 localized on a cell
membrane (anchored on a cell membrane) in a sample (e.g.,
blood, a tissue, and urine) using the t antibody.
Specific examples of the detection method can include
immunofluorescent staining, Western blotting, and ELISA
using the present antibody.
The present kit for detection is a kit comprising
the present antibody or a labeled form thereof and is
limited by the purpose of "detecting GPC3". The kit
usually comprises components generally used in this kind
of kit, for e, a carrier, a pH ing agent, and
a stabilizer as well as an attached document such as a
manual and an instruction for detecting GPC3.
The organism species of GPC3 to be detected in the
present detection method or the present kit for ion
may be a nonhuman animal such as a mouse or a rat and is
usually a human.
es of the labeling al for the labeled
form of the present antibody can include: an enzyme such
as peroxidase (e.g., horseradish peroxidase [HRP]),
alkaline atase, β-D-galactosidase, glucose oxidase,
glucosephosphate dehydrogenase, alcohol dehydrogenase,
malate dehydrogenase, penicillinase, catalase, apo-glucose
oxidase, urease, luciferase and acetylcholinesterase; a
fluorescent material such as fluorescein isothiocyanate,
phycobiliprotein, rare earth metal chelates, dansyl
chloride and tetramethylrhodamine isothiocyanate; a
fluorescence protein such as green fluorescence protein
(GFP), cyan scence protein (CFP), blue fluorescence
protein (BFP), yellow fluorescence protein (YFP), red
scence protein (RFP) and luciferase; a radioisotope
such as 3H, 14C, 125I and 131I; biotin; avidin; and a
chemiluminescence material.
References, such as scientific literatures, patents,
and patent applications, cited herein are incorporated
herein by reference in their entirety to the same extent
as if each dual reference was specifically described.
The present application claims the priority based on
Japanese Patent Application No. 2017-001732 (filed on
January 10, 2017), the contents of which are incorporated
herein by reference in their ty.
Hereinafter, the present invention will be bed
more specifically with reference to Examples. r,
the cal scope of the present invention is not
limited by these examples.
Example 1
1. Preparation of novel PC3 antibody recognizing N-
terminal polypeptide of human GPC3
[Summary]
SKG/Jcl mice were used as animals to be zed
for preparing an anti-human GPC3 antibody, and full-length
human GPC3 protein was used as an immunizing antigen. The
SKG/Jcl mice were autoimmune disease model mice that
spontaneously develop rheumatoid arthritis and are known
to produce antibodies in response even to self-components
depending on aging or a rearing environment. Meanwhile,
GPC3 is highly homologous between humans and mice and is
usually less likely to cause antibody production even by
the immunization of normal mice. Therefore, the l
mice were used as animals to be immunized. A scFv phage
library was prepared from cDNA derived from B cells of the
mice immunized with GPC3, and an anti-human GPC3 antibody
was isolated by the application of the phage display
method.
Although the antiserum of immunized mice contains
many types of antibodies, it is necessary to select mice
producing antibodies having specificity for the N-terminal
polypeptide of GPC3 by excluding mice producing antibodies
low specific for GPC3 or antibodies recognizing the C-
terminal polypeptide of GPC3. Accordingly, mouse
individuals that ted the production of an antibody
specifically binding to the inal polypeptide of GPC3
were selected by use of ELISA and FCM. Specifically, cDNA
was sized by reverse transcription reaction from
total RNA of the B cells derived from the immunized mice,
and antibody genes were amplified to prepare an antibody
gene library. A scFv phage library was ucted from
the antibody gene library, and E. coli was infected with
the library so that E. coli expressed scFv, ed by
biopanning using recombinant GPC3, the GPC3-expressing
cell line, and the C-terminal ptide of GPC3 to
enrich phages sing the target scFv, i.e., an
antibody against the N-terminal polypeptide of GPC3. In
order to further analyze the obtained scFv for binding
specificity for GPC3 in cells, i.e., GPC3 localized on
(bound to) a cell membrane (membrane-bound GPC3) via a GPI
(glycosylphosphatidylinositol) anchor, verification was
made by use of cell based-ELISA and FCM. rmore, the
nucleotide sequences of H chain and L chain variable
regions of clones having binding specificity were
sequenced, and the tide sequences of the anti-GPC3
antibodies produced by the B cells derived from the
immunized mice were determined on the basis of these
sequences. Finally, the mammalian display method which
involved expressing the N-terminal polypeptide nt
and the C-terminal ptide fragment of GPC3 on cell
surface was used to confirm that the epitope for the scFv
was the N-terminal polypeptide fragment of GPC3.
Hereinafter, ed methods and results will be shown.
1-1 Material and method
[Cell culture]
A JHH7 cell line, a HepG2 cell line, and a SK-Hep-1
cell line forced to express full-length human GPC3
(hereinafter, also referred to as a "GPC3-expressing cell
line") were used as human GPC3-expressing cells to perform
the biopanning and screening of an anti-GPC3 antibody.
The JHH7 cell line is a GPC3-expressing cell line derived
from hepatocellular carcinoma, and the cells
constitutively express GPC3 bound to a cell membrane
(membrane-bound GPC3) via a GPI
sylphosphatidylinositol) anchor. On the other hand,
the HepG2 cell line is a GPC3-expressing cell line derived
from hepatocellular oma, as in the JHH7 cell line,
but is a cell line in which the expression of secretory
GPC3 that is not bound to a cell membrane is dominant over
membrane-bound GPC3. The Sk-Hep-1 cell line is a
cellular carcinoma-derived cell line expressing no
GPC3. Hence, a cell line expressing only ne-bound
full-length GPC3 or membrane-bound GPC3 having a partial
length deficient in a portion of exons can be prepared by
forced expression.
The culture of 4 types of cell lines (JHH7 cell line,
HepG2 cell line, GPC3-expressing cell line, and human
nic kidney epithelium-derived 293T cell line) was
performed under conditions of 37°C and 5% CO2 in a DMEM
culture solution (manufactured by Sigma-Aldrich Co. LLC)
containing 10% FBS (manufactured by Gibco/Thermo Fisher
Scientific Inc.) and 1% penicillin-streptomycin
(manufactured by Gibco/Thermo Fisher Scientific Inc.)
(hereinafter, simply referred to as a "DMEM culture
on"). The culture of a CHO-K1 cell line was
med under conditions of 37°C and 5% CO2 in a Ham's
F12 culture solution (manufactured by Sigma-Aldrich Co.
LLC) containing 10% FBS (manufactured by Thermo
Fisher Scientific Inc.).
[Immunizing antigen]
C-terminally 6 × His-tagged recombinant GPC3
(manufactured by R & D Systems Inc.) was adjusted to 0.1
mg/mL with PBS and mixed with an artificial adjuvant
TiterMax Gold (manufactured by TiterMax USA, Inc.) or CFA
(Freund's Adjuvant Complete) (F5881, manufactured by
Sigma-Aldrich Co. LLC) in equal amounts to prepare an
emulsion, which was then used as an initial immunizing
antigen. Recombinant GPC3 was adjusted to a concentration
from 10 to 100 µg/mL with PBS and used as the second or
later immunizing antigens.
[Preparation of GPC3-expressing cell line]
A gene encoding full-length human GPC3 consisting of
the amino acid sequence represented by SEQ ID NO: 157
(full-length human GPC3 gene ting of the nucleotide
sequence represented by SEQ ID NO: 160) was inserted to a
pcDNA3.1 vector (manufactured by Thermo Fisher Scientific
Inc.) to prepare a GPC3 expression vector. A SK-Hep-1
cell line was transfected with the GPC3 sion vector
according to a rd method and then cultured in a DMEM
culture solution ning G418 (manufactured by Roche
Diagnostics K.K.) to establish a SK-Hep-1 cell line stably
expressing full-length GPC3 (GPC3-expressing cell line).
[Immunization of mouse]
SKG/Jcl mice (CLEA Japan, Inc., -old female,
SPF) were used as animals to be immunized, and immunized
through footpads with inant GPC3 a total of 4 times
on 1-week intervals. On 5 weeks from the start of
immunization, blood was collected, and serum was prepared
according to a standard method and used as a specimen for
the confirmation of an antibody titer.
[Serum antibody titer of rum using ELISA]
In order to confirm the response of the immunized
mice to produce an anti-GPC3 antibody, a serum antibody
titer was measured by use of antigen-immobilized ELISA.
0.5 or 2 µg/mL inant GPC3 was added at 50 µL/well to
a 96-well microplate (manufactured by Nalge Nunc
International), and the plate was incubated at room
temperature for 1 hour or at 4°C for 12 hours. Then, 2%
Block ACE (manufactured by DS Pharma Biomedical Co., Ltd.)
was added at 200 µL/well to perform ng treatment.
The serum derived from the GPC3-immunized mice was
serially diluted from 100-fold to 16500-fold with 0.1%
Block ACE/PBS solution. Each diluted serum sample was
added at 50 µL/well, and the plate was incubated at room
ature for 2 hours to perform n-antibody
reaction treatment. After washing of the wells with a
Tween 20-containing PBS (PBST) solution, goat anti-mouse
IgG (manufactured by Jackson ImmunoResearch Laboratories
Inc.) conjugated with 2 µg/mL dase was added thereto,
and the plate was incubated at room temperature for 2
hours to perform secondary antibody reaction ent.
After washing of the well five times with a PBST solution,
moisture was removed, and a TMB substrate (manufactured by
Thermo Fisher Scientific Inc.) was then added at 50
µL/well to perform color reaction. 15 minutes later, the
color reaction was terminated by the addition of 0.18 M
sulfuric acid at 50 µL/well, followed by the measurement
of absorbance at 450 nm and 540 nm using a plate reader
(manufactured by Bio-Rad Laboratories, Inc.).
Quantification was performed using a corrected value
obtained by cting the measurement value of 540 nm
from the measurement value of 450 nm.
[Specificity of antibody in antiserum using FCM]
In order to further confirm the specific binding
activity of the antiserum against membrane-bound GPC3 as
to the immunized mice, the mouse serum diluted 100-fold
and 5 × 105 cells of the GPC3-expressing cell line were
mixed and incubated for 30 minutes on ice. A FACS buffer
(1% BSA/PBS solution) was added thereto, and the mixture
was centrifuged to remove a supernatant. Then, 100 µL of
1 µg/mL goat ouse IgG (H + L) Alexa Fluor 488
(manufactured by Thermo Fisher ific Inc.) was added
as a secondary antibody, and the mixture was incubated for
minutes on ice to perform ary antibody reaction
treatment. The detection of Alexa Fluor 488 and the
measurement of a fluorescence level were performed using a
flow cytometer (FACSCanto) (manufactured by BD
ences).
[Preparation of scFv phage library]
B derived total RNA was extracted according to
a standard method as to the mice shown to e an
antibody binding to membrane-bound GPC3 by the method
described above in the section [Flow cytometer]. RT-PCR
with the total RNA as a template was performed according
to a standard method to prepare cDNA. Antibody H chain
and L chain variable region genes were amplified by PCR.
A nucleotide sequence encoding a fusion protein of scFv
having the H chain and L chain variable s linked via
a flexible linker, and coat protein g3p (cp3) of s
bacteriophage M13 was inserted to the multicloning site of
a pTZ19R phagemid vector to prepare a scFv expression
vector. The scFv library size was calculated from the
transformation efficiency of an E. coli DH12S strain
(manufactured by Invitrogen Corp.). The transformed DH12S
strain was infected with a helper phage M13KO7
(manufactured by Invitrogen Corp.) to prepare a phage
library expressing scFv.
[Biopanning and g of phage scFv]
The biopanning of phage scFv using a combination of
inant GPC3 immobilized on Dynabeads His-Tag
Isolation & Pulldown magnetic beads (manufactured by
VERITAS Corp.) via 6 × His tag, and the GPC3-expressing
cell line as a bait was performed ing to the method
described in a document such as "J Mol Biol. 1991 Dec 5;
222 (3): 581-97", "J Med Virol. 2007 Jun; 79 (6): 852-62",
"Proc Natl Acad Sci U S A. 2008 May 20; 105 (20): 7287-92",
or "JOURNAL OF GY, Apr. 2004, p. 3325-3332 Vol. 78,
No. 7". In each round (step) of biopanning consisting of
types of series (A to E series) (see Figure 1), an
aliquot of polyclonal phage antibodies was sampled. In
order to confirm the binding specificity of scFv, antigenimmobilized
ELISA was performed according to the method
described above in the section [Serum antibody titer of
antiserum using ELISA] (method using the culture
supernatant of E. coli containing a phage instead of the
serum), while cell-based ELISA was med according to
the method described below in the section ning of
scFv by ased ELISA]. Each step of this biopanning
was devised so as not to select a scFv phage binding to
the same portion as the C-terminal epitope of GPC3
recognized by existing antibodies, by binding in advance
the existing anti-GPC3 antibodies GC33 (manufactured by
Chugai Pharmaceutical Co., Ltd.) and GC199 (manufactured
by Chugai Pharmaceutical Co., Ltd.) to the bait.
Specifically, this competition method enables selective
panning of a novel antibody recognizing a GPC3 epitope
different from that for the existing anti-GPC3 antibodies.
E. coli DH12S was transformed with the phages enriched by
biopanning and inoculated to an LB agarose agar medium to
separate single es. The E. coli was further
cultured in a small-scale LB liquid medium, followed by
the extraction and purification of plasmids. The purified
plasmids were subjected to DNA sequencing to determine the
tide sequences of scFv H chain and L chain variable
[Screening of scFv by FCM]
100 µL of the culture supernatant in which scFv
phages were secreted was added to a GPC3-expressing cell
line (5 × 105 cells per sample) and mixed therewith, and
the mixture was then incubated for 30 minutes on ice. A
FACS buffer (1% BSA/PBS solution) was added thereto, and
the mixture was centrifuged and . Then, 1 µg/mL
anti-mouse antibody-Alexa 488 actured by Thermo
Fisher Scientific Inc.) was added thereto as a secondary
antibody, and the mixture was incubated for 30 minutes on
ice. Then, the fluorescent staining of the cells was
ed using a flow ter (FACSCanto, manufactured
by BD Biosciences).
[Screening of scFv by cell-based ELISA]
After removal of a DMEM culture solution from a 96-
well microplate in which 2 × 105 xpressing cells
were attached per well, 2% S solution was added for
the purpose of preventing the nonspecific binding of scFv
to the cells or the plate, and the plate was incubated for
minutes on ice. Then, 100 µL of the culture
supernatant of E. coli in which scFv phages were secreted
was added to each well, and the plate was incubated for 45
minutes on ice. Then, 5 µg/mL rabbit anti-cp3 dy
actured by l & Biological Laboratories Co.,
Ltd.) against cp3 fused on the C-terminal side of scFv was
added at 100 µL per well, and the plate was further
ted for 45 minutes on ice. A HRP-labeled antirabbit
IgG antibody (manufactured by Medical & Biological
Laboratories Co., Ltd.) diluted old was added at 100
µL per well as a tertiary antibody for anti-cp3 antibody
detection, and the plate was incubated for 45 minutes on
ice. Then, o-phenylenediamine (OPD) and hydrogen de
were added as substrates of HRP for color development.
Quantification was performed using a numeric value
obtained by subtracting absorbance at 620 nm as a
background from absorbance at 492 nm. When cell-based
ELISA was carried out using an antibody already converted
to an IgG type antibody, not scFv, a HRP-labeled antimouse
IgG antibody (manufactured by Medical & Biological
Laboratories Co., Ltd.) diluted 2000-fold was used as a
secondary antibody for the detection of the IgG type
antibody instead of the anti-cp3 antibody and the HRP-
labeled anti-rabbit IgG antibody among the conditions
described above.
[Determination of variable region gene sequences of scFv]
The variable region gene sequences of phage scFv
binding to membrane-bound GPC3 were decoded in a sequencer
(CEQ2000XL, manufactured by Beckman Coulter, Inc.) using a
T7 primer (primer consisting of the nucleotide ce
ented by SEQ ID NO: 176), which is a universal
primer, and a cp3R primer (primer consisting of the
nucleotide sequence represented by SEQ ID NO: 177) as a
forward primer for H chain V region (VH) decoding and a
reverse primer for L chain V region (VL) decoding,
tively.
[Preparation of cell line for use in antibody epitope
In order to identify an epitope for the cloned scFv,
the mammalian display method was applied. A gene
consisting of human GPC3 exons 1 to 7 and encoding a GPC3
N-terminal fragment (polypeptide consisting of the amino
acid sequence represented by SEQ ID NO: 155), and a gene
consisting of human GPC3 exons 8 and 9 and encoding a GPC3
C-terminal fragment (polypeptide consisting of the amino
acid sequence represented by SEQ ID NO: 156) were
amplified by PCR and each ed to the multicloning
site (MSC) of a ay expression vector (manufactured
by Thermo Fisher Scientific Inc.). The pDisplay
expression vector is an expression vector capable of
fusing a transmembrane domain of platelet-derived growth
factor receptor (PDGFR) to the C terminus of the target
protein and displaying the fusion product on the cell
surface of arbitrary mammalian cells. Also, the pDisplay
expression vector is constituted so as to add a HA tag to
the N terminus of the target protein and to add a myc tag
to the C terminus of the PDGFR. The pDisplay expression
vector for expressing the GPC3 N-terminal fragment or the
GPC3 C-terminal fragment was gene-transferred to a SK-Hep-
1 cell line or a 293T cell line, and a cell line
expressing the GPC3 N-terminal nt or the GPC3 C-
terminal fragment on the cell surface (GPC3 N-terminal
fragment-expressing cell line and GPC3 C-terminal
nt-expressing cell line) was isolated and used in
the epitope mapping of scFv.
[Antibody epitope mapping by FCM]
The GPC3 inal fragment-expressing cell line,
the GPC3 C-terminal nt-expressing cell line, and the
GPC3-expressing cell line (5 × 105 cells each per sample)
were each mixed with 100 µL of the culture supernatant in
which scFv phages were secreted, and the mixture was
incubated for 30 minutes on ice. A FACS buffer (1%
BSA/PBS solution) was added thereto, and the mixture was
centrifuged and washed. Then, 1 µg/mL anti-mouse
antibody-Alexa 488 (manufactured by Thermo Fisher
Scientific Inc.) was added thereto as a secondary dy,
and the mixture was incubated for 30 minutes on ice. Then,
the fluorescent staining of the cells was ed using a
flow ter (FACSCanto, manufactured by BD Biosciences).
[Construction of recombinant IgG expression vector]
In order to convert scFv to IgG, an sion
vector of Mammalian PowerExpress system (manufactured by
Toyobo Co., Ltd.) was used. A nucleotide sequence
encoding a fusion protein of the H chain variable region
of scFv and a mouse IgG2a H chain-derived constant region
was inserted to MSC of a pEH1.1 vector (pEH1.1-H). Also,
a tide sequence encoding a fusion protein of the L
chain variable region of scFv and a mouse IgG2a L chainderived
constant region was inserted to MSC of a pELX2.2
vector (pEH2.2-L). Then, a polynucleotide nt from
EF1α er to the L chain gene was excised from pEH2.2-
L with ction enzymes (BglII and SalI) and ligated
with pEH1.1-H treated with restriction enzymes (BglII and
SalI) to construct a vector for coexpressing the antibody
H chain and L chain.
[Expression of recombinant IgG]
32.6 µg of the dy H chain and L chain
coexpression vector prepared by the method described above
in [Construction of recombinant IgG expression ] was
diluted with 1.6 mL of opti-MEM (manufactured by
Gibco/Thermo Fisher Scientific Inc.) and mixed with 65 µL
of Transficient Transfection Reagent (manufactured by
Medical & Biological Laboratories Co., Ltd.) diluted with
1.6 mL of opti-MEM, and the mixture was incubated at room
temperature for 10 minutes. Then, the mixture was mixed
with CHO-K1 cells (1 × 107 cells) suspended in 10 mL of a
DMEM culture solution, followed by culture. 4 hours later,
a serum-free medium (Free Style expression CHO media
[manufactured by Gibco/Thermo Fisher Scientific Inc.]) was
added thereto, and the mixture was further cultured for 4
to 6 days to recover a culture supernatant containing a
recombinant antibody.
[Affinity purification of antibody]
An empty column (manufactured by Bio-Rad
Laboratories, Inc.) was packed with Protein G Sepharose 4
Fast Flow actured by GE Healthcare Japan Corp.) or
Bipo Resin Protein L (manufactured by Protein Express) at
1 mL bed volume. Then, the column resin was washed with
PBS in an amount of 10 times the bed volume. The culture
supernatant filtered through a 0.22 micron filter was
added to the column so that the antibody was entrapped to
protein G or protein L within the column. Then, the
column was washed with PBS in an amount of 10 times the
bed volume to wash off nonspecifically adsorbed
inants. The antibody was eluted using a 100 mM
glycine-HCl (pH 2.7) solution, and pH of the eluate was
neutralized with 1 M Tris-HCl (pH 8.5). Absorbance at 280
nm was measured with an absorbance meter nanoDrop
actured by Thermo Fisher ific Inc.), and the
antibody concentration was calculated. sion vectors
were also designed and prepared by the same method as
above as to the GC33 antibody and the GC199 antibody used
as competitive antibodies.
1-2 Results
[Antiserum evaluation of immunized mouse]
Blood was collected from SKG/Jcl mice immunized four
times with inant GPC3, and the production of an
antibody against GPC3 in serum was confirmed. As a result,
an antibody having binding activity against GPC3 was
detected by experiments of ELIS on recombinant GPC3 and
FCM on GPC3-expressing cells. Two mice having a
ularly high antibody titer (individual Nos. 1413 #2
and 1413 #3) among the mice were used as sources for the
preparation of an dy library.
[Construction of phage library]
The number of members in a scFv library estimated by
calculation from transformation efficiency was 5.8 × 107
for mouse 1413 #2 and 4.3 × 108 for mouse 1413 #3. The
immunoglobulin library prepared in this Example was a
library prepared from the mice found to produce antibodies
in response to the target antigen by immunization with the
antigen GPC3. ore, a e of this library is the
high possibility of containing the target antibody gene
even if the library size is small. Another advantageous
feature f is that the library contains an antibody
that forms a correct conformation in vivo, as compared
with a random synthetic antibody y.
[Classification of clone by sequence analysis of
monoclonal scFv]
The DNA sequence analysis of picked up onal
scFv was conducted to perform clone classification
excluding overlap. As a result, candidate clones were
identified as 7 types from D series of the mouse 1413 #2
library, 5 types from E series thereof, 3 types from D
series of the mouse 1413 #3 library, and 9 types from E
series thereof. The tide sequences of heavy chain
and light chain le regions of these candidate clones
were analyzed to exclude overlapping identical clones. As
a result, a total of 18 types of scFv clones, i.e., 9
types of scFv clones derived from the mouse 1413 #2
library, and 9 types of scFv clones derived from the mouse
1413 #3 library, were identified.
[Epitope mapping is of anti-GPC3 scFv clone]
18 types of scFv clones fied according to the
method described above in the section [Classification of
clone by sequence analysis of monoclonal scFv] were used
to analyze binding to each GPC3 by FCM using 3 types of
cell lines (GPC3 N-terminal fragment-expressing cell line,
GPC3 C-terminal fragment-expressing cell line, and GPC3-
expressing cell line). As a , among the 18 types of
scFv clones, 14 types (TF1413-02d028, 02d030, 02d039,
02e004, 02e014, 02e030, 02e040, 03e001, 03e004, 03e005,
03e015, 03e019, 03e027, and 03e034) bound to full-length
GPC3 and the GPC3 N-terminal fragment (polypeptide
ting of the amino acid sequence represented by SEQ
ID NO: 155), but did not bind to the GPC3 C-terminal
fragment (polypeptide consisting of the amino acid
sequence represented by SEQ ID NO: 156) (see Figure 2).
On the other hand, the existing anti-GPC3 antibodies GC33
(manufactured by Chugai Pharmaceutical Co., Ltd.) and
GC199 (manufactured by Chugai Pharmaceutical Co., Ltd.)
bound to full-length GPC3 and the GPC3 C-terminal nt,
but did not bind to the GPC3 N-terminal fragment.
From these results, the 14 types of novel scFv
clones described above recognizing a GPC3 N-terminal
epitope different from a GPC3 C-terminal epitope for the
existing anti-GPC3 antibodies (GC33 and GC199) were
identified.
Among the 14 types of scFv clones thus fied,
top 11 scFv clones (TF1413-02d028, 02d039, , 02e014,
02e030, 02e040, 03e001, 03e004, , 03e015, and
03e034) having particularly high binding strength were
selected. Table 1 shows the correspondence of SEQ ID NOs
to the H chain and L chain V regions of these 11 types of
scFv clones. Table 2 shows the correspondence of SEQ ID
NOs to the H chain CDR1 to CDR3 of these 11 types of scFv
clones. Table 3 shows the correspondence of SEQ ID NOs to
the L chain CDR1 to CDR3 of these 11 types of scFv clones.
[Table 1]
scFv clone name and V region SEQ ID NO
TF1413-02d028 H chain V region 7
TF1413-02d039 H chain V region 17
TF1413-02e004 H chain V region 27
TF1413-02e014 H chain V region 37
TF1413-02e030 H chain V region 47
TF1413-02e040 H chain V region 57
TF1413-03e001 H chain V region 67
TF1413-03e004 H chain V region 77
TF1413-03e005 H chain V region 87
TF1413-03e015 H chain V region 97
TF1413-03e034 H chain V region 107
TF1413-02d028 L chain V region 8
TF1413-02d039 L chain V region 18
TF1413-02e004 L chain V region 28
TF1413-02e014 L chain V region 38
TF1413-02e030 L chain V region 48
TF1413-02e040 L chain V region 58
TF1413-03e001 L chain V region 68
TF1413-03e004 L chain V region 78
-03e005 L chain V region 88
-03e015 L chain V region 98
TF1413-03e034 L chain V region 108
[Table 2]
Clone name and CDR SEQ ID NO
-02d028 H chain CDR1 1
H chain CDR2 2
H chain CDR3 3
TF1413-02d039 H chain CDR1 11
H chain CDR2 12
H chain CDR3 13
TF1413-02e004 H chain CDR1 21
H chain CDR2 22
H chain CDR3 23
TF1413-02e014 H chain CDR1 31
H chain CDR2 32
H chain CDR3 33
TF1413-02e030 H chain CDR1 41
H chain CDR2 42
H chain CDR3 43
TF1413-02e040 H chain CDR1 51
H chain CDR2 52
H chain CDR3 53
TF1413-03e001 H chain CDR1 61
H chain CDR2 62
H chain CDR3 63
TF1413-03e004 H chain CDR1 71
H chain CDR2 72
H chain CDR3 73
TF1413-03e005 H chain CDR1 81
H chain CDR2 82
H chain CDR3 83
TF1413-03e015 H chain CDR1 91
H chain CDR2 92
H chain CDR3 93
TF1413-03e034 H chain CDR1 101
H chain CDR2 102
H chain CDR3 103
[Table 3]
Clone name and CDR SEQ ID NO
TF1413-02d028 L chain CDR1 4
L chain CDR2 5
L chain CDR3 6
TF1413-02d039 L chain CDR1 14
L chain CDR2 15
L chain CDR3 16
TF1413-02e004 L chain CDR1 24
L chain CDR2 25
L chain CDR3 26
TF1413-02e014 L chain CDR1 34
L chain CDR2 35
L chain CDR3 36
TF1413-02e030 L chain CDR1 44
L chain CDR2 45
L chain CDR3 46
TF1413-02e040 L chain CDR1 54
L chain CDR2 55
L chain CDR3 56
TF1413-03e001 L chain CDR1 64
L chain CDR2 65
L chain CDR3 66
TF1413-03e004 L chain CDR1 74
L chain CDR2 75
L chain CDR3 76
TF1413-03e005 L chain CDR1 84
L chain CDR2 85
L chain CDR3 86
-03e015 L chain CDR1 94
L chain CDR2 95
L chain CDR3 96
TF1413-03e034 L chain CDR1 104
L chain CDR2 105
L chain CDR3 106
[Conversion of anti-GPC3 scFv antibody to IgG and its
ability to bind]
The H chain and L chain le regions of the 11
types of scFv clones selected as described above were
bound to mouse IgG constant regions, and full-length
recombinant antibodies were expressed using a vector for
recombinant IgG expression and affinity-purified. The
ability of these IgG antibodies to bind to the GPC3 N-
terminal fragment was analyzed using the GPC3 N-terminal
fragment-expressing cell line. As a result, 9 types of
IgG clones (TF1413-02d028, 02d039, 02e004, 02e014, 02e030,
02e040, 03e004, 03e005, and 03e034) maintained binding
activity against the GPC3 N-terminal fragment, whereas the
ing two types of IgG clones (TF1413-03e001 and
03e015) lacked g activity against the GPC3 N-
al fragment (see Figure 3). The 9 types of IgG
clones described above did not bind to the GPC3 C-terminal
fragment (see Figure 3).
These results indicate that among the 11 types of
scFv clones, 9 types (TF1413-02d028, , 02e004,
02e014, 02e030, , 03e004, 03e005, and 03e034) are
convertible to IgG type. Table 4 shows the correspondence
of SEQ ID NOs to the H chains and the L chains of the 11
types of IgG clones.
[Table 4]
IgG clone name and region SEQ ID NO
TF1413-02d028 H chain 9
TF1413-02d039 H chain 19
TF1413-02e004 H chain 29
TF1413-02e014 H chain 39
TF1413-02e030 H chain 49
TF1413-02e040 H chain 59
TF1413-03e001 H chain 69
TF1413-03e004 H chain 79
TF1413-03e005 H chain 89
TF1413-03e015 H chain 99
TF1413-03e034 H chain 109
TF1413-02d028 L chain 10
TF1413-02d039 L chain 20
TF1413-02e004 L chain 30
TF1413-02e014 L chain 40
TF1413-02e030 L chain 50
TF1413-02e040 L chain 60
-03e001 L chain 70
TF1413-03e004 L chain 80
TF1413-03e005 L chain 90
TF1413-03e015 L chain 100
TF1413-03e034 L chain 110
Example 2
2. Binding ty of novel anti-GPC3 antibody t
GPC3 treated with EDTA (ethylenediaminetetraacetic acid),
trypsin or enase
[Preparation of cell treated with EDTA or trypsin]
A SK-Hep-1 cell line forced to express GPC3 was
cultured in two T-75 flasks. The culture supernatant of
each flask was aspirated, and the flask was washed with 3
mL of PBS. Then, 3 mL of 0.02% EDTA/PBS solution
(hereinafter, simply referred to as "EDTA") or 0.05%
trypsin solution (hereinafter, simply referred to as
"trypsin") was added to each flask. Each flask was
incubated at 37°C for 5 minutes (EDTA) or 2 minutes and 30
seconds (trypsin) to dissociate the cells from the flask.
Then, 7 mL of a DMEM culture solution was added to each
flask. After pipetting, the cell suspension was recovered
into each 50 mL conical tube. Each flask was further
washed with 10 mL of a DMEM culture on. Then, the
red washes were also recovered into the 50 mL
conical tube containing each cell sion, followed by
centrifugation (1,500 rpm, 4°C, 4 min). After tion
of the supernatant from each conical tube, 10 mL of a DMEM
culture solution was added to the pellet, and the number
of cells dissociated with EDTA or trypsin was counted.
The cells treated with EDTA or trypsin were adjusted
to 2 × 105 cells/tube and subjected to FACS (EC800)
analysis. The FACS analysis employed 3 types of
antibodies escently APC-labeled anti-mouse IgG
antibody [5 µg/tube; manufactured by end, Inc.],
GC33 antibody [1.0 µg/tube; manufactured by Medical &
Biological Laboratories Co., Ltd. Life Science], and scFv
clone [TF1413-02d028] antibody described above [1.0
[Preparation of cell treated with collagenase]
1 × 106 cells dissociated with EDTA as described
above were placed in a 50 mL conical tube and centrifuged
(1,500 rpm, 4°C, 4 min), and the supernatant was aspirated
to prepare a cell mass (pellet). 5 mL of a collagenase P
solution was added to the pellet, and the mixture was
incubated at 37°C for 30 minutes to prepare a cell
suspension. Then, the cell suspension was passed through
a 100 µm cell strainer while washed with 30 mL of a DMEM
culture solution. The cell suspension was passed again
through a 100 µm cell er and centrifuged (300 g, 4°C,
min), and the atant was aspirated. The pellet
was washed by the addition of 20 mL of PBS and then
centrifuged (300 g, 4°C, 5 min), and the supernatant was
aspirated. The cells were suspended by the addition of 5
mL of a DMEM culture solution. Then, the number of cells
was counted, and 2 × 105 tube were analyzed by FACS
(EC800). The FACS analysis employed 3 types of antibodies
(fluorescently APC-labeled ouse IgG antibody [5
e; ctured by BioLegend, Inc.], GC33 antibody
[1.0 µg/tube; manufactured by Medical & Biological
Laboratories Co., Ltd. Life Science], and scFv clone
[TF1413-02d028] antibody described above [1.0 µg/tube]),
as in the cells treated with EDTA or trypsin. The results
are shown in Figure 4. In Figure 4, the right peak on the
abscissa represents that the GC33 antibody or the scFv
clone [TF1413-02d028] antibody bound to the GPC3 protein.
[Results]
As shown in Figure 4, the binding activity of the
antibody of the present invention (TF1413-02d028) against
the GPC3 protein treated with trypsin or collagenase was
markedly decreased. These results indicate that the
dy of the present invention specifically recognizes
the conformation of the GPC3 protein, suggesting that the
antibody of the present invention has high specificity in
vivo.
3. Development of GPC3 CAR-T cell using novel anti-GPC3
antibody
[Summary]
GPC3 is a cell surface molecule, the expression of
which is not observed in human adult tissues except for
placenta, but is observed in tissues of various cancers
such as hepatocellular carcinoma, ma, n clear
cell adenocarcinoma, and lung squamous cell carcinoma.
Thus, GPC3 is capable of g as a target molecule in
CAR-T cell therapy exploiting a chimeric antigen receptor
(CAR). Accordingly, GPC3 CAR-T cells were prepared using
11 types of scFv clones prepared in e 1 and analyzed
for cancer xic activity and the ability to produce
eron γ (IFN-γ).
[Preparation of GPC3 CAR vector]
scFv having a VH-linker-VL sequence was designed as
to 11 types of scFv clones (TF1413-02d028, 02d039, 02e004,
02e014, 02e030, 02e040, 03e001, 03e004, 03e005, 03e015,
and 03e034) prepared in Example 1, on the basis of their
respective amino acid sequences of VH and VL (see Table 5).
The linker used consisted of 15 amino acid residues with 3
repeats of a polypeptide "GGGGS". A human globulin
H chain-derived signal sequence consisting of the amino
acid sequence represented by SEQ ID NO: 188 was added to
the N terminus of VH.
[Table 5-1]
SEQ ID NO: 165: -derived scFv
SEQ ID NO: 166: -derived scFv
SEQ ID NO: 167: -derived scFv
SEQ ID NO: 168: -derived scFv
SEQ ID NO: 169: -derived scFv
SEQ ID NO: 170: -derived scFv
[Table 5-2]
SEQ ID NO: 171: ed scFv
SEQ ID NO: 172: -derived scFv
SEQ ID NO: 173: -derived scFv
SEQ ID NO: 174: -derived scFv
SEQ ID NO: 175: -derived scFv
In the tables, the linker is boxed in a double line,
VH is underlined with a single line, and VL is underlined
with a double line.
A nucleotide sequence encoding each anti-GPC3 scFv
of Table 5 was synthesized by optimization for human
codons and inserted to a CAR expression vector. The CAR
gene used had a gene encoding a fusion peptide de
consisting of the amino acid sequence represented by SEQ
ID NO: 185) consisting of a human CD8-derived
transmembrane region and a human CD28/4-1BB/CD3 zeta-
derived immunocompetent cell activation signal
transduction region, a 2A self-cleaving sequence, human
IL-7 gene, a 2A self-cleaving sequence, human CCL19 gene,
a 2A self-cleaving ce, and HSV-TK gene, downstream
of the scFv gene, and the whole was incorporated into a
MSGV1 retrovirus vector (see International Publication No.
[Preparation of GPC3 CAR-T cell]
The GPC3 CAR vectors derived from the 11 types of
scFv clones described above were each transiently
introduced to GP2 packaging cells to prepare retrovirus
vectors. T cells were ed with these vectors for
gene transfer to induce GPC3 CAR-T cells. The ratio of
GPC3 CAR-expressing cells to the gene-transferred T cells
varied from 5.3 to 39.2%. Accordingly, the following
function assay was carried out using GPC3 CAR-T cells
derived from 5 types of scFv clones 3-02d028,
TF1413-02d039, TF1413-02e014, TF1413-02e030, and TF1413-
03e005) that exhibited 25% or more of the ratio.
ing activity of GPC3 CAR-T cell against GPC3-
expressing cell line]
In order to study the damaging activity of the GPC3
CAR-T cells against cancer cells, ure assay was
carried out using the GPC3 CAR-T cells and a GPC3-
expressing cell line, i.e., a hepatocellular carcinomaderived
cell line Sk-HEP-1 caused to express GPC3 (Sk-HEP-
1 GPC3 cell line), or a cell line expressing no GPC3 (Sk-
HEP-1 mock cell line). The GPC3 CAR-T cells were mixed
with the target cancer cells (Sk-HEP-1 GPC3 cell line or
Sk-HEP-1 mock cell line) at a ratio of 1:1 (1 × 105
cells/well) and cultured in a 24-well plate. 48 hours
later, the cells were red, stained with an anti-CD45
antibody, and analyzed by FCM with CD45-positive cells as
GPC3 CAR-T cells and CD45-negative cells as residual
cancer cells [Sk-HEP-1 GPC3 cells]. As a result, all the
GPC3 CAR-T cells derived from the 5 types of scFv clones
described above almost completely damaged the Sk-HEP-1
GPC3 cells, but did not exhibit damaging ty against
the Sk-HEP-1 mock cells (see Figures 5 and 6). In the
case of using cells uninfected with the virus vector (nongene-transferred
cells ["Non infection" in Figures 5 and
6]) as a negative control for the GPC3 CAR-T cells, these
cells exhibited damaging ty r against the Sk-
HEP-1 GPC3 cells nor against the Sk-HEP-1 mock cells.
From these s, the GPC3CAR-T cells derived from
the selected 5 types of anti-GPC3 scFv clones (TF1413-
02d028, TF1413-02d039, TF1413-02e014, TF1413-02e030, and
TF1413-03e005) were shown to specifically exert cytotoxic
activity against cancer cells expressing GPC3.
ty of GPC3 CAR-T cell to produce IFN-γ by
recognizing GPC3-expressing cell]
In addition to the damaging activity against GPC3-
expressing (positive) cancer cells, the ability of the
GPC3 CAR-T cells to e IFN-γ was analyzed. The GPC3
CAR-T cells were mixed with the target cancer cells (Sk-
HEP-1 GPC3 cell line or Sk-HEP-1 mock cell line) at a
ratio of 1:1 (1 × 105 cells/well) and cultured for 48
hours in a 24-well plate, and the concentration of IFN-γ
produced into the culture supernatant was measured by
ELISA. As a result, all the GPC3 CAR-T cells derived from
the 5 types of scFv clones described above ted the
ability to produce IFN-γ in a manner dependent on the
expression of GPC3. Particularly, the GPC3 CAR-T cells
derived from clone -02d028 exhibited the highest
y to e IFN-γ (see Figure 7).
Example 4
4. Preparation of humanized antibody
scFv humanized antibodies were designed on the basis
of two types of scFv clones (TF1413-02d028 and 02d039)
prepared in Example 1 (see Table 6). The linker used
consisted of 15 amino acid residues with 3 repeats of a
polypeptide "GGGGS". A human immunoglobulin H chainderived
signal sequence consisting of the amino acid
sequence represented by SEQ ID NO: 188 was added to the N
terminus of VH.
[Table 6-1]
SEQ ID NO: 178: (TF1413-02d028-derived scFv humanized antibody 1)
SEQ ID NO: 179: 3-02d028-derived scFv humanized antibody 2)
SEQ ID NO: 180: (TF1413-02d028-derived scFv humanized antibody 3)
SEQ ID NO: 181: (TF1413-02d039-derived scFv humanized antibody 1)
SEQ ID NO: 182: (TF1413-02d039-derived scFv humanized antibody 2)
SEQ ID NO: 183: 3-02d039-derived scFv humanized antibody 3)
[Table 6-2]
SEQ ID NO: 184: (TF1413-02d039-derived scFv humanized antibody 4)
In the tables, the linker is boxed in a double line,
VH is underlined with a single line, and VL is underlined
with a double line.
Industrial ability
The present invention contributes to the field of
cancer immunotherapy.
Claims (17)
1. An antibody specifically binding to a human GPC3 (glypican-3)-derived polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 155, wherein the antibody (1-1) comprises a heavy chain complementarity determining region (CDR) 1 ting of the amino acid ce ented by SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3, and a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6; or (2-1) comprises a heavy chain CDR1 ting of the amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 12, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 13, and a light chain CDR1 ting of the amino acid sequence represented by SEQ ID NO: 14, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 15, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 16; or (3-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 23, and a light chain CDR1 consisting of the amino acid ce represented by SEQ ID NO: 24, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 25, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 26; or (4-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 33, and a light chain CDR1 ting of the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 36; or (5-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 consisting of the amino acid ce ented by SEQ ID NO: 42, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 43, and a light chain CDR1 consisting of the amino acid ce represented by SEQ ID NO: 44, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 45, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 46; or (6-1) comprises a heavy chain CDR1 consisting of the amino acid ce represented by SEQ ID NO: 51, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 52, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 53, and a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 54, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 55, and a light chain CDR3 consisting of the amino acid sequence ented by SEQ ID NO: 56; or (7-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 61, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 62, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 63, and a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 65, and a light chain CDR3 ting of the amino acid sequence ented by SEQ ID NO: 66; or (8-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 71, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 72, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 73, and a light chain CDR1 consisting of the amino acid ce represented by SEQ ID NO: 74, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 75, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 76; or (9-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 81, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 82, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 83, and a light chain CDR1 consisting of the amino acid ce represented by SEQ ID NO: 84, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 ting of the amino acid sequence represented by SEQ ID NO: 86; or (10-1) comprises a heavy chain CDR1 consisting of the amino acid sequence ented by SEQ ID NO: 91, a heavy chain CDR2 consisting of the amino acid ce represented by SEQ ID NO: 92, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 93, and a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 94, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 95, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 96; or (11-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 101, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 102, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 103, and a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 104, a light chain CDR2 ting of the amino acid sequence represented by SEQ ID NO: 105, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 106.
2. The antibody according to claim 1, wherein the dy (1-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 7, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher ce identity to the amino acid sequence ented by SEQ ID NO: 8; or (2-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence ented by SEQ ID NO: 17, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 18; or (3-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 27, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 28; or (4-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher ce identity to the amino acid sequence represented by SEQ ID NO: 37, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 38; or (5-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 47, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 48; or (6-2) comprises a heavy chain le region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 57, and a light chain variable region ting of an amino acid sequence having at least 80% or higher ce identity to the amino acid sequence represented by SEQ ID NO: 58; or (7-2) ses a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 67, and a light chain variable region consisting of an amino acid ce having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 68; or (8-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence ty to the amino acid sequence ented by SEQ ID NO: 77, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher ce identity to the amino acid sequence represented by SEQ ID NO: 78; or (9-2) ses a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid ce represented by SEQ ID NO: 87, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 88; or (10-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 97, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 98; or (11-2) comprises a heavy chain variable region consisting of an amino acid ce having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 107, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 108.
3. The antibody according to claim 1 or 2, wherein the antibody is a single chain antibody.
4. The antibody according to claim 3, wherein the single chain antibody (1-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence ented by SEQ ID NO: 165; or (2-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid ce represented by SEQ ID NO: 166; or (3-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 167; or (4-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence ented by SEQ ID NO: 168; or (5-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 169; or (6-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid ce represented by SEQ ID NO: 170; or (7-3) comprises an amino acid sequence having at least 80% or higher ce identity to the amino acid sequence represented by SEQ ID NO: 171; or (8-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 172; or (9-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 173; or (10-3) comprises an amino acid sequence having at least 80% or higher ce identity to the amino acid sequence represented by SEQ ID NO: 174; or (11-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 175.
5. The antibody ing to claim 3, n the single chain antibody (1-3'-1) comprises an amino acid sequence having at least 80% or higher sequence ty to the amino acid sequence represented by SEQ ID NO: 178; or (1-3'-2) comprises an amino acid ce having at least 80% or higher sequence identity to the amino acid sequence ented by SEQ ID NO: 179; or (1-3'-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 180; or (2-3'-1) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 181; or (2-3'-2) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 182; or (2-3'-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 183; or (2-3'-4) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 184.
6. The antibody according to claim 1 or 2, wherein the antibody (1-4) comprises a heavy chain ting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence ented by SEQ ID NO: 9, and a light chain consisting of an amino acid sequence having at least 80% or higher ce identity to the amino acid sequence represented by SEQ ID NO: 10; (2-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid ce represented by SEQ ID NO: 19, and a light chain consisting of an amino acid sequence having at least 80% or higher ce identity to the amino acid sequence represented by SEQ ID NO: 20; (3-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 29, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 30; (4-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 39, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 40; (5-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence ty to the amino acid ce represented by SEQ ID NO: 49, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 50; (6-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 59, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence ty to the amino acid sequence represented by SEQ ID NO: 60; (7-4) comprises a heavy chain ting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 69, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 70; (8-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 79, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 80; (9-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 89, and a light chain consisting of an amino acid ce having at least 80% or higher sequence identity to the amino acid ce represented by SEQ ID NO: 90; (10-4) ses a heavy chain ting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 99, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 100; (11-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 109, and a light chain consisting of an amino acid sequence having at least 80% or higher ce identity to the amino acid sequence represented by SEQ ID NO: 110.
7. A chimeric antigen receptor (CAR) comprising the antibody according to any one of claims 3 to 5, a transmembrane region fused with a carboxyl terminus of the antibody, and an immunocompetent cell activation signal transduction region fused with a carboxyl terminus of the transmembrane region.
8. The CAR according to claim 7, comprising the amino acid sequence represented by any of SEQ ID NOs: 185 to 187.
9. An immunocompetent cell expressing the CAR according to claim 7 or 8.
10. The immunocompetent cell ing to claim 9, further expressing eukin 7 (IL-7) and chemokine ligand 19 (CCL19).
11. An antibody gene encoding an antibody according to any one of claims 1 to 6, or a CAR gene encoding the CAR according to claim 7 or 8.
12. An antibody gene encoding an antibody according to any one of claims 1 to 4 and 6.
13. A vector comprising a promoter, and the antibody gene ing to claim 11 or the CAR gene encoding the CAR ing to claim 11 operably linked downstream of the promoter.
14. A vector comprising a promoter, and the antibody gene according to claim 12 operably linked downstream of the promoter.
15. A host cell in which the vector according to claim 13 or 14 has been introduced.
16. A method for detecting GPC3 (glypican-3), comprising the step of detecting GPC3 using the antibody according to any one of claims 1 to 6.
17. A kit for the detection of GPC3 (glypican-3), comprising the antibody according to any one of claims 1 to 6, or a d form thereof. [
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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JP2017-001732 | 2017-01-10 |
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NZ795720A true NZ795720A (en) | 2022-12-23 |
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