EA043515B1 - ANTI-GPC3-ANTIBODY - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to an antibody that specifically binds to GPC3 (glypican-3) (anti-GPC3 antibody); to a chimeric antigen receptor (also referred to herein as CAR) containing an anti-GPC3 single chain antibody, a transmembrane region fused to the carboxyl (C) terminus of the anti-GPC3 single chain antibody, and an immunocompetent cell signal transduction activation region fused to the C terminus transmembrane region; to an immunocompetent cell expressing CAR; to the anti-GPC3 antibody gene or CAR gene; to a vector containing an anti-GPC3 antibody gene or a CAR gene; to the host cell into which the vector was introduced; to a method for detecting GPC3; and to the GPC3 detection kit.

State of the art

Glypican-3 (GPC3) is an extracellular matrix protein that is expressed in embryonic tissues, particularly the liver or kidney, and is associated with organogenesis. GPC3 expression is not observed in adult tissues except the placenta, but is observed in tissues of various types of malignant tumors, such as hepatocellular carcinoma, melanoma, clear cell ovarian adenocarcinoma, and squamous cell lung cancer. Thus, GPC3 is a protein that is expressed in embryonic tissues, for example, as is the case with proteins such as α-fetoprotein (AFP) and carcinoembryonic antigen (CEA), and therefore they are classified as embryonal carcinoma antigens. In particular, GPC3 can be used as a target molecule in the treatment of malignancy, as a tumor marker and a diagnostic marker, since its distinguishing feature is the absence of protein expression in normal tissue cells and specific expression in malignant cells.

GPC3 is a member of a family of proteoglycans that act as an extracellular matrix in cell adhesion during organogenesis or as a cell growth factor receptor. A GPI (glycosylphosphatidylinositol) anchor is added to the serine at position 560, located at the carboxyl (C) terminus of GPC3. The GPI anchor plays a role in localizing GPC3 to the cell surface through covalent binding to a cell membrane lipid. In addition, serine at position 495 and serine at position 509 of GPC3 are modified with a heparan sulfate chain (HS chain). The HS chain is known to regulate multiple growth-related signal transduction pathways, such as the Wnt, FGF, and BMP signaling pathway. The growth-related signal transduction pathway is known to differ among different types of malignancies. For example, in hepatocellular carcinoma (HCC), cells grow when the Wnt signaling pathway is stimulated. A common feature of the glypican family is an excess of 16 cysteines in the extracellular region, and these cysteine residues are thought to contribute to the formation of a stable conformation by forming multiple intramolecular disulfide bonds. It has been reported that furin convertase GPC3 can cleave at the surface of the cell membrane between arginine (R) at position 358 and serine (S) at position 359 (R358/S359). However, since the amino (N)-terminal subunit of GPC3 is cross-linked through intramolecular disulfide bonds, even when cleaved by furin convertase into two subunits, an N-terminal subunit and a C-terminal subunit, GPC3 can likely retain its full-length structure without dissociation of these subunits . The structure of soluble GPC3 remains questionable. Thus, there are many questions regarding the conformation of GPC3 localized to the cell membrane, including also the structures of GPC3 isoforms.

GPC3 on the cell membrane has a complex structure. Therefore, to produce an anti-GPC3 antibody, it would be desirable for the simplest structural region to be an epitope. An existing example of an anti-GPC3 antibody is the monoclonal antibody 1G12, which is available from BioMosaics, Inc. This antibody is an antibody produced by immunizing Balb/c mice with an antigen (C-terminal 70-residue polypeptide of GPC3) designed to bypass the complex structure or localization of GPC3 to produce hybridomas and to screen hybridomas using this antigen. Antibodies GC33 and GC199, developed by a Japanese pharmaceutical manufacturer, are also monoclonal antibodies created based on the same concept as above, and are antibodies produced by using the C-terminal fragment of GPC3 as an antigen (Patent Document 1).

State of the Art Documents

Patent Document/

Patent Document 1: Japanese Patent No. 4011100.

The essence of the invention

The problem solved by the invention.

The present invention provides: an anti-GPC3 antibody that recognizes an epitope different from that of existing antibodies (eg, GC33 and GC199) and that can specifically bind, even in the form of a single chain antibody, to GPC3 located on the cell membrane; CAR containing anti-GPC3 single chain antibody; an immunocompetent cell expressing CAR; anti-GPC3 antibody gene or CAR gene; a vector containing an anti-GPC3 antibody gene or a CAR gene; a host cell into which said vector is introduced; method for specific detection of GPC3; and set for

- 1 043515 specific detection of GPC3.

Means for solving the problem.

The inventors have conducted a series of careful studies and created the present invention. During these studies, the inventors produced a new anti-GPC3 antibody using a phage display method, which differs from conventional methods for producing monoclonal antibodies, which include the production of hybridomas. Specifically, using B cells derived from mice immunized with full-length human GPC3, an immune antibody gene library was synthesized and the genes were converted into a single-chain antibody (scFv) library, which was then incorporated into phage display and expressed on the phage surface. and then selected by biopanning with recombinant full-length human GPC3 and GPC3-expressing cell lines, and then, if necessary, a competitive GPC3 C-terminal polypeptide as an epitope of existing antibodies, to produce an anti-GPC3 antibody. It was also confirmed that the resulting anti-GPC3 antibody can be used for immunotherapy of malignancies using chimeric antigen receptor (CAR) expressing T cells (also called CAR-T cells). The present invention has been carried out based on these findings.

In particular, the present invention is as follows.

[1] An antibody that specifically binds to human GPC3 (a polypeptide derived from (glycan-3)) consisting of the amino acid sequence SEQ ID NO: 155 (herein also referred to as the antibody of the present invention), wherein the antibody (1-1 ) contains a heavy chain complementarity determining region (CDR) 1 consisting of the amino acid sequence SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence SEQ ID NO: 3 , and a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6; or (2-1) contains a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 12, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 13, and a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 14, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 15, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 16; or (3-1) contains a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 21, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 22, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 23, and a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 25, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 26; or (4-1) contains a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 31, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 32, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 33, and a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 34, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 35, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 36; or (5-1) contains a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 42, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 43, and a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 44, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 45, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 46; or (6-1) contains a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 51, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 52, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 53, and a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 54, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 55, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 56; or (7-1) contains a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 61, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 62, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 63, and a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 64, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 65, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 66; or (8-1) contains a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID

- 2 043515

NO: 71, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 72, and

Heavy chain CDR3 consisting of the amino acid sequence SEQ ID NO: 73, and light chain CDR1 consisting of amino acid sequence SEQ ID NO: 74, light chain CDR2 consisting of the amino acid sequence SEQ ID NO: 75, and light chain CDR3 consisting of amino acid sequence SEQ ID NO: 76; or (9-1) contains a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 81, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 82, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 83, and a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 84, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 85, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 86; or (10-1) contains a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 91, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 92, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 93, and a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 94, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 95, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 96; or (11-1) contains a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 101, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 102, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 103, and a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 104, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 105, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 106.

[2] The antibody of [1], wherein the antibody (1-2) comprises a heavy chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 7, and a variable region a light chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 8; or (2-2) contains a heavy chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 17, and a light chain variable region consisting of an amino acid sequence that is at least 80% or greater sequence identity to the amino acid sequence of SEQ ID NO: 18; or (3-2) contains a heavy chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 27, and a light chain variable region consisting of an amino acid sequence that is at least 80% or greater sequence identity to the amino acid sequence of SEQ ID NO: 28; or (4-2) contains a heavy chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 37, and a light chain variable region consisting of an amino acid sequence that is at least 80% or greater sequence identity to the amino acid sequence of SEQ ID NO: 38; or (5-2) contains a heavy chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 47, and a light chain variable region consisting of an amino acid sequence that is at least 80% or greater sequence identity to the amino acid sequence of SEQ ID NO: 48; or (6-2) contains a heavy chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 57, and a light chain variable region consisting of an amino acid sequence that at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 58; or (7-2) contains a heavy chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 67, and a light chain variable region consisting of an amino acid sequence that is at least 80% or greater sequence identity to the amino acid sequence of SEQ ID NO: 68; or (8-2) contains a heavy chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 77, and a light chain variable region consisting of an amino

- 3 043515 an acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 78; or (9-2) contains a heavy chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 87, and a light chain variable region consisting of an amino acid sequence that is at least 80% or greater sequence identity to the amino acid sequence of SEQ ID NO: 88; or (10-2) contains a heavy chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 97, and a light chain variable region consisting of an amino acid sequence that is at least 80% or greater sequence identity to the amino acid sequence of SEQ ID NO: 98; or (11-2) contains a heavy chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 107, and a light chain variable region consisting of an amino acid sequence that is is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 108.

[3] The antibody of [1] or [2], where the antibody is a single chain antibody.

[4] The antibody of [3], wherein the single chain antibody (1-3) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 165; or (2-3) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 166; or (3-3) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 167; or (4-3) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 168; or (5-3) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 169; or (6-3) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 170; or (7-3) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 171; or (8-3) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 172; or (9-3) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 173; or (10-3) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 174; or (11-3) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 175.

[5] The antibody of [3], wherein the single chain antibody (1-3'-1) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 178; or (1-3'-2) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 179; or (1-3'-3) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 180; or (2-3'-1) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 181; or (2-3'-2) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 182; or (2-3'-3) contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 183; or (2-3'-4) contains an amino acid sequence that is at least 80% or greater identical in sequence to the amino acid sequence of SEQ ID NO: 184.

[6] The antibody of [1] or [2], wherein the antibody (1-4) contains a heavy chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 9, and a light chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 10; or

- 4 043515 (2-4) contains a heavy chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 19, and a light chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 20; or (3-4) contains a heavy chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 29, and a light chain consisting of an amino acid sequence that is at least 8 0% or greater sequence identity to the amino acid sequence of SEQ ID NO: 30; or (4-4) contains a heavy chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 39, and a light chain consisting of an amino acid sequence that is at least 80% identical in sequence to the amino acid sequence of SEQ ID NO: 39 % or more identical in sequence to the amino acid sequence of SEQ ID NO: 40; or (5-4) contains a heavy chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 49, and a light chain consisting of an amino acid sequence that is at least 80% identical in sequence to the amino acid sequence of SEQ ID NO: 49 % or more identical in sequence to the amino acid sequence of SEQ ID NO: 50; or (6-4) contains a heavy chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 59, and a light chain consisting of an amino acid sequence that is at least 80% identical % or more identical in sequence to the amino acid sequence of SEQ ID NO: 60; or (7-4) contains a heavy chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 69, and a light chain consisting of an amino acid sequence that is at least 80% identical in sequence to the amino acid sequence of SEQ ID NO: 69 % or more identical in sequence to the amino acid sequence of SEQ ID NO: 70; or (8-4) contains a heavy chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 79, and a light chain consisting of an amino acid sequence that is at least 80% identical in sequence to the amino acid sequence of SEQ ID NO: 79 % or more identical in sequence to the amino acid sequence of SEQ ID NO: 80; or (9-4) contains a heavy chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 89, and a light chain consisting of an amino acid sequence that is at least 80% identical in sequence to the amino acid sequence of SEQ ID NO: 89 % or more identical in sequence to the amino acid sequence of SEQ ID NO: 90; or (10-4) contains a heavy chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 99, and a light chain consisting of an amino acid sequence that is at least 80% identical % or more identical in sequence to the amino acid sequence of SEQ ID NO: 100; or (11-4) contains a heavy chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 109, and a light chain consisting of an amino acid sequence that is at least 80% identical % or more identical in sequence to the amino acid sequence of SEQ ID NO: 110.

[7] A CAR comprising an antibody according to any one of [3]-[5] (herein also referred to as a single chain antibody of the present invention), a transmembrane region fused to the carboxyl terminus of the single chain antibody of the present invention, and a signal transduction activation region of an immunocompetent cells fused to the carboxyl terminus of the transmembrane region (also referred to herein as a CAR of the present invention).

[8] CAR according to [7], containing any amino acid sequence SEQ ID NO: 185-187.

[9] An immunocompetent cell expressing the CAR of [7] or [8] (herein also referred to as an immunocompetent cell of the present invention).

[10] An immunocompetent cell according to [9], also expressing interleukin 7 (IL-7) and chemokine ligand 19 (CCL19).

[11] An antibody gene encoding an antibody according to any one of [1] to [6] (herein also referred to as an antibody gene of the present invention), or a CAR gene encoding a CAR according to [7] or [8] (in

- 5 043515 is also referred to herein as the CAR gene of the present invention).

[12] An antibody gene encoding an antibody according to any one of [1] to [4] and [6].

[13] A vector containing a promoter and an antibody gene according to [11] or a CAR gene encoding a CAR according to [11] operably linked to a downstream promoter (also referred to herein as a vector of the present invention).

[14] A vector containing a promoter and an antibody gene according to [12], functionally linked to a promoter located downstream in the signaling pathway.

[15] A host cell into which the vector of [13] or [14] has been introduced (also referred to herein as the host cell of the present invention).

[16] A method for detecting GPC3 (glypican-3), including the step of detecting GPC3 using an antibody according to any one of [1] to [6] (herein also referred to as the detection method of the present invention).

[17] A GPC3 (glypican-3) detection kit containing an antibody according to any one of [1]-[6], or a labeled form thereof (also referred to herein as the detection kit of the present invention).

Examples of other embodiments of the present invention include an antibody of the present invention for use in detecting GPC3 and a method of producing an antibody of the present invention, comprising the steps of: immunizing non-human animals (eg, mice and rats) with full-length human GPC3 consisting of the amino acid sequence of SEQ ID NO: 157; synthesizing cDNA by reverse transcription reaction from total RNA of B cells obtained from immunized animals other than humans, and amplifying antibody genes to produce an antibody gene library; and constructing a scFv phage library from the antibody gene library, and infecting E. coli with the library so that the cells express the scFv, followed by biopanning using full-length human GPC3 and a GPC3-expressing lineage, and, if necessary, additionally, a competitive C-terminal polypeptide GPC3 (human GPC3 polypeptide consisting of the amino acid sequence SEQ ID NO: 156).

The invention effect.

The antibody of the present invention is an antibody that specifically binds to GPC3 localized on the cell membrane, not only in the form of IgA, but also in the form of scFv. CAR-T cells using the antibody of the present invention as a scFc in CAR have excellent cytotoxic activity and IFN-γ production ability. Therefore, the antibody of the present invention can be used in the immunotherapy of malignancies.

Brief description of the figures

In fig. Figure 1 shows a diagram showing each round (stage) of biopanning, consisting of 5 types of series (A-E series). Series A involves performing 3 rounds of biopanning with recombinant GPC3 immobilized on magnetic beads as a seed, and conducting biopanning in the 4th and 5th rounds with a GPC3-expressing cell line as a seed (the 5th round is carried out only for 1413 # 3). In rounds 1–4, existing anti-GPC3 antibodies (GC33 and GC199) were added as competition antibodies. Series B involves biopanning with GPC3-expressing cells as a primer in the presence of competitive antibodies after the 2nd round of series A. Series E involves biopanning with recombinant GPC3 immobilized on magnetic beads as a seed in the absence of a competitive antibody after the 3rd round of series A In series C, in the absence of competitive antibodies, 4 rounds of biopanning were performed: 2 rounds with a GPC3-expressing cell line as a seed and 2 rounds with recombinant GPC3 immobilized on magnetic beads as a seed. Series D involves the same biopanning as in series A in the absence of competitive antibodies.

In fig. Figure 2 is a chart showing flow cytometry (FCM) results using 18 types of anti-GPC3 scFv clones (TF1413-02d023, 02d028, 02d030, 02d039, 02e003, 02e004, 02e014, 02e030, 02e040, 03e001 , 03e004, 03e005, 03e015, 03e016 , 03e019, 03e027, 03e034 and 03e045) and existing anti-GPC3 antibodies (GC33 and GC199), and 3 types of cell lines (a cell line expressing the N-terminal fragment of GPC3, a cell line expressing the C-terminal fragment of GPC3, and cell line expressing [full-length] GPC3). Numerical values in the graph indicate relative values, whereas the fluorescence intensity of a non-GPC3 expressing cell line (SK-Hep-1 cell line) was determined to be 1 in FCM.

In fig. Figure 3 is a chart showing flow cytometry (FCM) results using 11 scFv clone types (TF1413-02d028, 02d039, 02e004, 02e014, 02e030, 02e040, 03e001, 03e004, 03e005, 03e015 and 03e0 34) and existing anti-GPC3 antibodies (GC33 and GC199), and 3 types of cell lines (a cell line expressing the N-terminal fragment of GPC3, a cell line expressing the C-terminal fragment of GPC3, and a cell line expressing [full-length] GPC3).

In fig. Figure 4 is a graph showing the results of fluorescence-activated FASC cell sorting using GPC3-expressing cell lines treated

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3 methods (EDTA, trypsin and EDTA+collagenase), 3 antibody combinations (anti-mouse IgG antibody labeled with APC [herein also referred to as APC anti-mouse IgG antibody] and a combination of APC anti-mouse IgG antibody and scFv clone antibody

[TF1413-02d028]).

In fig. 5 is a graph showing the results of the analysis of GPC3 CAR-T cells (T cells expressing CAR from scFv that recognizes GPC3) derived from 5 types of scFv clones (TF1413-02d028, TF1413-02d039, TF1413-02e014, TF1413-02e030 and TF1413- 03e005) for cytotoxic activity against the GPC3 cell line Sk-HEP-1. In each graph, the right peak depicts CD45-positive cells (GPC3 CAR-T cells) and the left peak depicts CD45-negative cells (remaining malignant cells [GPC3 Sk-HEP-1 cells]). The ordinate of each graph corresponds to the number of cells. The numerical value in each graph shows the ratio (%) of the number of CD45 positive cells to the total number of cells (CD45 positive cells and CD45 negative cells). T cells not expressing GPC3 CAR (uninfected in the diagram) were used as a control.

In fig. 6 is a graph showing the ratio of CD45 negative cells in FIG. 5 (Fig. 6A) and the number of CD45-negative cells (Fig. 6B). In a pair of histograms: the left histogram shows mock (Sk-HEP-1 mock cell line) and the right histogram shows GPC3 (GPC3 Sk-HEP-1 cell line).

In fig. 7 is a graph showing the results of analyzing GPC3 CAR-T cells derived from 5 types of scFv clones (TF1413-02d028, TF1413-02d039, TF1413-02e014, TF1413-02e030 and TF1413-03e005) for the ability to produce IFN-γ against the cell line GPC3 Sk-HEP-1. T cells not expressing GPC3 CAR (uninfected in the diagram) were used as a control.

Embodiments of the Invention

The antibody of the present invention is an antibody containing CDR1 to CDR3 of a heavy (H) chain and a light (L) chain as described above in any of (1-1) to (11-1), and specifically binds as an epitope according to at least a portion (typically in the range of 3-30 amino acid residues, preferably 4-20 amino acid residues, more preferably 5-15 amino acid residues) of a human GPC3 polypeptide consisting of the amino acid sequence SEQ ID NO: 155 (amino [N] terminal a polypeptide consisting of amino acid residues 32-471 [exons 1-7] of full-length human GPC3, consisting of the amino acid sequence SEQ ID NO: 157). This antibody specifically binds not only in the form of immunoglobulin A, but also in the form of scFv to GPC3 located on the cell membrane, and, as a rule, contains an H chain variable region containing CDR1-CDR3 of the H chains, as described above in any of (1-1)-(11-1), and an L chain variable region containing L chain CDR1-CDR3, as described above in any of (1-1)-(11-1). In this context, the phrase specifically binds means that the antibody recognizes and binds to the polypeptide of SEQ ID NO: 155 through a highly specific antigen-antibody recognition mechanism. Thus, the antibody of the present invention does not specifically bind to the human GPC3 polypeptide consisting of the amino acid sequence SEQ ID NO: 156 (carboxyl [C] terminal polypeptide consisting of amino acid residues 472-580 [exons 8 and 9] of full-length human GPC3 consisting from the amino acid sequence SEQ ID NO: 157).

The antibody of the present invention is not particularly limited by origin, type, class, morphology and the like. The antibody of the present invention includes, for example: a human antibody; an antibody from an animal such as a mouse or rat; polyclonal antibody, oligoclonal antibody (a mixture of several to several dozen antibodies) and monoclonal antibody; and a chimeric antibody or a humanized antibody in which part of a region (e.g., constant regions) of the antibody is replaced by a region obtained from organisms that differ in species, an antibody fragment, such as an F(ab') ₂ antibody fragment obtained by pepsin cleavage of a monoclonal antibody, an antibody fragment Fab' obtained by reducing the antibody fragment F(ab') ₂ , and a Fab obtained by cleaving a monoclonal antibody with papain, and a recombinant antibody such as scFv containing an antibody heavy (H) chain variable region and a light (H) chain variable region antibody chains linked through amino acid cross-links. In case of using the antibody of the present invention as a CAR, scFv is preferred.

The antibody of the present invention is preferably in the form of an isolated antibody. In this context, the term isolated means that the antibody is in a state different from the state in which the antibody was originally present, that is, the antibody is removed from the environment in which it was originally present, or expressed into an environment different from the environment in which the antibody was originally expressed , through artificial manipulation. In particular, an isolated antibody excludes an antibody that is obtained from a specific individual and is present in the form in which it is found in the individual's body, without external manipulation (artificial manipulation) or in body tissue or fluid (blood, plasma, serum, etc.) ) taken from the body. The antibody of the present invention is preferably a manipulative antibody (eg, a recombinant antibody as described above). Such a cell-derived, artificially manipulated, or cell-derived antibody excludes an antibody that has not been artificially manipulated, such as an antibody derived from a natural B cell.

In the antibody of the present invention, a framework region (FR) is typically linked to the N-terminus and/or C-terminus of each of the CDR1-CDR3 regions of the H chain and the L chain. Among such FRs, for example, the H chain FRs 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), FR3 of the H chain linked to the C-terminus of the CDR2 of the H chain (N-terminus of the CDR3 of the H chain) and FR4 of the H chain linked to the C-terminus of the CDR3 of the H chain. Among the FRs, examples of L chain FR may 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 (L chain N terminus CDR2), FR3 L-chain linked to the C-terminus of CDR2 L-chain (N-terminus of CDR3 L-chain) and FR4 L-chain linked to the C-terminus of CDR3 L-chain.

Examples of H chain FR1 may specifically include: (1-HFR1) a polypeptide consisting of the amino acid sequence 1-30 of amino acid sequence SEQ ID NO: 7, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in the amino acid sequence of this polypeptide; (2-HFR1) a polypeptide consisting of the amino acid sequence 1-30 of amino acid sequence SEQ ID NO: 17, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (3-HFR1) a polypeptide consisting of the amino acid sequence 1-30 of amino acid sequence SEQ ID NO: 27, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (4-HFR1) a polypeptide consisting of the amino acid sequence 1-30 of amino acid sequence SEQ ID NO: 37, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (5-HFR1) a polypeptide consisting of the amino acid sequence 1-30 of amino acid sequence SEQ ID NO: 47, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (6-HFR1) a polypeptide consisting of the amino acid sequence 1-30 of amino acid sequence SEQ ID NO: 57, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (7-HFR1) a polypeptide consisting of the amino acid sequence 1-30 of amino acid sequence SEQ ID NO: 67, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (8-HFR1) a polypeptide consisting of the amino acid sequence 1-30 of amino acid sequence SEQ ID NO: 77, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (9-HFR1) a polypeptide consisting of the amino acid sequence 1-30 of amino acid sequence SEQ ID NO: 87, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (10-HFR1) a polypeptide consisting of the amino acid sequence 1-30 of amino acid sequence SEQ ID NO: 97, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; and (11-HFR1) a polypeptide consisting of the amino acid sequence 1-30 of amino acid sequence SEQ ID NO: 107, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide.

Examples of H chain FR2 may specifically include: (1-HFR2) a polypeptide consisting of the amino acid sequence 36-49 of amino acid sequence SEQ ID NO: 7, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in the amino acid sequence of this polypeptide; (2-HFR2) a polypeptide consisting of the amino acid sequence 36-49 of amino acid sequence SEQ ID NO: 17, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (3-HFR2) a polypeptide consisting of the amino acid sequence 36-49 of amino acid sequence SEQ ID NO: 27, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (4-HFR2) a polypeptide consisting of amino acid sequence 36-49 of amino acid sequence SEQ ID NO: 37, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of that polypeptide ; (5-HFR2) a polypeptide consisting of the amino acid sequence 36-49 of amino acid sequence SEQ ID NO: 47, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (6-HFR2) a polypeptide consisting of the amino acid sequence 36-49 of amino acid sequence SEQ ID NO: 57, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (7-HFR2) a polypeptide consisting of the amino acid sequence 36-49 of amino acid sequence SEQ ID NO: 67, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (8-HFR2) a polypeptide consisting of the amino acid sequence 36-49 of amino acid sequence SEQ ID NO: 77, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (9-HFR2) a polypeptide consisting of the amino acid sequence 36-49 of amino acid sequence SEQ ID NO: 87, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (10-HFR2) a polypeptide consisting of the amino acid sequence 36-49 of amino acid sequence SEQ ID NO: 97, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; and (11-HFR2) a polypeptide consisting of the amino acid sequence 36-49 of amino acid sequence SEQ ID NO: 107, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide.

Examples of H chain FR3 may specifically include: (1-HFR3) a polypeptide consisting of the amino acid sequence 67-98 of amino acid sequence SEQ ID NO: 7, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in the amino acid sequence of this polypeptide; (2-HFR3) a polypeptide consisting of the amino acid sequence 67-98 of amino acid sequence SEQ ID NO: 17, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (3-HFR3) a polypeptide consisting of the amino acid sequence 67-98 of amino acid sequence SEQ ID NO: 27, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (4-HFR3) a polypeptide consisting of the amino acid sequence 67-99 of amino acid sequence SEQ ID NO: 37, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (5-HFR3) a polypeptide consisting of the amino acid sequence 67-99 of amino acid sequence SEQ ID NO: 47, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (6-HFR3) a polypeptide consisting of the amino acid sequence 67-98 of amino acid sequence SEQ ID NO: 57, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (7-HFR3) a polypeptide consisting of the amino acid sequence 67-98 of amino acid sequence SEQ ID NO: 67, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (8-HFR3) a polypeptide consisting of the amino acid sequence 67-98 of amino acid sequence SEQ ID NO: 77, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (9-HFR3) a polypeptide consisting of the amino acid sequence 67-99 of amino acid sequence SEQ ID NO: 87, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (10-HFR3) a polypeptide consisting of the amino acid sequence 67-98 of amino acid sequence SEQ ID NO: 97, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; and (11-HFR3) a polypeptide consisting of the amino acid sequence 67-98 of amino acid sequence SEQ ID NO: 107, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence

- 9 043515 identity of this polypeptide.

Examples of H chain FR4 may specifically include: (1-HFR4) a polypeptide consisting of the amino acid sequence 109-118 of amino acid sequence SEQ ID NO: 7, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in the amino acid sequence of this polypeptide; (2-HFR4) a polypeptide consisting of the amino acid sequence 108-117 of amino acid sequence SEQ ID NO: 17, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (3-HFR4) a polypeptide consisting of the amino acid sequence 106115 of amino acid sequence SEQ ID NO: 27, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (4-HFR4) a polypeptide consisting of the amino acid sequence 111-120 of amino acid sequence SEQ ID NO: 37, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (5HFR4) a polypeptide consisting of the amino acid sequence 108-117 of amino acid sequence SEQ ID NO: 47, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (6-HFR4) a polypeptide consisting of the amino acid sequence 107-116 of amino acid sequence SEQ ID NO: 57, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (7-HFR4) a polypeptide consisting of the amino acid sequence 106-115 of amino acid sequence SEQ ID NO: 67, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (8HFR4) a polypeptide consisting of the amino acid sequence 106-115 of amino acid sequence SEQ ID NO: 77, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (9-HFR4) a polypeptide consisting of the amino acid sequence 111-120 of amino acid sequence SEQ ID NO: 87, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (10-HFR4) a polypeptide consisting of the amino acid sequence 110-119 of amino acid sequence SEQ ID NO: 97, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; and (11-HFR4) a polypeptide consisting of the amino acid sequence 109-118 of amino acid sequence SEQ ID NO: 107, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide.

An example of an FR1 L chain may specifically include: (1-LFR1) a polypeptide consisting of the amino acid sequence 1-23 of amino acid sequence SEQ ID NO: 8, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in the amino acid sequence of this polypeptide; (2-LFR1) a polypeptide consisting of the amino acid sequence 1-23 of amino acid sequence SEQ ID NO: 18, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (3-LFR1) a polypeptide consisting of the amino acid sequence 1-23 of amino acid sequence SEQ ID NO: 28, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (4-LFR1) a polypeptide consisting of the amino acid sequence 1-23 of amino acid sequence SEQ ID NO: 38, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (5-LFR1) a polypeptide consisting of the amino acid sequence 1-23 of amino acid sequence SEQ ID NO: 48, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (6-LFR1) a polypeptide consisting of the amino acid sequence 1-23 of amino acid sequence SEQ ID NO: 58, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (7-LFR1) a polypeptide consisting of the amino acid sequence 1-23 of amino acid sequence SEQ ID NO: 68, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence

- 10 043515 the amino acid sequence of this polypeptide; (8-LFR1) a polypeptide consisting of the amino acid sequence 1-23 of amino acid sequence SEQ ID NO: 78, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (9-LFR1) a polypeptide consisting of the amino acid sequence 1-23 of amino acid sequence SEQ ID NO: 88, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (10-LFR1) a polypeptide consisting of the amino acid sequence 1-23 of amino acid sequence SEQ ID NO: 98, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; and (11-LFR1) a polypeptide consisting of the amino acid sequence 1-23 of amino acid sequence SEQ ID NO: 108, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide.

An example of an FR2 L chain may specifically include: (1-LFR2) a polypeptide consisting of the amino acid sequence 35-49 of amino acid sequence SEQ ID NO: 8, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in the amino acid sequence of this polypeptide; (2-LFR2) a polypeptide consisting of the amino acid sequence 40-54 of amino acid sequence SEQ ID NO: 18, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (3-LFR2) a polypeptide consisting of the amino acid sequence 35-49 of amino acid sequence SEQ ID NO: 28, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (4-LFR2) a polypeptide consisting of the amino acid sequence 35-49 of amino acid sequence SEQ ID NO: 38, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (5-LFR2) a polypeptide consisting of the amino acid sequence 41-55 of amino acid sequence SEQ ID NO: 48, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (6-LFR2) a polypeptide consisting of the amino acid sequence 35-49 of amino acid sequence SEQ ID NO: 58, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (7-LFR2) a polypeptide consisting of the amino acid sequence 35-49 of amino acid sequence SEQ ID NO: 68, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (8-LFR2) a polypeptide consisting of the amino acid sequence 35-49 of amino acid sequence SEQ ID NO: 78, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (9-LFR2) a polypeptide consisting of the amino acid sequence 35-49 of amino acid sequence SEQ ID NO: 88, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (10-LFR2) a polypeptide consisting of the amino acid sequence 35-49 of amino acid sequence SEQ ID NO: 98, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; and (11-LFR2) a polypeptide consisting of the amino acid sequence 35-49 of amino acid sequence SEQ ID NO: 108, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide.

An example L chain FR3 may specifically include: (1-LFR3) a polypeptide consisting of the amino acid sequence 57-88 of amino acid sequence SEQ ID NO: 8, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in the amino acid sequence of this polypeptide; (2-LFR3) a polypeptide consisting of the amino acid sequence 62-93 of amino acid sequence SEQ ID NO: 18, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (3-LFR3) a polypeptide consisting of the amino acid sequence 57-88 of amino acid sequence SEQ ID NO: 28, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in amino acid sequence

- 11 043515 acid sequence of this polypeptide; (4-LFR3) a polypeptide consisting of the amino acid sequence 57-88 of amino acid sequence SEQ ID NO: 38, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (5-LFR3) a polypeptide consisting of the amino acid sequence 63-94 of amino acid sequence SEQ ID NO: 48, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (6-LFR3) a polypeptide consisting of the amino acid sequence 57-88 of amino acid sequence SEQ ID NO: 58, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (7-LFR3) a polypeptide consisting of the amino acid sequence 57-88 of amino acid sequence SEQ ID NO: 68, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (8-LFR3) a polypeptide consisting of the amino acid sequence 57-88 of amino acid sequence SEQ ID NO: 78, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (9-LFR3) a polypeptide consisting of the amino acid sequence 57-88 of amino acid sequence SEQ ID NO: 88, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (10-LFR3) a polypeptide consisting of the amino acid sequence 57-88 of amino acid sequence SEQ ID NO: 98, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; and (11-LFR3) a polypeptide consisting of the amino acid sequence 57-88 of amino acid sequence SEQ ID NO: 108, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide.

An example of an FR4 L chain may specifically include: (1-LFR4) a polypeptide consisting of the amino acid sequence 98-108 of amino acid sequence SEQ ID NO: 8, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in the amino acid sequence of this polypeptide; (2-LFR4) a polypeptide consisting of the amino acid sequence 103-113 of amino acid sequence SEQ ID NO: 18, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (3-LFR4) a polypeptide consisting of the amino acid sequence 97-107 of amino acid sequence SEQ ID NO: 28, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (4-LFR4) a polypeptide consisting of the amino acid sequence 98-108 of amino acid sequence SEQ ID NO: 38, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (5-LFR4) a polypeptide consisting of the amino acid sequence 104-114 of amino acid sequence SEQ ID NO: 48, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (6-LFR4) a polypeptide consisting of the amino acid sequence 98108 of amino acid sequence SEQ ID NO: 58, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (7-LFR4) a polypeptide consisting of the amino acid sequence 98-108 of amino acid sequence SEQ ID NO: 68, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (8-LFR4) a polypeptide consisting of the amino acid sequence 98-108 of amino acid sequence SEQ ID NO: 78, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (9-LFR4) a polypeptide consisting of the amino acid sequence 98-108 of amino acid sequence SEQ ID NO: 88, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; (10-LFR4) a polypeptide consisting of the amino acid sequence 98-108 of amino acid sequence SEQ ID NO: 98, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide; and (11-12 043515

LFR4) a polypeptide consisting of the amino acid sequence 98-108 of amino acid sequence SEQ ID NO: 108, or a polypeptide consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of this polypeptide.

The FR antibodies of the present invention are preferably FRs of known human antibodies. Examples of such known human antibody FRs may include a human antibody FR registered in a sequence database known in the art, such as GenBank, and the FR selected from a conventional sequence (the consensus sequence of the most homologous human sequence; Rabat, E. A. et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991), obtained from each human antibody subset.

The H chain CDR1 in the antibody of the present invention generally corresponds to residues at positions H31-H35 based on Rabat numbering (see Rabat, E.A. et al., (1991) NIH Publication No. 91-3242, sequence of proteins of immunological interest) . The H chain CDR2 in the antibody of the present invention generally corresponds to residues at positions H50-H52, H52A and H53H65 based on Rabat numbering. The H chain CDR3 in the antibody of the present invention generally corresponds to residues at positions H95-H100, H100A, H100B, H101 and H102 based on Rabat numbering. The L chain CDR1 in the antibody of the present invention generally corresponds to residues at positions L24-L34 based on Rabat numbering. The L chain CDR2 in the antibody of the present invention generally corresponds to residues at positions L50-L56 based on Rabat numbering. The L chain CDR3 in the antibody of the present invention generally corresponds to residues at positions L89-L97 based on Rabat numbering.

Examples of the antibody containing CDR1 to CDR3 of H chains and L chains as described in (1-1) above, as the antibody of the present invention, may include an antibody containing variable (V) regions of an H chain and an L chain, as described above in (1-2), and may specifically include: a single chain antibody, as described above in (1-3); single chain antibody as described above in (1-3'-1), single chain antibody as described above in (1-3'-2), and single chain antibody as described above in (1-3'-3); and an antibody containing an H chain and an L chain as described above in (1-4). Examples of an antibody containing CDR1CDR3 H chains and L chains as described above in (2-1) may include an antibody containing H chain and L chain V regions as described above in (2-2), and may specifically include: single chain antibody as described above in (2-3); single chain antibody as described above in (2-3'-1), single chain antibody as described above in (2-3'-2), single chain antibody as described above in (2-3'-3), and single chain antibody as described above in (2-3'-4); and an antibody containing an H chain and an L chain as described above in (24). Examples of an antibody containing H chain and L chain CDR1 to CDR3 as described above in (3-1) may include an antibody containing H chain and L chain V regions as described above in (3-2) and may specifically include: a single chain antibody as described above in (3-3); and an antibody containing an H chain and an L chain as described above in (3-4). Examples of an antibody containing H chain and L chain CDR1 to CDR3 as described above in (4-1) may include an antibody containing H chain and L chain V regions as described above in (4-2) and may specifically include: a single chain antibody as described above in (4-3); and an antibody containing an H chain and an L chain as described above in (4-4). Examples of an antibody containing CDR1-CDR3 H chains and L chains as described above in (5-1) may include an antibody containing H chain and L chain V regions as described above in (5-2) and may specifically include : single chain antibody as described above in (5-3); and an antibody containing an H chain and an L chain as described above in (5-4). Examples of an antibody containing H chain and L chain CDR1 to CDR3 as described above in (6-1) may include an antibody containing H chain and L chain V regions as described above in (6-2) and may specifically include: a single chain antibody as described above in (6-3); and an antibody containing an H chain and an L chain as described above in (6-4). Examples of an antibody containing H chain and L chain CDR1 to CDR3 as described above in (7-1) may include an antibody containing H chain and L chain V regions as described above in (7-2) and may specifically include: a single chain antibody as described above in (7-3); and an antibody containing an H chain and an L chain as described above in (7-4). Examples of an antibody containing H chain and L chain CDR1 to CDR3 as described above in (8-1) may include an antibody containing H chain and L chain V regions as described above in (8-2) and may specifically include: a single chain antibody as described above in (8-3); and an antibody containing an H chain and an L chain as described above in (8-4). Examples of an antibody containing CDR1-CDR3 H chains and L chains as described above in (9-1) may include an antibody containing H chain and L chain V regions as described above in (9-2) and may specifically include : single chain antibody as described above in (9-3); and an antibody containing an H chain and an L chain as described above in (9-4). Examples of an antibody containing H chain and L chain CDR1 to CDR3 as described above in (10-1) may include an antibody containing H chain and L chain V regions as described above in (10-2) and may specifically include: a single chain antibody as described above in (10-3); and an antibody containing an H chain and an L chain as described above in (10-4). Examples of an antibody containing H chain and L chain CDR1 to CDR3 as described above in (111) may include an antibody containing H chain and L chain V regions as described above in (11-2) and may specifically include: a single chain antibody as described above in (11-3); and an antibody containing an H chain and an L chain as described above in (11-4). Heavy chain variable region and variable region

- 13 043515 light chains in a single chain antibody are usually linked via a peptide linker.

A CAR of the present invention may comprise a single chain antibody of the present invention, a transmembrane region fused to the C-terminus of the single chain antibody of the present invention, and a signal transduction activation region of an immunocompetent cell fused to the C-terminus of the transmembrane region. In this context, the fusion of a single chain antibody of the present invention and a transmembrane region, or a transmembrane region and a signal transduction activation region of an immunocompetent cell, may be mediated by a peptide linker or an IgG4 hinge region.

Examples of the length of the peptide linker in the antibody of the present invention may include 1-100 amino acid residues, preferably 10-50 amino acid residues. Examples of a peptide linker in an antibody of the present invention may include a contiguous linkage of 3 amino acid sequences, each consisting of 1-4 glycine residues and 1 serine residue.

The transmembrane region can be any peptide that can penetrate the cell membrane. Examples may include transmembrane region derived from CD8, α- or β-chain T cell receptor, CD3Z, 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 may specifically include the human CD8 transmembrane region consisting of amino acid residues 1-83 of amino acid sequence SEQ ID NO: 185. Alternatively, the transmembrane region may be derived from a peptide that can penetrate the cell membrane by truncation of the C-terminal 1-10 amino acid residues, preferably 6 or 7 amino acid residues. Examples may include engineered human CD8 transmembrane region form 1 consisting of amino acid residues 1-77 of amino acid sequence SEQ ID NO: 186, and engineered human CD8 transmembrane region form 2 consisting of amino acid residues 1-76 of amino acid sequence SEQ ID NO: 187.

The signal transduction activation region of an immunocompetent cell may be any region capable of transmitting a signal to immunocompetent cells upon binding of the single chain antibody of the present invention to human GPC3. The signal transduction activation region of an immunocompetent cell preferably contains at least one or more members selected from the intracellular region polypeptides CD28, 4-1BB (CD137), GITR, CD27, OX40, HVEM, CD3Z and Fc receptor γ chain, and, more preferably, contains three intracellular region polypeptides CD28, 4-1BB and CD3Z. Examples of such a CD28 intracellular region polypeptide may specifically include a human CD28 intracellular region polypeptide consisting of amino acid residues 85-124 of amino acid sequence SEQ ID NO: 185. Examples of a 4-1BB intracellular region polypeptide may specifically include a human 4-1BB intracellular region polypeptide consisting of amino acid residues 125-170 of amino acid sequence SEQ ID NO: 185. Examples of such a CD3Z intracellular region polypeptide may specifically include a human CD3Z intracellular region polypeptide consisting of amino acid residues 172283 of amino acid sequence SEQ ID NO: 185.

Arginine (Arg) at position 84 of amino acid sequence SEQ ID NO: 185, arginine at position 78 of amino acid sequence SEQ ID NO: 186, and arginine at position 77 of amino acid sequence SEQ ID NO: 187 are the general sequence of a transmembrane region polypeptide derived from human CD8, and human CD28 intracellular region polypeptide. Leucine (Leu) at position 171 of amino acid sequence SEQ ID NO: 185, leucine at position 165 of amino acid sequence SEQ ID NO: 186 and leucine at position 164 of amino acid sequence SEQ ID NO: 187 are the common sequence of the human intracellular region 4-1BB polypeptide and polypeptide intracellular region of human CD3Z.

As used herein, an immunocompetent cell means a cell responsible for immune functions in a living body. Examples of immunocompetent cells may include: lymphoid cells such as T cells, natural killer cells (NK cells) and B cells; antigen-presenting cells such as monocytes, macrophages and dendritic cells; and granulocytes, such as neutrophils, eosinophils, basophils and mast cells. Specific examples may preferably include a T cell derived from a mammal such as a human, dog, cat, pig or mouse, preferably a T cell derived from a human. The T cell can be obtained by isolating or purifying from an immunocompetent cell infiltrating a body fluid such as blood or bone marrow fluid, tissue of the spleen, thymus, lymph node or the like, or malignant tissue of a primary tumor, metastatic tumor, cancerous ascites or the like similar. Alternatively, a T cell derived from an ES cell or an iPS cell can be used. Examples of such a T cell may include an alpha beta T cell, a gamma delta T cell, a CD8+ T cell, a CD4+ T cell, a tumor infiltrating lymphocyte, a memory T cell, a naïve T cell, and an NKT cell. An immunocompetent cell has the same origin as an individual or its nature may differ. If the individual being administered is a human, then as an immunocompetent cell

- 14 043515 an autologous cell obtained from a patient as the individual being administered may be used, or any other cell obtained from a person other than the individual being administered may be used as the immunocompetent cell. In particular, the donor and recipient may be the same or different, and preferably they are the same.

Examples of the subject to be administered preferably include a mammal and a mammalian cell. Examples of mammals preferably include human, mouse, dog, rat, guinea pig, rabbit, bird, sheep, pig, cattle, horse, cat, monkey and chimpanzee, especially preferably human.

The CARs of the present invention are preferably used for ex vivo expression on the cell surface of an immunocompetent cell collected from a patient suffering from a cancer during treatment. In the case of using a T cell as an immunocompetent cell, examples of a peptide consisting of a transmembrane region and a signal transduction activation region of an immunocompetent cell fused to the C-terminus of the transmembrane region in the CAR of the present invention may specifically include a peptide consisting of any amino acid sequence of SEQ ID NO: 185-187. Examples of the CARs of the present invention may specifically include a CAR comprising a single chain antibody selected from the group consisting of a single chain antibody as described above in (1-3), a single chain antibody as described above in (2-3), a single chain antibody as described above above in (1-3'-1), single chain antibody as described above in (1-3'-2), single chain antibody as described above in (1-3'-3), single chain antibody as described above in (2-3'-1), single chain antibody as described above in (2-3'-2), single chain antibody as described above in (23'-3), and single chain antibody as described above in (2- 3'-4), and a peptide consisting of any amino acid sequence of SEQ ID NO: 185-187 fused to the C-terminus of a single chain antibody.

Specifically, examples of the CARs of the present invention may include:

A CAR comprising a single chain antibody as described above in (1-3) and a peptide consisting of the amino acid sequence SEQ ID NO: 185,

A CAR comprising a single chain antibody as described in (1-3) above and a peptide consisting of the amino acid sequence SEQ ID NO: 186,

A CAR comprising a single chain antibody as described in (1-3) above and a peptide consisting of the amino acid sequence SEQ ID NO: 187,

A CAR comprising a single chain antibody as described above in (1-3'-1) and a peptide consisting of the amino acid sequence SEQ ID NO: 185,

A CAR comprising a single chain antibody as described above in (1-3'-1) and a peptide consisting of the amino acid sequence SEQ ID NO: 186,

A CAR comprising a single chain antibody as described above in (1-3'-1) and a peptide consisting of the amino acid sequence SEQ ID NO: 187,

A CAR comprising a single chain antibody as described above in (1-3'-2) and a peptide consisting of the amino acid sequence SEQ ID NO: 185,

A CAR comprising a single chain antibody as described above in (1-3'-2) and a peptide consisting of the amino acid sequence SEQ ID NO: 186,

A CAR comprising a single chain antibody as described above in (1-3'-2) and a peptide consisting of the amino acid sequence SEQ ID NO: 187,

A CAR comprising a single chain antibody as described above in (1-3'-3) and a peptide consisting of the amino acid sequence SEQ ID NO: 185,

A CAR comprising a single chain antibody as described above in (1-3'-3) and a peptide consisting of the amino acid sequence SEQ ID NO: 186,

A CAR comprising a single chain antibody as described above in (1-3'-3) and a peptide consisting of the amino acid sequence SEQ ID NO: 187,

A CAR comprising a single chain antibody as described in (2-3) above and a peptide consisting of the amino acid sequence SEQ ID NO: 185,

A CAR comprising a single chain antibody as described in (2-3) above and a peptide consisting of the amino acid sequence SEQ ID NO: 186,

A CAR comprising a single chain antibody as described in (2-3) above and a peptide consisting of the amino acid sequence SEQ ID NO: 187,

A CAR comprising a single chain antibody as described above in (2-3'-1) and a peptide consisting of the amino acid sequence SEQ ID NO: 185,

A CAR comprising a single chain antibody as described above in (2-3'-1) and a peptide consisting of the amino acid sequence SEQ ID NO: 186,

A CAR comprising a single chain antibody as described above in (2-3'-1) and a peptide consisting of the amino acid sequence SEQ ID NO: 187,

A CAR comprising a single chain antibody as described above in (2-3'-2) and a peptide consisting of the amino acid sequence SEQ ID NO: 185,

A CAR containing a single chain antibody as described above in (2-3'-2) and a peptide consisting of

- 15 043515 amino acid sequence SEQ ID NO: 186,

A CAR comprising a single chain antibody as described above in (2-3'-2) and a peptide consisting of the amino acid sequence SEQ ID NO: 187,

A CAR comprising a single chain antibody as described above in (2-3'-3) and a peptide consisting of the amino acid sequence SEQ ID NO: 185,

A CAR comprising a single chain antibody as described above in (2-3'-3) and a peptide consisting of the amino acid sequence SEQ ID NO: 186,

A CAR comprising a single chain antibody as described above in (2-3'-3) and a peptide consisting of the amino acid sequence SEQ ID NO: 187,

A CAR comprising a single chain antibody as described above in (2-3'-4) and a peptide consisting of the amino acid sequence SEQ ID NO: 185,

A CAR comprising a single chain antibody as described above in (2-3'-4) and a peptide consisting of the amino acid sequence SEQ ID NO: 186, and

A CAR comprising a single chain antibody as described above in (2-3'-4) and a peptide consisting of the amino acid sequence SEQ ID NO: 187.

The immunocompetent cell of the present invention can be any immunocompetent cell expressing a CAR. Because CAR does not typically occur naturally, the immunocompetent cell expresses the foreign rather than the endogenous CAR. The immunocompetent cell of the present invention preferably also expresses IL-7 and/or CCL19. If the immunocompetent cell is a cell that does not express IL-7 and/or CCL19, such as a T cell, or if the immunocompetent cell is a cell other than a T cell that expresses IL-7 and/or CCL19 at a low level, then the immunocompetent cell of the present invention preferentially expresses foreign IL-7 and/or CCL19.

The immunocompetent cell of the present invention can be obtained by introducing the vector of the present invention containing the CAR gene of the present invention and the vector containing the IL-7 and/or CCL19 gene into the immunocompetent cell. The route of administration may be any method of introducing DNA into mammalian cells. Examples of such methods may include, for example, electroporation (Cytotechnology, 3, 133 (1990)), the calcium phosphate method (unexamined Japanese Patent Application No. 2-227075), lipofection (Proc. Natl. Acad. Sci. U.S.A., 84, 7413 (1987)), and methods based on virus infection. Examples of such a virus infection-based method may include a method that involves transfecting a packaging cell such as GP2-293 cell (manufactured by Takara Bio Inc.), Plat-GP cell (manufactured by Cosmo Bio Co., Ltd.), PG13 cell (ATCC CRL-10686) or PA317 cell (ATCC CRL-9078) with a CAR expression vector (International Publication No. WO 2016/056228) and a packaging plasmid to produce a recombinant virus and infect a T cell with the recombinant virus.

An immunocompetent cell of the present invention can be obtained by inserting a nucleotide encoding a CAR of the present invention and a nucleotide encoding IL-7 and/or CCL19 into the genome of the cell by a gene editing method known in the art, allowing the nucleotides to be expressed under the control of an appropriate promoter . Examples of gene editing techniques known in the art include techniques using an endonuclease such as zinc finger nuclease, TALEN (transcription activator-like effector nuclease) or the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system.

The immunocompetent cell of the present invention can be used in combination with an additional antitumor agent. Examples of the additional antitumor agent may include: an alkylating drug such as cyclophosphamide, bendamustine, ifosfamide and dacarbazine; an antimetabolite such as pentostatin, fludarabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine and enocytabine; 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 bortezomib; a calcineurin inhibitory drug such as cyclosporine and tacrolimus; anticancer antibiotic such as idarubicin and doxorubicin mexomicin C; plant alkaloid such as irinotecan and etoposide; a platinum-based drug such as cisplatin, oxaliplatin and carboplatin; a hormone therapy such as tamoxifen and bicalutamide; and an immunosuppressive drug such as interferon, nivolumab and pembrolizumab.

Examples of a method of using an immunocompetent cell of the present invention in combination with an additional antitumor agent may include a method using treatment with an additional antitumor agent followed by use of an immunocompetent cell of the present invention, a method using an immunocompetent cell of the present invention and an additional antitumor agent simultaneously, and a method using treatment with an immunocompetent cell of the present invention followed by the use of an additional antitumor agent. The use of an immunocompetent cell of the present invention in combination with an additional antitumor agent can

- 16 043515 improve the therapeutic effect against malignancy, as well as reduce adverse reactions by reducing the number of administrations or dose.

The antibody gene of the present invention is not particularly limited as long as the antibody gene (nucleotide) encodes the antibody of the present invention. Example antibody genes include:

(1-1D) an antibody gene comprising: an H chain CDR1 gene consisting of nucleotide residues 91-105 of an H chain V region gene consisting of nucleotide residues SEQ ID NO: 111 (the gene encoding the CDR1 H chain as described above in (1-1)), or a variant, based on codon degeneracy, of the CDR1 H chain gene; an H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 111 (the gene encoding the CDR2 H chain as described above in (1-1)), or a variant, based on codon degeneracy, of the H chain CDR2 gene; and an H chain CDR3 gene consisting of nucleotide residues 295 to 324 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 111 (the gene encoding the CDR3 H chain as described above in (1-1)) , or a variant, based on codon degeneracy, of the H-chain CDR3 gene; and an L chain CDR1 gene consisting of nucleotide residues 70102 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 112 (the gene encoding L chain CDR1 as described above in (1-1)), or variant, based on codon degeneracy, of the L-chain CDR1 gene; an L chain CDR2 gene consisting of nucleotide residues 148-168 of the L chain V region gene consisting of the nucleotide sequence SEQ ID NO: 112 (the gene encoding L chain CDR2 as described above in (1-1)), or a variant , based on codon degeneracy, L-chain CDR2 gene; and an L chain CDR3 gene consisting of nucleotide residues 265 to 291 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 112 (the gene encoding L chain CDR3 as described above in (11)), or variant, based on codon degeneracy, L chain CDR3 gene, (2-1D) antibody gene containing: H chain CDR1 gene, consisting of nucleotide residues 91-105 of the H chain V region gene, consisting of nucleotide residues SEQ ID NO: 115 (the gene encoding the CDR1 H chain as described above in (2-1)), or a variant, based on codon degeneracy, of the H chain CDR1 gene; an H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 115 (the gene encoding the CDR2 H chain as described above in (2-1)), or a variant, based on codon degeneracy, of the H chain CDR2 gene; and an H chain CDR3 gene consisting of nucleotide residues 295 to 321 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 115 (the gene encoding the CDR3 H chain as described above in (2-1)) , or a variant, based on codon degeneracy, of the H-chain CDR3 gene; and an L chain CDR1 gene consisting of nucleotide residues 70117 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 116 (the gene encoding L chain CDR1 as described above in (2-1)), or variant, based on codon degeneracy, of the L-chain CDR1 gene; an L chain CDR2 gene consisting of nucleotide residues 163-183 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 116 (the gene encoding L chain CDR2 as described above in (2-1)), or a variant , based on codon degeneracy, L-chain CDR2 gene; and an L chain CDR3 gene consisting of nucleotide residues 280-306 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 116 (the gene encoding L chain CDR3 as described above in (21)), or variant, based on codon degeneracy, L-chain CDR3 gene, (3-1D) antibody gene containing: H-chain CDR1 gene, consisting of nucleotide residues 91-105 of the H-chain V-region gene, consisting of nucleotide residues SEQ ID NO: 119 (the gene encoding the CDR1 H chain as described above in (3-1)), or a variant, based on codon degeneracy, of the H chain CDR1 gene; an H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 119 (the gene encoding the CDR2 H chain as described above in (3-1)), or a variant, based on codon degeneracy, of the H chain CDR2 gene; and an H chain CDR3 gene consisting of nucleotide residues 295 to 315 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 119 (the gene encoding the CDR3 H chain as described above in (3-1)) , or a variant, based on codon degeneracy, of the H-chain CDR3 gene; and an L chain CDR1 gene consisting of nucleotide residues 70102 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 120 (the gene encoding L chain CDR1 as described above in (3-1)), or variant, based on codon degeneracy, of the L-chain CDR1 gene; an L chain CDR2 gene consisting of nucleotide residues 148 to 168 of the L chain V region gene consisting of the nucleotide sequence SEQ ID NO: 120 (the gene encoding L chain CDR2 as described above in (3-1)), or a variant , based on codon degeneracy, L-chain CDR2 gene; and an L chain CDR3 gene consisting of nucleotide residues 265-288 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 120 (the gene encoding L chain CDR3 as described above in (31)), or variant, based on codon degeneracy, L chain CDR3 gene, (4-1D) antibody gene containing: H chain CDR1 gene, consisting of nucleotide residues 91-105 of the H chain V region gene, consisting of nucleotide residues SEQ ID NO: 123 (the gene encoding the CDR1 H chain as described above in (4-1)), or a variant, based on codon degeneracy, of the H chain CDR1 gene; an H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 123 (the gene encoding the CDR2 H chain as described above in (4-1)), or a variant, based on codon degeneracy, of the H chain CDR2 gene; and the H chain CDR3 gene, consisting of nucleotide residues 298-330 of the H chain V region gene, consisting of nucleotide residues

- 17 043515

SEQ ID NO: 123 (the gene encoding the CDR3 H chain as described above in (4-1)), or a codon degeneracy variant of the H chain CDR3 gene; and an L chain CDR1 gene consisting of nucleotide residues 70102 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 124 (the gene encoding L chain CDR1 as described above in (4-1)), or variant, based on codon degeneracy, of the L-chain CDR1 gene; an L chain CDR2 gene consisting of nucleotide residues 148-168 of the L chain V region gene consisting of the nucleotide sequence SEQ ID NO: 124 (the gene encoding L chain CDR2 as described above in (4-1)), or a variant , based on codon degeneracy, L-chain CDR2 gene; and an L chain CDR3 gene consisting of nucleotide residues 265-291 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 124 (the gene encoding L chain CDR3 as described above in (41)), or variant, based on codon degeneracy, L chain CDR3 gene, (5-1D) antibody gene containing: H chain CDR1 gene, consisting of nucleotide residues 91-105 of the H chain V region gene, consisting of nucleotide residues SEQ ID NO: 127 (the gene encoding the CDR1 H chain as described above in (5-1)), or a variant, based on codon degeneracy, of the H chain CDR1 gene; an H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 127 (the gene encoding the CDR2 H chain as described above in (5-1)), or a variant, based on codon degeneracy, of the H chain CDR2 gene; and an H chain CDR3 gene consisting of nucleotide residues 298 to 321 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 127 (the gene encoding the CDR3 H chain as described above in (5-1)) , or a variant, based on codon degeneracy, of the H-chain CDR3 gene; and an L chain CDR1 gene consisting of nucleotide residues 70120 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 128 (the gene encoding L chain CDR1 as described above in (5-1)), or variant, based on codon degeneracy, of the L-chain CDR1 gene; an L chain CDR2 gene consisting of nucleotide residues 166 to 186 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 128 (the gene encoding L chain CDR2 as described above in (5-1)), or a variant , based on codon degeneracy, L-chain CDR2 gene; and an L chain CDR3 gene consisting of nucleotide residues 283-309 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 128 (the gene encoding L chain CDR3 as described above in (51)), or variant, based on codon degeneracy, L chain CDR3 gene, (6-1D) antibody gene containing: H chain CDR1 gene, consisting of nucleotide residues 91-105 of the H chain V region gene, consisting of nucleotide residues SEQ ID NO: 131 (the gene encoding the CDR1 H chain as described above in (6-1)), or a variant, based on codon degeneracy, of the H chain CDR1 gene; an H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 131 (the gene encoding the CDR2 H chain as described above in (6-1)), or a variant, based on codon degeneracy, of the H chain CDR2 gene; and an H chain CDR3 gene consisting of nucleotide residues 295 to 318 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 131 (the gene encoding the CDR3 H chain as described above in (6-1)) , or a variant, based on codon degeneracy, of the H-chain CDR3 gene; and an L chain CDR1 gene consisting of nucleotide residues 70102 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 132 (the gene encoding L chain CDR1 as described above in (6-1)), or variant, based on codon degeneracy, of the L-chain CDR1 gene; an L chain CDR2 gene consisting of nucleotide residues 148 to 168 of the L chain V region gene consisting of the nucleotide sequence SEQ ID NO: 132 (the gene encoding L chain CDR2 as described above in (6-1)), or a variant , based on codon degeneracy, L-chain CDR2 gene; and an L chain CDR3 gene consisting of nucleotide residues 265-291 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 132 (the gene encoding L chain CDR3 as described above in (61)), or variant, based on codon degeneracy, L chain CDR3 gene, (7-1D) antibody gene containing: H chain CDR1 gene, consisting of nucleotide residues 91-105 of the H chain V region gene, consisting of nucleotide residues SEQ ID NO: 135 (the gene encoding the CDR1 H chain as described above in (7-1)), or a variant, based on codon degeneracy, of the H chain CDR1 gene; an H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 135 (the gene encoding the CDR2 H chain as described above in (7-1)), or a variant, based on codon degeneracy, of the H chain CDR2 gene; and an H chain CDR3 gene consisting of nucleotide residues 295 to 315 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 135 (the gene encoding the CDR3 H chain as described above in (7-1)) , or a variant, based on codon degeneracy, of the H-chain CDR3 gene; and an L chain CDR1 gene consisting of nucleotide residues 70102 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 136 (the gene encoding L chain CDR1 as described above in (7-1)), or variant, based on codon degeneracy, of the L-chain CDR1 gene; an L chain CDR2 gene consisting of nucleotide residues 148 to 168 of the L chain V region gene consisting of the nucleotide sequence SEQ ID NO: 136 (the gene encoding L chain CDR2 as described above in (7-1)), or a variant , based on codon degeneracy, L-chain CDR2 gene; and an L chain CDR3 gene consisting of nucleotide residues 265-291 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 136 (the gene encoding L chain CDR3 as described above in (71)), or variant, based on codon degeneracy, L-chain CDR3 gene, (8-1D) antibody gene containing: H-chain CDR1 gene, consisting of nucleotide residues 91-105

- 18 043515 of the H chain V region gene consisting of the nucleotide residues of SEQ ID NO: 139 (the gene encoding the H chain CDR1, as described above in (8-1)), or a variant, based on codon degeneracy, of the H chain CDR1 gene; an H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 139 (the gene encoding the CDR2 H chain as described above in (8-1)), or a variant, based on codon degeneracy, of the H chain CDR2 gene; and an H chain CDR3 gene consisting of nucleotide residues 295 to 315 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 139 (the gene encoding the CDR3 H chain as described above in (8-1)) , or a variant, based on codon degeneracy, of the H-chain CDR3 gene; and an L chain CDR1 gene consisting of nucleotide residues 70102 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 140 (the gene encoding L chain CDR1 as described above in (8-1)), or variant, based on codon degeneracy, of the L-chain CDR1 gene; an L chain CDR2 gene consisting of nucleotide residues 148-168 of the L chain V region gene consisting of the nucleotide sequence SEQ ID NO: 140 (the gene encoding L chain CDR2 as described above in (8-1)), or a variant , based on codon degeneracy, L-chain CDR2 gene; and an L chain CDR3 gene consisting of nucleotide residues 265-291 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 140 (the gene encoding L chain CDR3 as described above in (81)), or variant, based on codon degeneracy, L chain CDR3 gene, (9-1D) antibody gene containing: H chain CDR1 gene, consisting of nucleotide residues 91-105 of the H chain V region gene, consisting of nucleotide residues SEQ ID NO: 143 (the gene encoding the CDR1 H chain as described above in (9-1)), or a variant, based on codon degeneracy, of the H chain CDR1 gene; an H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 143 (the gene encoding the CDR2 H chain as described above in (9-1)), or a variant, based on codon degeneracy, of the H chain CDR2 gene; and an H chain CDR3 gene consisting of nucleotide residues 298 to 330 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 143 (the gene encoding the CDR3 H chain as described above in (9-1)) , or a variant, based on codon degeneracy, of the H-chain CDR3 gene; and an L chain CDR1 gene consisting of nucleotide residues 70102 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 144 (the gene encoding L chain CDR1 as described above in (9-1)), or variant, based on codon degeneracy, of the L-chain CDR1 gene; an L chain CDR2 gene consisting of nucleotide residues 148-168 of the L chain V region gene consisting of the nucleotide sequence SEQ ID NO: 144 (the gene encoding L chain CDR2 as described above in (9-1)), or a variant , based on codon degeneracy, L-chain CDR2 gene; and an L chain CDR3 gene consisting of nucleotide residues 265-291 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 144 (the gene encoding L chain CDR3 as described above in (91)), or variant, based on codon degeneracy, L-chain CDR3 gene, (10-1D) antibody gene containing: H-chain CDR1 gene, consisting of nucleotide residues 91-105 of the H-chain V-region gene, consisting of nucleotide residues SEQ ID NO: 147 (the gene encoding the CDR1 H chain as described above in (10-1)), or a variant, based on codon degeneracy, of the H chain CDR1 gene; an H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 147 (the gene encoding the CDR2 H chain as described above in (10-1)), or a variant, based on codon degeneracy, of the H chain CDR2 gene; and an H chain CDR3 gene consisting of nucleotide residues 295 to 327 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 147 (the gene encoding the CDR3 H chain as described above in (10-1)) , or a variant, based on codon degeneracy, of the H-chain CDR3 gene; and an L chain CDR1 gene consisting of nucleotide residues 70 to 102 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 148 (the gene encoding L chain CDR1 as described above in (10-1)) , or a variant, based on codon degeneracy, of the L chain CDR1 gene; an L chain CDR2 gene consisting of nucleotide residues 148 to 168 of the L chain V region gene consisting of the nucleotide sequence SEQ ID NO: 148 (the gene encoding L chain CDR2 as described above in (10-1)), or a variant , based on codon degeneracy, L-chain CDR2 gene; and an L chain CDR3 gene consisting of nucleotide residues 265 to 291 of the L chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 148 (the gene encoding L chain CDR3 as described above in (10-1)) , or a variant, based on codon degeneracy, of the L-chain CDR3 gene, and (11-1D) an antibody gene containing: the H-chain CDR1 gene, consisting of nucleotide residues 91-105 of the H-chain V-region gene, consisting of nucleotide residues of SEQ ID NO: 151 (the gene encoding the CDR1 H chain as described above in (11-1)), or a variant, based on codon degeneracy, of the H chain CDR1 gene; an H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of nucleotide residues of SEQ ID NO: 151 (the gene encoding the CDR2 H chain as described above in (11-1)), or a variant, based on codon degeneracy, of the H chain CDR2 gene; and an H chain CDR3 gene consisting of nucleotide residues 295 to 324 of the H chain V region gene consisting of the nucleotide sequence of SEQ ID NO: 151 (the gene encoding the CDR3 H chain as described above in (11-1)) , or a variant, based on codon degeneracy, of the H-chain CDR3 gene.

Other examples of the antibody gene of the present invention may include a (1-2D) antibody gene comprising an H chain variable region gene consisting of a nucleotide sequence that is at least 80% or greater sequence identical to

- 19 043515 leotide sequence SEQ ID NO: 111 (a gene encoding an H chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence SEQ ID NO: 7), and a gene variable L chain variable region consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 112 (a gene encoding an L chain variable region consisting of an amino acid sequence that is at least 80% identical or more identical in amino acid sequence to the amino acid sequence of SEQ ID NO: 8), (2-2D) an antibody gene comprising an H chain variable region gene consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 115 (a gene encoding an H chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 17), and a gene encoding an L chain variable region consisting of of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 116 (a gene encoding an L chain variable region consisting of an amino acid sequence that is at least 80% or greater identical in sequence to the amino acid sequences SEQ ID NO: 18), (3-2D) an antibody gene containing an H chain variable region gene consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence SEQ ID NO: 119 (gene encoding an H chain variable region gene consisting of an amino acid sequence that is at least 80% identical in sequence to the amino acid sequence of SEQ ID NO: 27), and an L chain variable region gene consisting of a nucleotide sequence that is at least 80% or greater sequence identical to the nucleotide sequence of SEQ ID NO: 120 (a gene encoding an L chain variable region consisting of an amino acid sequence that is at least 80% or greater sequence identical to the amino acid sequence of SEQ ID NO: 28 ), (4-2D) an antibody gene comprising an H chain variable region gene consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 123 (gene encoding the H chain variable region chain consisting of an amino acid sequence that is at least 80% or greater identical in sequence to the amino acid sequence of SEQ ID NO: 37), and an L chain variable region gene consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 124 (a gene encoding an L chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 38), (5-2D ) an antibody gene containing an H chain variable region gene consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 127 (a gene encoding an H chain variable region consisting of an amino acid sequence which is at least 80% or greater sequence identical to the amino acid sequence of SEQ ID NO: 47), and an L chain variable region gene consisting of a nucleotide sequence that is at least 80% or greater sequence identical to the nucleotide sequence of SEQ ID NO: 128 (a gene encoding an L chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 48), (6-2D) an antibody gene containing a gene H chain variable region consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 131 (a gene encoding an H chain variable region consisting of an amino acid sequence that is at least 80% or greater sequence identity to the amino acid sequence of SEQ ID NO: 57), and an L chain variable region gene consisting of a nucleotide sequence that is at least 80% or greater sequence identity to the nucleotide sequence of SEQ ID NO: 132 (gene encoding an L chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 58), (7-2D) an antibody gene containing an H chain variable region gene, consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 135 (a gene encoding an H chain variable region consisting of an amino acid sequence that is at least 80% or greater identical

- 20 043515 is identical in sequence to the amino acid sequence of SEQ ID NO: 67), and an L chain variable region gene consisting of a nucleotide sequence that is at least 80% or greater sequence identical to the nucleotide sequence of SEQ ID NO: 136 (gene, encoding an L chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 68), (8-2D) an antibody gene comprising an H chain variable region gene consisting of a nucleotide sequence that is at least 80% or greater sequence identity to the nucleotide sequence of SEQ ID NO: 139 (a gene encoding an H chain variable region consisting of an amino acid sequence that is at least 80% or greater sequence identity amino acid sequence SEQ ID NO: 77), and an L chain variable region gene consisting of a nucleotide sequence that is at least 80% or greater sequence identical to the nucleotide sequence SEQ ID NO: 140 (L chain variable region gene encoding consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 78), (9-2D) an antibody gene comprising an H chain variable region gene, consisting of a nucleotide sequence that is at least 80% or greater sequence identical to the nucleotide sequence of SEQ ID NO: 143 (a gene encoding an H chain variable region consisting of an amino acid sequence that is at least 80% or greater sequence identical to the amino acid sequence of SEQ ID NO: 87), and an L chain variable region gene consisting of a nucleotide sequence that is at least 80% or greater sequence identity to the nucleotide sequence of SEQ ID NO: 144 (a gene encoding an L chain variable region consisting of an amino acid sequence which is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 88), (10-2D) an antibody gene containing an H chain variable region gene consisting of a nucleotide sequence that is at least 80% or above is identical in sequence to the nucleotide sequence of SEQ ID NO: 147 (a gene encoding an H chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 97), and a gene, an L chain variable region consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 148 (a gene encoding an L chain variable region consisting of an amino acid sequence that is at least 80% identical % or more identical in sequence to the amino acid sequence of SEQ ID NO: 98), and (11-2D) an antibody gene comprising an H chain variable region gene consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to sequences of SEQ ID NO: 151 (a gene encoding an H chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 107), and a gene encoding an L chain variable region , consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 152 (a gene encoding an L chain variable region consisting of an amino acid sequence that is at least 80% or greater identical in amino acid sequence SEQ ID NO: 108).

In particular, examples of the antibody gene of the present invention may specifically include a (1-4D) antibody gene comprising an H chain gene consisting of a nucleotide sequence that is at least 80% or higher sequence identical to the nucleotide sequence of SEQ ID NO: 113 ( a gene encoding an H chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 9), and an L chain gene consisting of a nucleotide sequence that is at least 80% identical or higher in sequence identical to the nucleotide sequence of SEQ ID NO: 114 (a gene encoding an L chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 10), (2-4D ) an antibody gene containing an H chain gene consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 117 (a gene encoding an H chain consisting of an amino acid sequence that is at least 80% or greater sequence identity to the amino acid sequence of SEQ ID NO: 19), and an L chain gene consisting of a nucleotide sequence that is at least 80% or greater sequence identity to the nucleotide sequence

- 21 043515

SEQ ID NO: 118 (a gene encoding an L chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 20), (3-4D) an antibody gene containing an H gene -chain consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 121 (a gene encoding an H chain consisting of an amino acid sequence that is at least 80% or greater identical in amino acid sequence of SEQ ID NO: 29), and an L-chain gene consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 122 (a gene encoding an L-chain consisting of an amino acid sequence that is at least 80% or greater identical in sequence to the amino acid sequence of SEQ ID NO: 30), (4-4D) an antibody gene containing an H chain gene consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 125 (a gene encoding an H chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 39), and an L chain gene, consisting of a nucleotide sequence that is at least 80% or greater sequence identical to the nucleotide sequence of SEQ ID NO: 126 (a gene encoding an L chain consisting of an amino acid sequence that is at least 80% or greater sequence identical to the amino acid sequence SEQ ID NO: 40), (5-4D) an antibody gene containing an H-chain gene consisting of a nucleotide sequence that is at least 80% or greater sequence identical to the nucleotide sequence of SEQ ID NO: 129 (the gene encoding the H-chain a chain consisting of an amino acid sequence that is at least 80% or greater identical in sequence to the amino acid sequence of SEQ ID NO: 49), and an L chain gene consisting of a nucleotide sequence that is at least 80% or greater identical in sequence nucleotide sequence SEQ ID NO: 130 (a gene encoding an L chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence SEQ ID NO: 50), (6-4D) an antibody gene containing an H chain gene consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 133 (a gene encoding an H chain consisting of an amino acid sequence that is at least 80% or greater identical in sequence to the amino acid sequence of SEQ ID NO: 59), and an L chain gene consisting of a nucleotide sequence that is at least 80% or greater sequence identical to the nucleotide sequence of SEQ ID NO: 134 (a gene encoding an L chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 60), (7-4D) an antibody gene comprising an H chain gene consisting of a nucleotide sequence that is at least 80% identical or higher sequence identical to the nucleotide sequence of SEQ ID NO: 137 (a gene encoding an H chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 69), and the L- gene chain consisting of a nucleotide sequence that is at least 80% or more identical in sequence to the nucleotide sequence of SEQ ID NO: 138 (a gene encoding an L chain consisting of an amino acid sequence that is at least 80% or more identical in sequence amino acid sequence SEQ ID NO: 70), (8-4D) an antibody gene containing an H chain gene consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence SEQ ID NO: 141 (the gene encoding An H chain consisting of an amino acid sequence that is at least 80% or greater identical in sequence to the amino acid sequence of SEQ ID NO: 79), and an L chain gene consisting of a nucleotide sequence that is at least 80% or greater identical by nucleotide sequence SEQ ID NO: 142 (a gene encoding an L chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence SEQ ID NO: 80), (9-4D) antibody gene containing an H chain gene consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 145 (a gene encoding an H chain consisting of an amino acid sequence that is at least 80% identical or more identical in sequence to the amino acid sequence of SEQ ID NO: 89), and an L chain gene consisting of a nucleotide sequence that

- 22 043515 at least 80% or greater sequence identity to the nucleotide sequence

SEQ ID NO: 146 (gene encoding an L chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 90), (10-4D) antibody gene containing an H gene -chain consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 149 (a gene encoding an H chain consisting of an amino acid sequence that is at least 80% or greater identical in amino acid sequence of SEQ ID NO: 99), and an L chain gene consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 150 (a gene encoding an L chain consisting of an amino acid a sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 100), and (11-4D) an antibody gene comprising an H chain gene consisting of a nucleotide sequence that is at least 80% or above is identical in sequence to the nucleotide sequence of SEQ ID NO: 153 (a gene encoding an H chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 109), and an L chain gene , consisting of a nucleotide sequence that is at least 80% or greater identical in sequence to the nucleotide sequence of SEQ ID NO: 154 (a gene encoding an L chain consisting of an amino acid sequence that is at least 80% or greater identical in sequence to the amino acid sequence SEQ ID NO: 110).

The CAR gene of the present invention gene is not particularly limited as long as the gene (nucleotide) encodes the CAR of the present invention. Examples of a CAR gene may specifically include a (1-3D) CAR gene containing a gene encoding a single-chain antibody as described above in (13), or a codon degeneracy-based gene variant, (2-3D) a CAR gene containing a gene encoding a single-chain an antibody as described above in (23), or a codon degeneracy-based gene variant, (3-3D) a CAR gene containing a gene encoding a single-chain antibody, as described above in (33), or a codon degeneracy-based gene variant, ( 4-3D) a CAR gene containing a gene encoding a single-chain antibody, as described above in (43), or a gene variant based on codon degeneracy, (5-3D) a CAR gene containing a gene encoding a single-chain antibody, as described above in ( 53), or a codon degeneracy-based gene variant, (6-3D) a CAR gene containing a gene encoding a single-chain antibody as described in (b3) above, or a codon degeneracy-based gene variant, (7-3D) a CAR gene, containing a gene encoding a single chain antibody, as described above in (73), or a gene variant based on codon degeneracy, (8-3D) a CAR gene containing a gene encoding a single chain antibody, as described above in (83), or a gene variant on codon degeneracy-based, (9-3D) a CAR gene containing a gene encoding a single-chain antibody, as described above in (93), or a codon-degeneracy-based gene variant, (10-3D) a CAR gene containing a gene encoding a single-chain antibody, as described above in (103), or a codon degeneracy-based gene variant, (11-3D) a CAR gene containing a gene encoding a single chain antibody, as described above in (113), or a codon degeneracy-based gene variant, (1- 3'-1D) CAR gene containing a gene encoding a single chain antibody as described above in (13'-1), or a gene variant based on codon degeneracy, (1-3'-2D) CAR gene containing a gene encoding a single chain an antibody as described above in (13'-2), or a gene variant based on codon degeneracy, (1-3'-3D) a CAR gene containing a gene encoding a single chain antibody, as described above in (13'-3), or a codon degeneracy-based gene variant, (2-3'-1D) a CAR gene containing a gene encoding a single chain antibody as described above in (23'-1), or a codon degeneracy-based gene variant, (2-3' -2D) a CAR gene containing a gene encoding a single chain antibody as described above in (23'-2), or a variant of the gene based on codon degeneracy, (2-3'-3D) a CAR gene containing a gene encoding a single chain antibody, as described above in (23'-3), or a gene variant based on codon degeneracy, and (2-3'-4D) a CAR gene containing a gene encoding a single chain antibody, as described above in (23'-4), or gene variant based on codon degeneracy.

As used herein, the phrase at least 80% identical or greater means that the identity is 80% or greater, preferably 85% or greater, more preferably 88% or

- 23 043515 higher, even more preferably 90% or higher, more preferably 93% or higher, particularly preferably 95% or higher, particularly more preferably 98% or higher, most preferably 100%.

As used herein, the term identity refers to the degree of similarity of polypeptide or polynucleotide sequences (this degree is determined by comparing the searched sequence with another sequence, preferably of the same type (nucleic acid or protein sequence)). Examples of a preferred computer program method for scoring and determining 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: 3389-3402; and Devereux et al., Nucleic 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: 24442448), and GCG GelMerge, which identifies and aligns pairs 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).

As used herein, an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: X, in other words, is an amino acid sequence derived from the amino acid sequence of SEQ ID NO: X by deletion, substitution, insertion and/or the addition of 0, 1 or more amino acid residues and has functions equivalent to those of the amino acid sequence of SEQ ID NO: X. In this context, an amino acid sequence obtained by deletion, substitution, insertion and/or addition of 1 or more amino acid residues means an amino acid sequence wherein amino acid residues have been deleted, substituted, inserted and/or added, for example, within 1-30 residues, preferably within 1-20 residues, more preferably within 1-15 residues, even more preferably within 1-10 residues, even more preferably in the range of 1-5 residues, even more preferably in the range of 1-3 residues, even more preferably in the range of 1 or 2 residues. Mutational processing of these amino acid residues can be performed by any method known to those skilled in the art, such as chemical synthesis, genetic engineering approach or mutagenesis.

The promoter in the vector of the present invention can be any region that drives transcription of the mRNA encoded by the antibody gene of the present invention and located downstream of the promoter. A promoter typically contains a transcription start site (TSS).

The type of promoter or vector in the vector of the present invention may be suitably selected according to the type of host cell (or host organism) into which the vector of the present invention is introduced.

A host cell may express an antibody of the present invention by transcribing the antibody gene of the present invention, or may express a CAR of the present invention by transcribing the mRNA of the CAR gene of the present invention. In the case of introducing a vector containing an antibody of the present invention, yeast, mammalian cells, insect cells or plant cells below can be used as the host cell as the vector of the present invention. In the case of introducing a vector containing the CAR gene of the present invention as the vector of the present invention, an immunocompetent cell can be used as the host cell.

In the case of using yeast (eg, Saccharomyces cerevisiae and Schizosaccharomyces pombe) as the host cell, examples of the vector may actually include a vector such as YEP13 (ATCC37115), YEp24 (ATCC37051) and YCp50 (ATCC37419), and any vector derived from this vector.

Examples of the promoter may include a glycolysis gene promoter (eg, a hexosine kinase gene), a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, a gal1 promoter, a gal10 promoter, a heat shock protein promoter, an MFa1 promoter, and a CUP1 promoter.

In the case of using a mammalian cell (for example, a Namalwa cell isolated from a human, a COS cell isolated from a monkey, a Chinese hamster ovary CHO cell, and a human or mouse T cell) as the host cell, and in the case of using a vector containing the gene antibodies as the vector of the present invention, examples of the vector of the present invention may include a vector such as pcDNAI, pcDM8 (manufactured by Funakoshi Co., Ltd.), pAGE107 (Unexamined Japanese Patent Application Publication No. 3-22979; and Cytotechnology, 3, 133 ((1990)), pAS3-3 (Unexamined Japanese Patent Application Publication No. 2-227075), pCDM8 (Nature, 329, 840, (1987)), pcDNAI/Amp (produced by Invitrogen Corp.), pREP4 (manufactured by Invitrogen Corp.), pAGE103 (J. Biochemistry, 101, 1307 (1987)) and pAGE210, and any vector derived from this vector. On the other hand, in the case of using a mammalian cell (for example, human immunocompetent cell described above) as a host cell and using a vector containing a CAR gene as a vector of the present invention, examples of the vector of the present invention may include a retroviral vector such as pMSGV vector (Tamada k et

- 24 043515 al., Clin Cancer Res 18: 6436-6445 (2002)) and the pMSCV vector (manufactured by Takara Bio Inc.) and any vector derived from this vector.

Examples of the promoter in the vector of the present invention may include cytomegalovirus (CMV) IE gene promoter (immediate early), SV40 early promoter, retroviral promoter, metallothionein promoter, heat shock promoter, SRa promoter, NFAT promoter and HIF promoter.

In the case of using an insect cell (for example, Sf9 cells and Sf21 cells, which are the ovary cells of Spodoptera frugiperda, and the High5 cell, which is the ovarian cell of Trichoplusia ni) as the host cell, examples of the vector of the present invention may include a transfer vector for use in methods for producing recombinant baculovirus, in particular a vector such as pVL1392, pVL1393 and pBlueBacIII (all manufactured by Invitrogen Corp.), and any vector derived from this vector. Examples of the promoter may include a polyhedrin promoter and a p10 promoter.

In case of using a plant cell (for example, tobacco, potato, tomato, carrot, soybean, canola, alfalfa, rice, wheat and barley cells) as the host cell, examples of the expression vector may include a vector such as Ti vector and tobacco virus vector mosaics, and any vector obtained from this vector. Examples of the promoter may include the cauliflower mosaic virus (CaMV) 35S promoter and the rice actin 1 promoter.

The vector of the present invention preferably also contains enhancer region and ribosome binding site (RBS) nucleotide sequences to further enhance the efficiency of gene expression and also contains a drug resistance gene (eg, spectinomycin resistance gene, chloramphenicol resistance gene, tetracycline resistance gene). , kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, hygromycin resistance gene, blastidin resistance gene and geneticin resistance gene) suitable for the host cell type for screening for the host cell of the present invention. The enhancer region is typically located upstream of a promoter, and the RBS is typically located between the promoter and the gene of the present invention. The nucleotide sequence of the antibody gene of the present invention, which is introduced into the vector of the present invention, may in fact be subjected to codon sequence optimization depending on the host cell for expression. The vector of the present invention can be produced by a method known in the art using gene recombination techniques.

The host cell of the present invention can be obtained by introducing the vector of the present invention into a host cell (host cell transfection) by a method suitable for the type of host cell.

In the case of using the yeast described above as the host cell, the method of introducing the vector of the present invention into the yeast may be any method of introducing DNA into the yeast. Examples include a method such as electroporation (Methods Enzymol., 194, 182 (1990)), the spheroplast method (Proc. Natl. Acad. Sci. U.S.A., 84, 1929 (1978)), and the lithium acetate method (J. Bacteriology , 153, 163 (1983).

In the case of using the mammalian cells described above as the host cell, the method of introducing the vector of the present invention into mammalian cells may be any method of introducing DNA into mammalian cells. Examples include a method such as electroporation (Cytotechnology, 3, 133 (1990)), the calcium phosphate method (Pending Japanese Patent Application Publication No. 2227075), lipofection (Proc. Natl. Acad. Sci. U.S.A., 84, 7413 (1987)) and the method of viral infection as above. Examples of such a viral infection method may include a method that involves transfecting 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), a CAR expression vector (International Publication WO 2016/056228) and a packaging plasmid to produce a recombinant virus, and infecting the T cell with the recombinant virus as above.

In the case of using the insect cells described above as a host cell, examples of the method of introducing the vector of the present invention into insect cells include a method that includes cotransfecting insect cells with the vector of the present invention (transfer vector) and a baculovirus-derived genomic DNA to produce recombinant baculovirus according to the method described in Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, W.H. Freeman and Company, New York (1992), Bio/Technology, 6, 47 (1988), and the like. Examples of such a cotransfection method may include a method such as the calcium phosphate method (pending Japanese Patent Application Publication No. 2-227075) and lipofection (Proc. Natl. Acad. Sci. U.S.A. 84, 7413 (1987).

In the case of using the plant cells described above as the host cell, examples of the method of introducing the vector of the present invention into plant cells may include a method such as the method using Agrobacterium (Japanese Patent Application Publication No. 59140885 and 50-70080, filed pending; and Cytotechnology, 3, 133 ((1990)), pAS3-3 (Pending Japanese Patent Application Publication No. 60-251887) and method using

- 25 043515 particle gun (gene gun) (Japanese patents No. 2606856 and 2517813).

The antibody of the present invention can be obtained by culturing a host cell of the present invention obtained by the above method in a culture solution suitable for the host cell.

A transgenic animal such as a mouse, cattle, goat, sheep, chicken or pig into which the antibody gene of the present invention has been introduced (vector of the present invention) is produced using a transgenic animal preparation technique, and an antibody produced from the antibody gene according to the present number, can also be obtained in large quantities from the blood, milk or the like of a transgenic animal.

Non-human animals (eg, mice and rats) are immunized with a substance containing a human GPC3 polypeptide consisting of the amino acid sequence SEQ ID NO: 155 (GPC3 polypeptide antigen). A phage library of scFv genes is obtained by the phage display method. The scFv of the present invention can be produced by a biopanning method using a GPC3 polypeptide antigen and/or a cell line expressing the GPC3 polypeptide antigen (preferably a cell line not expressing endogenous GPC3), and further preferably a competitive C-terminal GPC3 polypeptide consisting from the amino acid sequence SEQ ID NO: 159. Antibody-producing hybridomas are prepared from non-human animals immunized with the antigen using a cell fusion method. A culture supernatant containing the antibody of the present invention can also be obtained by ELISA screening using a plate in which the antigen is immobilized on a solid phase. The antibody of the present invention can be separated and purified from the culture supernatant using an antibody purification method known in the art.

The detection method of the present invention may be any method including the step of detecting GPC3 located on a cell membrane (cell membrane anchored) in a sample (eg, blood, tissue and urine) using an antibody of the present invention. Specific examples of the detection method may include immunofluorescence staining, Western blotting and ELISA using the antibody of the present invention.

The detection kit of the present invention is a kit containing the antibody of the present invention or a labeled form thereof, and is limited to the purpose of detecting GPC3. The kit typically contains the components typically used in a kit of this type, such as carrier, pH buffer and stabilizer, as well as an accompanying document such as a manual and GPC3 detection instructions.

The species of GPC3 organisms to be detected in the detection method of the present invention may be a non-human animal such as a mouse or a rat, and generally a human.

Examples of a label for obtaining a labeled form of the antibody of the present invention may include: an enzyme such as peroxidase (for example, horseradish peroxidase [HRP]), alkaline phosphatase, βD-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apo-glucose oxidase, urease, luciferase and acetylcholinesterase; fluorescent material such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride and tetramethylrhodamine isothiocyanate; fluorescence protein such as green fluorescence protein (GFP), cyan fluorescence protein (CFP), blue fluorescence protein (BFP), yellow fluorescence protein (YFP), red fluorescence protein (RFP) and luciferase; a radioisotope such as ³ H, ¹⁴ C, ¹²⁵ I and ¹³¹ I; biotin; avidin; and a chemiluminescent agent.

References, such as scientific literature, patents and patent applications cited herein, are incorporated herein by reference in their entirety to the same extent as if each individual reference were specifically described. This application claims priority to Japanese Patent Application No. 2017-001732 (filed January 10, 2017), the contents of which are incorporated herein by reference in their entirety.

In the present description, the present invention will be described in more detail with reference to examples. However, the technical scope of the present invention is not limited to these examples.

Examples

Example 1. Production of a new anti-GPC3 antibody recognizing the N-terminal polypeptide of human GPC3.

Protocol.

SKG/Jcl mice were used as immunization animals to produce anti-human GPC3 antibodies, and full-length human GPC3 protein was used as the immunizing antigen. The SKG/Jcl mouse is a mouse model of autoimmune disease that spontaneously develops rheumatoid arthritis and is known to produce antibodies in response to even its own components depending on age or environmental conditions. Meanwhile, GPC3 is highly homologous in humans and mice and is generally less likely to induce antibodies even when immunized in normal mice. Therefore, as animals for

- 26 043515 immunization used SKG/Jcl mice. The scFv phage library was prepared from cDNA derived from GPC3-immunized mouse B cells, and human anti-GPC3 antibody was isolated by phage display.

Although antiserum from immunized mice contains many types of antibodies, it is necessary to select for mice that produce antibodies that are specific for the N-terminal GPC3 polypeptide by excluding mice that produce antibodies with low specificity for GPC3 or antibodies that recognize the C-terminal GPC3 polypeptide. Accordingly, mice that produced antibodies specifically binding to the N-terminal polypeptide of GPC3 were selected by ELISA and FCM. Specifically, cDNA was synthesized by reverse transcription from total RNA of B cells obtained from immunized mice, and antibody genes were amplified to generate an antibody gene library. A scFv phage library was constructed from the antibody gene library, and E. coli cells were infected with the library to express the scFv, and then biopanned with recombinant GPC3, a GPC3-expressing cell line, and a GPC3 C-terminal polypeptide to enrich for phages expressing the target scFv. , that is, antibodies against the N-terminal polypeptide of GPC3. To further analyze the resulting scFv for specificity of binding to GPC3 in cells, that is, GPC3 localized to (bound to) the cell membrane (membrane-bound GPC3) via a GPI (glycosylphosphatidylinositol) anchor, was tested using cellular ELISA and FCM. In addition, the nucleotide sequences of the H chain and L chain variable regions of the clones having binding specificity were sequenced, and based on these sequences, the nucleotide sequences of anti-GPC3 antibodies produced by B cells of immunized mice were determined. Finally, a mammalian display method, which involves expression of the N-terminal polypeptide fragment and the C-terminal polypeptide fragment of GPC3 on the cell surface, was used to confirm that the epitope for the scFv is the N-terminal polypeptide fragment of GPC3. A detailed description of the methods and results will be presented herein.

1-1. Materials and methods.

Cell culture.

The JHH7 cell line, the HepG2 cell line, and the SK-Hep-1 cell line activated to express full-length human GPC3 (also referred to as a GPC3-expressing cell line in the description) were used as GPC3-expressing cells to perform biopanning and anti-GPC3 screening. GPC3 antibodies. The JHH7 cell line is a GPC3-expressing cell line derived from hepatocellular carcinoma, and its cells constitutively express GPC3 associated with the cell membrane (membrane-bound GPC3) via a GPI (glycosylphosphatidylinositol) anchor. On the other hand, the HepG2 cell line is a GPC3-expressing hepatocellular carcinoma cell line, like the JHH7 cell line, but is a cell line in which the expression of secretory GPC3, not associated with the cell membrane, predominates over the expression of membrane-bound GPC3. The Sk-Hep-1 cell line is a hepatocellular carcinoma cell line that does not express GPC3. Therefore, a cell line expressing only full-length membrane-bound GPC3 or exon-deficient partial-length membrane-bound GPC3 can be generated by forced expression.

Cultivation of 4 types of cell lines (JHH7 cell line, HepG2 cell line, cell line expressing GPC3, and 293T cell line obtained from human embryonic kidney epithelium) was carried out under conditions of 37°C and 5% CO ₂ in a DMEM culture (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.) (simply referred to as DMEM culture solution). CHO-K1 cell line was cultivated under conditions of 37°C and 5% CO ₂ in Ham's F12 culture solution (manufactured by Sigma-Aldrich Co. LLC) containing 10% FBS (manufactured by Gibco/Thermo Fisher Scientific Inc.).

Immunizing antigen.

C-terminal 6xHis-tagged recombinant GPC3 (manufactured by R&D Systems Inc.) was adjusted to 0.1 mg/ml with PBS and mixed with artificial adjuvant TiterMax Gold (manufactured by TiterMax USA, Inc.) or CFA (Freund's complete adjuvant) (F5881 , manufactured by Sigma-Aldrich Co. LLC) in equal quantities to obtain an emulsion, which was then used as the original immunizing antigen. Recombinant GPC3 was adjusted to a concentration of 10-100 μg/ml with PBS and used as a second or later immunizing antigen.

Generation of GPC3-expressing cell line.

The gene encoding the full-length human GPC3 consisting of SEQ ID NO: 157 (the full-length human GPC3 gene consisting of the nucleotide sequence of SEQ ID NO: 160) was inserted into the pcDNA3.1 vector (manufactured by Thermo Fisher Scientific Inc.) to obtain the GPC3 expression vector . The SK-Hep-1 cell line was transfected with the GPC3 expression vector in a standard manner and then cultured in DMEM culture solution containing G418 (manufactured by Roche Diagnos- 27 043515 tics KK) to obtain the SK-Hep-1 cell line stably expressing full-length GPC3 (GPC3 -expressing cell line).

Mouse immunization.

SKG/Jcl mice (CLEA Japan, Inc., 8-week female, SPF) were used as immunization animals and were immunized with recombinant GPC3 for a total of 4 times at 1-week intervals in the footpad. 5 weeks after the start of immunization, blood was collected and serum prepared according to a standard method and used as a sample to confirm antibody titer.

Serum antibody titer in antiserum using ELISA.

To confirm the response of immunized mice with anti-GPC3 antibody production, serum antibody titer was measured using an enzyme-linked immunosorbent ELISA. 0.5 or 2 μg/ml recombinant GPC3 was added to a 96-well microplate (manufactured by Nalge Nunc International) in an amount of 50 μl/well, 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 for blocking. Serum from GPC3-immunized mice was serially diluted 100- to 160-fold in 0.1% Block ACE/PBS solution. Each diluted serum sample was added at 50 μl/well, and the plate was incubated at room temperature for 2 hours to allow the antigen-antibody reaction to occur. After the wells were washed with PBS containing Tween 20 (PBST), goat anti-mouse IgG (manufactured by Jackson ImmunoResearch Laboratories Inc.) conjugated to 2 μg/ml peroxidase was added and the plate was incubated at room temperature for 2 hours for a second antibody reaction. . After washing the well five times with PBST, the moisture was removed and TMB substrate (manufactured by Thermo Fisher Scientific Inc.) was added at 50 μl/well for the staining reaction. After 15 minutes, the staining reaction was stopped by adding 0.18 M sulfuric acid at 50 μl/well, and then the absorbance was measured at 450 nm and 540 nm using a plate reader (manufactured by Bio-Rad Laboratories, Inc.). Quantification was performed using the corrected value obtained by subtracting the value measured at 540 nm from the value measured at 450 nm.

Antibody specificity in antiserum using FCM.

To further confirm the specific binding of antiserum against membrane-bound GPC3 in immunized mice, mouse serum was diluted 100-fold and 5x105 GPC3-expressing cell line cells were mixed and incubated for 30 minutes on ice. FACS buffer (1% BSA/PBS solution) was added to the solution, and the mixture was centrifuged to remove the supernatant. Next, 100 μl of 1 μg/ml goat anti-mouse IgG (H+L) Alexa Fluor 488 (manufactured by Thermo Fisher Scientific Inc.) was added as a second antibody, and the mixture was incubated for 30 minutes on ice to allow the second antibody reaction to occur.

Alexa Fluor 488 detection and fluorescence level measurements were performed using a flow cytometer (FACSCanto) (manufactured by BD Biosciences).

Preparation of scFv phage library.

Total RNA obtained from B cells was extracted using standard techniques for mice that have been shown to produce antibody binding to membrane-bound GPC3 using the method described above in the [Flow Cytometer] section. RT-PCR was carried out with total RNA as a template according to the standard protocol for cDNA preparation. The H chain and L chain variable region genes of the antibody were amplified by PCR. The nucleotide sequence encoding the fusion protein scFv with the variable regions of the H chain and L chain connected by a flexible linker, and the g3p coat protein (cp3) of fibrous bacteriophage M13 was inserted into the multicloning site of the phagemidine vector pTZ19R to obtain the scFv expression vector. The size of the scFv library was calculated based on the transformation efficiency of E. coli strain DH12S (manufactured by Invitrogen Corp.). The transformed strain DH12S was infected with helper phage M13KO7 (manufactured by Invitrogen Corp.) to obtain a phage library expressing scFv.

Biopanning and cloning of scFv phage.

Phage scFv was biopanned with a combination of recombinant GPC3 immobilized on Dynabeads His-Tag Isolation & Pulldown magnetic beads (manufactured by VERITAS Corp.) with a 6xHis tag, and a GPC3-expressing cell line as bait, according to the method described in J Mol Biol. 1991 Dec 5; 222 (3): 581-97, J Med Virol. 2007 Jun; 79 (6): 852-62, Proc Natl Acad Sci USA. 2008 May 20; 105 (20): 7287-92, or JOURNAL OF VIROLOGY, Apr. 2004, p. 3325-3332 Vol. 78, No. 7. In each round (stage) of biopanning, consisting of 5 types of series (series AE) (see Fig. 1), an aliquot of polyclonal phage antibodies was selected. To confirm the binding specificity of the scFv, a captive antigen ELISA was performed using the method described above in [Serum antibody titer using ELISA] (method using E. coli culture supernatant containing phage instead of serum), and a cell-based ELISA was performed using the method described below in [ScFv Screening by Cell-Based ELISA]. Each biopanning step was designed without selection to bind the phage scFv to the same part as the C-terminal epitope

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GPC3 recognized by existing antibodies by pre-binding existing aHTu-GPC3 antibodies GC33 (manufactured by Chugai Pharmaceutical Co., Ltd.) and GC199 (manufactured by Chugai Pharmaceutical Co., Ltd.) for bait. Specifically, this competitive method allows for the selective biopanning of a new antibody that recognizes a GPC3 epitope that is distinct from the epitopes of existing anti-GPC3 antibodies. DH12S E. coli were transformed with biopanning-enriched phages and inoculated into LB agarose medium to separate into individual colonies. E. coli was then cultured in small scale LB liquid medium and plasmids were then extracted and purified. Purified plasmids were subjected to DNA sequencing to determine the nucleotide sequences of the scFv H chain and L chain variable regions.

ScFv screening using FCM.

100 µl of the culture supernatant in which the scFv phages were secreted was added to a GPC3-expressing cell line (5x105 cells per sample) and mixed, and then the mixture was incubated for 30 minutes on ice. FACS buffer (1% BSA/PBS solution) was added to the mixture, and the mixture was centrifuged to remove the supernatant. Then, 1 μg/ml anti-mouse antibody-Alexa 488 (manufactured by Thermo Fisher Scientific Inc.) was added as a secondary antibody, and the mixture was incubated for 30 minutes on ice. Fluorescence staining of the cells was then measured using a flow cytometer (FACSCanto, manufactured by BD Biosciences).

Screening of scFvs by cell-based ELISA.

After removing the DMEM culture solution from the 96-well microplate, each well containing 2x105 GPC3-expressing cells, 2% BSA-PBS was added to prevent nonspecific scFv binding to the cells or plate, and the plate was incubated for 30 minutes on ice. . Then, 100 μl of E. coli culture supernatant in which the 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 antibody (manufactured by Medical & Biological Laboratories Co., Ltd.) against cp3 fused to the C-terminal side of the scFv was added at 100 μl per well, and the plate was incubated for an additional 45 minutes on ice. HRP-labeled anti-rabbit IgG antibody (manufactured by Medical & Biological Laboratories Co., Ltd.), 500-fold diluted, was added at 100 μl/well as a third antibody to detect anti-cp3 antibody, and the plate was incubated for 45 min on ice. . Then, o-phenylenediamine (OPD) and hydrogen peroxide were added as HRP substrates for the coloring reaction. Quantification was performed using the numerical value obtained by subtracting the absorbance at 620 nm as background from the absorbance at 492 nm. If a cellular ELISA was performed using an antibody already converted to an IgG antibody rather than a scFv, an HRP-labeled anti-mouse IgG antibody (manufactured by Medical & Biological Laboratories Co., Ltd.) was used at a 2000-fold dilution as the secondary antibody for detecting an IgG antibody instead of an anti-cp3 antibody and an HRP-labeled anti-rabbit IgG antibody, under the conditions described above.

Determination of scFv variable region gene sequences.

The variable region gene sequences of the phage scFv binding to membrane-bound GPC3 were decoded on a sequencer (CEQ2000XL, manufactured by Beckman Coulter, Inc.) using primer T7 (primer consisting of the nucleotide sequence SEQ ID NO: 176), which is a universal primer, and primer cp3R (a primer consisting of the nucleotide sequence of SEQ ID NO: 177) as a forward primer for decoding the H chain V region (VH) and a reverse primer for decoding the V region (VL) of the L chain, respectively.

Generation of cell line for antibody epitope mapping.

The mammalian display method was used to identify the epitope of the cloned scFv. A gene consisting of exons 1 to 7 of human GPC3 and encoding an N-terminal fragment of GPC3 (a polypeptide consisting of the amino acid sequence of SEQ ID NO: 155), and a gene consisting of exons 8 and 9 of human GPC3 and encoding a C-terminal fragment of GPC3 ( a polypeptide consisting of the amino acid sequence SEQ ID NO: 156) was amplified by PCR and each was inserted into the multicloning site (MSC) of the expression vector pDisplay (manufactured by Thermo Fisher Scientific Inc.). The expression vector pDisplay is an expression vector capable of condensing the transmembrane domain of platelet-derived growth factor receptor (PDGFR) and the C terminus of a target protein and presenting the fusion construct on the cell surface of arbitrary mammalian cells. In addition, the expression vector pDisplay is designed to add an HA tag to the N-terminus of the target protein and a myc tag to the C-terminus of PDGFR. Gene transfer was carried out using the pDisplay expression vector to express the N-terminal fragment of GPC3 or the C-terminal fragment of GPC3 into the SK-Hep-1 cell line or the 293T cell line, and a cell line expressing the N-terminal fragment of GPC3 or the C-terminal fragment of GPC3 cell surface (a cell line expressing the N-terminal fragment of GPC3 and a cell line expressing the C-terminal fragment of GPC3) were isolated and used for scFv epitope mapping.

Antibody epitope mapping using FCM.

A cell line expressing the N-terminal fragment of GPC3, a cell line expressing the C-terminal fragment of GPC3, and a GPC3-expressing cell line (in each case

5x105 cells per sample) were mixed with 100 μl of culture supernatant in which the scFv phages were secreted, and the mixture was incubated for 30 min on ice. Buffer was added to the mixture

FACS (1% BSA/PBS solution) and the mixture was centrifuged and washed. Then, 1 μg/ml anti-mouse antibody-Alexa 488 (manufactured by Thermo Fisher Scientific Inc.) was added as a secondary antibody, and the mixture was incubated for 30 min on ice. Fluorescence staining of the cells was then measured using a flow cytometer (FACSCanto, manufactured by BD Biosciences).

Construction of a recombinant IgG expression vector.

The expression vector of the Mammalian PowerExpress system (manufactured by Toyobo Co., Ltd.) was used to convert scFv to IgG. The nucleotide sequence encoding the fusion protein of the variable region of the H chain scFv and the constant region of the H chain of mouse IgG2a was inserted into MSCs of the vector pEN1.1 (pEN1.1-H). In addition, a nucleotide sequence encoding a fusion protein of the variable region of the L chain of scFv and the constant region of the L chain of mouse IgG2a was inserted into MSCs of the pELX2.2 vector (pEH2.2-L). Then, the polynucleotide fragment of the promoter, starting from EF1a to the L chain gene, was excised from pEH2.2-L using restriction enzymes (BglII and SalI) and ligated into pEN1.1-H, treated with restriction enzymes (BglII and SalI) to obtain the vector for simultaneous expression of the H chain and L chain of the antibody.

Expression of recombinant IgG.

32.6 μg of an antibody H chain and L chain coexpression vector prepared as described above in [Construction of a Recombinant IgG Expression Vector] was diluted in 1.6 ml opti-MEM (manufactured by Gibco/Thermo Fisher Scientific Inc.) and mixed with 65 μl of 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. The mixture was then mixed with CHO-K1 cells (1x10 ⁷ cells) suspended in 10 ml DMEM culture solution and then cultured. After 4 hours, serum-free medium (Free Style expression CHO medium [manufactured by Gibco/Thermo Fisher Scientific Inc.]) was added to the mixture, and the mixture was further cultured for 4-6 days to recover the culture supernatant containing the recombinant antibody.

Affinity purification of antibodies.

The empty column (manufactured by Bio-Rad Laboratories, Inc.) was filled with Protein G Sepharose 4 Fast Flow (manufactured by GE Healthcare Japan Corp.) or Bipo Resin Protein L (manufactured by Protein Express) with a bed volume of 1 ml. The column polymer was then washed with 10 times the bed volume of PBS. Culture supernatant, filtered through a 0.22 micron filter, was added to the column so that the antibody was captured by protein G or protein L within the column. The column was then washed with 10 times the bed volume of PBS to wash away nonspecifically adsorbed contaminants. The antibody was eluted using 100 mM glycine-HCl (pH 2.7), and the pH of the eluate was neutralized with 1 M Tris-HCl (pH 8.5). The absorbance was measured at 280 nm using a nanoDrop absorbance meter (manufactured by Thermo Fisher Scientific Inc.) and the antibody concentration was calculated. Expression vectors were designed and produced in the same manner as above for antibody GC33 and antibody GC199 used as competition antibodies.

1-2 Results.

Evaluation of immunized mouse antiserum.

Blood was collected from SKG/Jcl mice immunized four times with recombinant GPC3, and serum anti-GPC3 antibody production was confirmed. As a result, an antibody with binding activity against GPC3 was detected in ELIS experiments with recombinant GPC3 and FCM on GPC3-expressing cells. Two mice with particularly high antibody titres (numbered 1413 #2 and 1413 #3) among the mice were used as sources to generate the antibody library.

Phage library construction.

The number of scFv library members calculated by transformation efficiency was 5.8 x 10 ⁷ for mouse 1413 # 2 and 4.3 x 10 8 for mouse 1413 # 3. The immunoglobulin library obtained in this example was a library obtained from mice that had production of antibodies in response to the target antigen was detected during immunization with the GPC3 antigen. Therefore, a feature of this library is the high probability of containing the target antibody gene, even considering the small size of the library. Another advantage of the library is the content of an antibody that forms the correct conformation in vivo compared to a random library of synthetic antibodies.

Clone classification by monoclonal scFv sequence analysis.

DNA sequence analysis of the isolated monoclonal scFv was performed to classify the clones, excluding overlap. As a result, the candidate clones were identified as 7 types of the D series of the mouse 1413 #2 library, 5 types of the E series of the same library, 3 types of the D series of the mouse 1413 #3 library, and 9 types of the E series of the same library. The nucleotide sequences of the heavy chain and light chain variable regions of these candidate clones were analyzed to exclude overlapping identical clones. As a result, a total of 18 scFv clone types were identified, that is, 9 scFv clone types obtained from the mouse 1413 #2 library and 9 scFv clone types obtained from the mouse 1413 #3 library.

[Anti-GPC3 scFc clone epitope mapping analysis] The 18 types of scFv clones identified by the method described above in [Classification of clones by monoclonal scFv sequence analysis] were used to analyze binding to each GPC3 by FCM using 3 types of cell lines (cell a line expressing the N-terminal fragment of GPC3, a cell line expressing the C-terminal fragment of GPC3, and a cell line expressing GPC3). As a result, among the 18 types of scFv clones, there are 14 types (TF1413-02d028, 02d030, 02d039, 02e004, 02e014, 02e030, 02e040, 03e001, 03e004, 03e005, 03e015, 03e019, 03 e027 and 03e034) bind to full-length GPC3 and the N-terminal fragment of GPC3 (polypeptide consisting of the amino acid sequence SEQ ID NO: 155), but did not bind to the C-terminal fragment of GPC3 (polypeptide consisting of the amino acid sequence SEQ ID NO: 156) (see Fig. 2). On the other hand, 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 C-terminal fragment of GPC3, but did not bind to the N-terminal fragment GPC3.

The results identified 14 types of new scFv clones described above, recognizing an N-terminal GPC3 epitope different from the C-terminal GPC3 epitope for existing anti-GPC3 antibodies (GC33 and GC199).

From the 14 types of scFv clones identified in this way, the 11 best scFv clones were selected (TF1413-02d028, 02d039, 02e004, 02e014, 02e030, 02e040, 03e001, 03e004, 03e005, 03e015 and 03e03 4), which have a particularly high binding force. In table 1 shows the SEQ ID NO V region of the H chain and L chain of these 11 types of scFv clones. In table 2 shows the correspondence of SEQ ID NO CDR1CDR3 to the H chain of these 11 types of scFv clones. In table 3 shows the correspondence of SEQ ID NO CDR1-CDR3 L chain of these 11 types of scFv clones.

Table 1

ScFv clone name and V-regions 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 TF1413-03e005 L chain V region 88 TF1413-03e015 L chain V region 98 TF1413-03e034 L chain V region 108

- 31 043515

table 2

Clone name and CDR SEQ ID NO TF1413-02d028 CDR1 H-chain 1 CDR2 H-chain 2 CDR3 H chains 3 TF1413-02d039 CDR1 H-chain eleven CDR2 H-chain 12 CDR3 H chains 13 TF1413-02e004 CDR1 H-chain 21 CDR2 H-chain 22 CDR3 H chains 23 TF1413-02e014 CDR1 H-chain 31 CDR2 H-chain 32 CDR3 H chains 33 TF1413-02e030 CDR1 H-chain 41 CDR2 H-chain 42 CDR3 H chains 43 TF1413-02e040 CDR1 H-chain 51 CDR2 H-chain 52 CDR3 H chains 53 TF1413-03e001 CDR1 H-chain 61 CDR2 H-chain 62 CDR3 H chains 63 TF1413-03e004 CDR1 H-chain 71 CDR2 H-chain 72 CDR3 H chains 73 TF1413-03e005 CDR1 H-chain 81 CDR2 H-chain 82 CDR3 H chains 83 TF1413-03e015 CDR1 H-chain 91 CDR2 H-chain 92 CDR3 H chains 93 TF1413-03e034 CDR1 H-chain 101 CDR2 H-chain 102 CDR3 H chains 103

- 32 043515

Table 3

Clone name and CDR SEQ ID NO TF1413-02d028 CDR1 L-chain 4 CDR2 L-chains 5 CDR3 L-chains 6 TF1413-02d039 CDR1 L-chain 14 CDR2 L-chains 15 CDR3 L-chains 16 TF1413-02e004 CDR1 L-chain 24 CDR2 L-chains 25 CDR3 L-chains 26 TF1413-02e014 CDR1 L-chain 34 CDR2 L-chains 35 CDR3 L-chains 36 TF1413-02e030 CDR1 L-chain 44 CDR2 L-chains 45 CDR3 L-chains 46 TF1413-02e040 CDR1 L-chain 54 CDR2 L-chains 55 CDR3 L-chains 56 TF1413-03e001 CDR1 L-chain 64 CDR2 L-chains 65 CDR3 L-chains 66 TF1413-03e004 CDR1 L-chain 74 CDR2 L-chains 75 CDR3 L-chains 76 TF1413-03e005 CDR1 L-chain 84 CDR2 L-chains 85 CDR3 L-chains 86 TF1413-03e015 CDR1 L-chain 94 CDR2 L-chains 95 CDR3 L-chains 96 TF1413-03e034 CDR1 L-chain 104 CDR2 L-chains 105 CDR3 L-chains 106

Conversion of scFv aHmu-GPC3 antibody to IgG and its binding ability.

The H chain and L chain variable regions of 11 scFv clone types selected as described above were linked to mouse IgG constant regions, and full-length recombinant antibodies were expressed using a recombinant IgG expression vector and affinity purified. The ability of these IgG antibodies to bind to the N-terminal fragment of GPC3 was analyzed using a cell line expressing the N-terminal fragment of GPC3. According to the results, 9 types of IgG clones (TF1413-02d028, 02d039, 02e004, 02e014, 02e030, 02e040, 03e004, 03e005 and 03e034) retained binding activity against the N-terminal fragment of GPC3, while the remaining two types of IgG clones (TF1413-03 e001 and 03e015) there was no binding activity against the N-terminal fragment of GPC3 (see Fig. 3). The 9 types of IgG clones described above did not bind to the C-terminal fragment of GPC3 (see Fig. 3).

These results indicate that among the 11 types of scFv clones, 9 types (TF1413-02d028, 02d039, 02e004, 02e014, 02e030, 02e040, 03e004, 03e005, and 03e034) can be converted to the IgG type. In table 3 shows the correspondence of SEQ ID NO CDR1-CDR3 of the H chains and L chain of these 11 types of IgG clones.

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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 thirty TF1413-02e014 L-chain 40 TF1413-02e030 L-chain 50 TF1413-02e040 L-chain 60 TF1413-03e001 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 activity of a novel aHTU-GPC3 anti-GPC3 antibody treated with EDTA (ethylenediaminetetraacetic acid), trypsin or collagenase.

Preparation of cells treated with EDTA or trypsin.

The SK-Hep-1 cell line activated 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 PBS.

3 ml of 0.02% EDTA/PBS (herein referred to simply as EDTA) or 0.05% trypsin solution (herein referred to simply as trypsin) was then added to each flask. Each flask was incubated at 37°C for 5 minutes (EDTA) or 2 minutes and 30 seconds (trypsin) to remove cells from the flask. Then 7 ml of DMEM culture solution was added to each flask. After pipetting, the cell suspension was collected into each 50 mL conical tube. Each flask was then washed with 10 ml of DMEM culture solution. The resulting washes were then also returned to the 50 mL conical tube containing each cell suspension and then centrifuged (1500 rpm, 4°C, 4 min). After aspiration of the supernatant from each conical tube, 10 ml of DMEM culture solution was added to the pellet and the number of cells dissociated with EDTA or trypsin was counted.

Cells treated with EDTA or trypsin were adjusted to 2 x 10 ⁵ cells/tube and FACS analysis (EC800) was performed. Three types of antibodies were used in the FACS analysis (fluorescent APC-labeled anti-mouse IgG antibody [5 μg/tube; produced by BioLegend, Inc.], GC33 antibody [1.0 μg/tube; produced by Medical & Biological Laboratories Co., Ltd.). Life Science] and scFv clone antibody [TF1413-02d028] described above [1.0 μg/tube]).

Preparation of cells treated with EDTA or trypsin] In a conical tube, 50 ml of 1x10 ⁶ cells dissociated with EDTA as described above were placed and centrifuged (1500 rpm, 4°C, 4 min), and the supernatant was aspirated to obtain cell mass (sediment). 5 ml of collagenase P solution was added to the sediment, and the mixture was incubated at 37°C for 30 minutes to obtain a cell suspension. The cell suspension was then passed through a 100 μm cell strainer, washed with 30 ml of DMEM culture solution. The cell suspension was again passed through a 100 μm cell sieve and centrifuged (300 g, 4°C, 10 minutes), and the supernatant was aspirated. The precipitate was washed by adding 20 ml PBS and then centrifuged (300 g, 4°C, 5 minutes), and the supernatant was aspirated. The cells were suspended by adding 5 ml of DMEM culture solution. Cell numbers were then counted and 2 x 10 ⁵ cells/tube analyzed by FACS (EC800). FACS analysis used 3 types

- 34 043515 antibodies (fluorescent APC-labeled anti-mouse IgG antibody [5 μg/tube; manufactured by BioLegend, Inc.], antibody GC33 [1.0 μg/tube; manufactured by Medical & Biological Laboratories Co., Ltd.). Life Science] and scFv clone [antibody [TF1413-02d028] described above [1.0 μg/tube]), as in the case of cells treated with EDTA or trypsin. The results are shown in Fig. 4. In FIG. 4, the right peak on the x-axis indicates that the GC33 antibody or scFv clone [TF1413-02d028] binds to the GPC3 protein.

Results.

As shown in FIG. 4, the binding activity of the antibody of the present invention (TF141302d028) to GPC3 protein treated with trypsin or collagenase was noticeably lower. These results show that the antibody of the present invention specifically recognizes the conformation of the GPC3 protein, which suggests that the antibody of the present invention has high specificity in vivo.

Example 3

3. Development of GPC3 CAR-T cells using a new anti-GPC3 antibody.

Protocol.

GPC3 is a cell surface molecule whose expression is not observed in adult tissues other than the placenta, but is observed in tissues of various cancers, such as hepatocellular carcinoma, melanoma, ovarian clear cell adenocarcinoma, and squamous cell lung cancer. Thus, GPC3 may serve as a target molecule for CAR-T cell therapy using chimeric antigen receptor (CAR). Accordingly, GPC3 CAR-T cells were generated using the 11 types of scFv clones obtained in Example 1 and assayed for cancer cytotoxic activity and interferon-γ (IFN-γ) production capacity.

Preparation of the GPC3 CAR vector.

scFv with the VH-linker-VL sequence were constructed for 11 types of scFv clones (TF141302d028, 02d039, 02e004, 02e014, 02e030, 02e040, 03e001, 03e004, 03e005, 03e015 and 03e03 4) obtained in example 1, based on their corresponding amino acid sequences VH and VL (see Table 5). A linker consisting of 15 amino acid residues with 3 repeats of the GGGGS polypeptide was used. The human immunoglobulin H chain signal sequence SEQ ID NO: 188 was added to the N-terminus of VH.

Table 5-1

SEQ ID NO: 165: TF1413-02d028 scFv

QVQLKESGPELEKPGASVKISCKASGYSFTGYNMNWVKQSNGKSLEWIGNIDPYYGGTSYNQKF kgkatltvdkssstaymqlksltsedsavyycargdyrayyfdywgqgttltvs|ggggsggggs|| IggggsIdiomtqspkfmstsvgdrvsitckasqnvrtavawyqqkpgqspkaliylasnrhtgvp drftgsgsgtdftltisnvqsedladyfclqhwnypltfgagtklelkr

SEQ ID NO: 166: TF1413-02d039 scFv

EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISSGGGSTYYPDTVK IGRFTI8RDNAKNTLYLQMS8LK8EDTAMYYCARRGLRRAMDYWGQGT8VTVS|GGGG8GGGG8G| IgggsIIdwmtqtplslpvslgdqasiscrssqslvhsngntylhwylqkpgqspklliykvsnrf SGVPDRFSGSG8GTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKLELKR_____________ SEQ ID NO: 167: TF1413-02e004 scFv

QVQLQQSGAELVKPGAPVKLSCKASGYTFTSYWMNWVKQRPGRGLEWIGRIDPSDSETHYNQK fkdeatltvdkss8tayiqlssltsedsavyycargyyamdywgqgt8vtvs|ggggsggggsgg| IggsIdivltqspkfmstsvgdrvsitckasqdvstavawyqqkpgqspklliysasyrytgvpdrft gsgsgtdftftissvqaedlavyycqqhystptfgggtkleikr_______________________ SEQIDNO:168: TF1413-02e014 scFv

QVQLKQSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQGLEWIGWIDPENGDTEYAPKF QGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNAGYYDYDGYAMDYWGQGTSVTVSIgGGGSGGI IggsggggsIdivltqspkfmstsvgdrvsitckasqdvgtavawyq qkpgqspktijywastrhtg _ SEQ ID NO: 169: TF1413-02e030 scFv evqlqqsgaelvrpgalvklsckasgfnikdyymhwvkqrpeqglewigwidpengntitopkF QGKA8ITADTSSNTAYLQLS8LTSEDTAVYYCAISTMITTLDYWGQGTTLTVstGGGGSGG GG8GG| |GG|DIQMTQ.SPSSLAMSVGQKVTMSCKSSQ8LLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRE SGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELKR

SEQIDNO:170: TF1413-02e040 scFv

EVMLVESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNGGTSYNQN fkgkatltvdkssstaymellsltsedsavyycargyygrfdywgqgttltvs[ggggsggggs^ IgggsIIdilltqspkfmstsvgdrvsitckasonvrtavawyqqkpgqspkali ylasnrhtgvpdr ftgsgsgtdftltisnvqsedladyfclqhwnypltfgagtklelkr

- 35 043515

Table 5-2

SEQIDNO:171: TF1413-03e001 scFv QVQLKQSGPELVKPGASVKISCKASGYSFTGYYMHWVKQSHVKSLEWIGRINPYNGATSYNQNF KDKASLTVDKSSSTAYMELHSLTSEDSAVYYCARNYGYFDYWGQGTTLTVS|GGGGSGGGGSGG| |GGSjDIKMTQSPKFMSTSVGDRVSVTCEASQNVDNNWWYQQKPGQSPKALIYSASYRY8GVPDR FTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPLTFGAGTKLEIKR

SEQIDNO:172: TF1413-03e004 scFv

1QVQLKQSGAELVKPGAPVKLSCKASGYTFTSYWMNWVKQRPGRGLEWIGRIDPSDSETHYNQK FKDKATLTVDKSSSTAYIQLSSLTSEDSAVYYCARGYYGSNYWGQGTTLTVSlGGGGSGGGGSGGGi iGSlniKMTQSPKFMSTgVGnRVgVTCKAfiQNVGTNVAWYQQKPG QgPKAT.TYSASYRYSGVPDRF TGSGSGTDFTLTTSNVQSEDT AEYFCQQYNSYPT ,TFG AGTKT TJ.KR

SEQIDNO:173: TF1413-03e005 scFv

QVQLKESGAELVRSGASVKLSCTA3GFNIKDYYMHWVKQRPEQGLEWIGWIDPENGDTEYAPKF QGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNAFYYDYDGYAMDYWGQGTSVTVSlGGGGSGGlI IggsggggsIdwmtqtpsslsaslgervsltcrasqeisgylswlqqk pdgtikrliyaastldsg VPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPLTFGAGTKEEEKR

SEQIDNO:174: TF1413-03e015 scFv

EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNGGTSYNQK fkgkatltvdkssstaymellsltsedsavyycargdyyppyamdywgqgtsvtMggggsgggI |GSGGGGSjDTVMSQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGOSPKPT JYSASYRYSG VPDRFTGSGSGTDFTLTISNVOSEDLAEYFCQOYNRYPLTFGVGTKLEIKR________________ SEQIDNO:175: TF1413-03e034 scFv

EVQLQQ3GPELEKPGASVKISCKA3GYSFTGYNMNWVKQ3NGKSLEWIGNIDPYYGGT3YNQKF KGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARGNYGYYAMDYWGQGTSVTVS|GGGGSGGGG| |SGGGGS|DIVMSQSPKFMSTSVGDRVSITCKASQNVRTAVAWYQQKPGQSPKALIYLASNRHTGV PDRFTGSGSGTDFTLTISNVQSEDLADYFCLQHWNYPLTFGAGTKLELKR_______________

In the tables, the linker is shown in a double line box, VH is underlined by one line, and VL is underlined by two lines.

The nucleotide sequence encoding each anti-GPC3 scFv from table. 5 was synthesized by optimization for human codons and inserted into a CAR expression vector. The CAR gene used had a gene encoding a fusion peptide (a peptide consisting of the amino acid sequence SEQ ID NO: 185) consisting of the human CD8 transmembrane region and the immune cell signal transduction activation region CD28/4-1BB/CD3 zeta self-cleaving sequence 2A gene human IL-7, the 2A self-cleavage sequence, the human CCL19 gene, the 2A self-cleavage sequence and the HSV-TK gene located 5'-3' of the scFv gene, all of which were included in the MSGV1 retroviral vector (see international publication WO 2016/ 056228).

Generation of GPC3 CAR-T cells.

GPC3 CAR vectors derived from the 11 types of scFv clones described above were each transiently introduced into GP2 packing cells to produce retroviral vectors. T cells were infected with these gene transfer vectors to induce GPC3 CAR-T cells. The ratio of CARC-expressing GPC3 cells to gene-transferred T cells varied from 5.3 to 39.2%. Accordingly, the following functional analysis was performed using CAR-T GPC3 cells derived from 5 types of scFv clones (TF1413-02d028, TF1413-02d039, TF1413-02e014, TF1413-02e030 and TF1413-03e005) with a ratio of 25% or more.

Degrading activity of GPC3 CAR-T cells against a GPC3-expressing cell line.

To study the destructive activity of GPC3 CAR-T cells against malignant cells, we analyzed the simultaneous cultivation of GPC3 CAR-T cells and a GPCS-expressing cell line, that is, the Sk-HEP-1 hepatocellular carcinoma cell line activated for GPC3 expression (cell line GPC3 Sk-HEP-1), or a cell line that does not express GPC3 (Sk-HEP-1 mock cell line). GPC3 CAR-T cells were mixed with malignant target cells (GPC3 Sk-HEP-1 cell line or Sk-HEP-1 mock cell line) in a 1:1 ratio (1x10 ⁵ cells/well) and cultured in a 24-well plate. After 48 hours, cells were recovered, stained with anti-CD45 antibody, and FCM analyzed with CD45-positive cells as GPC3 CAR-T cells and CD45-negative cells as remaining malignant cells [GPC3 Sk-HEP-1 cells]. According to the results, all GPC3 CAR-T cells obtained from the 5 types of scFv clones described above almost completely destroyed GPC3 Sk-HEP-1 cells, but did not show destructive activity against Sk-HEP-1 mock cells (see Fig. 5 and 6). When cells uninfected with the viral vector (cells without gene transfer [Uninfected in Figures 5 and 6]) were used as a negative control for GPC3 CAR-T cells, these cells did not exhibit destructive activity

- 36 043515 neither against Sk-HEP-1 GPC3 cells nor against Sk-HEP-1 mock cells.

Based on these results, it was shown that GPC3 CAR-T cells derived from selected types of anti-GPC3 scFv clones (TF1413-02d028, TF1413-02d039, TF1413-02e014, TF1413-02e030 and

TF1413-03e005), exhibit specific cytotoxic activity against malignant

GPC3-expressing cells.

Ability of GP-3 CAR-T cells to produce IFN-γ upon recognition of GPC3-expressing cells.

In addition to the destructive activity against GPC3-expressing (positive) malignant cells, the ability of GPC3 CARC-T cells to produce IFN-γ was analyzed. GPC3 CAR T cells were mixed with malignant target cells (GPC3 Sk-HEP-1 cell line or Sk-HEP-1 mock cell line) in a 1:1 ratio (1x10 ⁵ cells/well) and cultured for 48 hours at 24- well plate and measured the concentration of IFN-γ produced in the culture supernatant using ELSIA. As a result, all GPC3 CAR-T cells derived from the 5 types of scFv clones described above exhibited the ability to produce IFN-γ in a GPC3 expression-dependent manner. In particular, GPC3 CAR-T cells derived from clone TF1413-02d028 showed the highest ability to produce IFN-γ (see Fig. 7).

Example 4.

4. Obtaining a humanized antibody.

Humanized scFv antibodies were constructed based on two types of scFv clones (TF141302d028 and 02d039) obtained in Example 1 (see Table 6). A linker consisting of 15 amino acid residues with 3 repeats of the GGGGS polypeptide was used. The human immunoglobulin H chain signal sequence SEQ ID NO: 188 was added to the N-terminus of VH.

Table 6

SEQ ID NO:178: #5 VH1-15-VL1 (TF1413-02d028 scFv humanized antibody 1)

QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYNMNWVRQAPGQGLEWIGNIDPYYGGTSYNQK fkgratltvdtststaymelrslrsddtavyycargdyrayyfdywgqgttvtvss|ggggsggg| ||GSGGGGS|tDIQMTQSPSSLSASVGDRVTITCKASQNVRTAVAWYQQKPGKAPKALIYLASNRHTGV PSRFSGSGSGTDFTLTI8SLQPEDFATYYCLOHWNYPLTFGGGTKVEIK

SEQ ID NO:179: #5 VH2-15-VL1 (TF1413-02d028 scFv humanized antibody 2)

QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYNMNWVRQAPGQGLEWIGNIDPYYGGTSYNQK IFKGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCARGDYRAYYFDYWGQGTTVTVSSjGGGGSGGG| ||GSGGGGS|IdIQMTQSPS3LSASVGDRVTITCKASQNVRTAVAWYQQKPGKAPKALIYLASNRHTGV PSRFSGSGSGTDFTLTTSSEQPRDFATYYCLQHWNYPLTFGGGTKVETK

SEQ ID NO:180: #5 VH3-15-VL1 (TF1413-02d028 scFv humanized antibody 3)

QVQLVQ8GAEVKKPGASVKV8CKASGYTFTGYNMNWVRQAPGQGLEWIGNIDPYYGGTSYNQK FKGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCARGDYRAYYFDYWGQGTTVTVSSjGGGGSGGGl IlGSGGGGSjDIQMTQSPSSLSASVGDRVTITCKASQNVRTAVAWYQQKPGKAPKALI YLASNRHTGV PSRFSGSGSGTDFTLTTSSLO PEDFATYYCLOHWNYPLTFGGGTKVEIK

SEQ ID NO:181: #6 VH1-15-VL1 (TF1413-02d039 scFv humanized antibody 1)

EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLEWVAYISSGGGSTYYPDTVK GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLRRAMDYWGQGTMVTVSS|GGGGSGGGGS| |GGGGS|DIVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGOSPQLLIYKV3NRF SGVPDRFSGSGSGTDDFTT.KT8RVEAEDVGVYYCSOSTHVPTTFGGGTKVETK

SEQ ID NO: 182: #6 VH115 VL2 (TF1413-02d039 scFv humanized antibody 2)

EVQLVESGGGLVQPGGSLRLSCAA8GFAFSSYDMSWVRQAPGKGLEWVAYISSGGGSTYYPDTVK GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLRRAMDYWGQGTMVTVSS|GGGGSGGGGS| tGGGGSjDIVMTQSPLSLPVTPGEPASISCRSSQSLVHSSGNTYLHWYLQKPGQSPQLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKYEIK______________ SEQ ID NO:183: #6 VH2-15-VL1 (TF1413-02d039 scFv gu manized antibody 3)

EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKRLEWVAYISSGGGSTYYPDTVK GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLRRAMDYWGQGTMVTVSS|GGGGSGGGGS| Ugggg^divmtqsplslpvtpgepasiscrssqslvhsngntylhwylqkpgospqlliykvsnrf SGVPDRFSGSG8GTDFTLKISRVE AEDVGVYYCSQSTHVPLTFGGGTKyEIK

In the tables, the linker is shown in a double line box, VH is underlined by one line, and VL is underlined by two lines.

Industrial applicability

The present invention contributes to the field of immunotherapy of malignant tumors.

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Claims (11)

CLAIM 1. An antibody that specifically binds to a human GPC3 (glypican-3)-derived polypeptide consisting of the amino acid sequence SEQ ID NO: 155, wherein the antibody contains a heavy chain complementarity determining region (CDR)1 consisting of the amino acid sequence SEQ ID NO: 1, Heavy chain CDR2 consisting of the amino acid sequence SEQ ID NO: 2, and heavy chain CDR3 consisting of the amino acid sequence SEQ ID NO: 3, and light chain CDR1 consisting of the amino acid sequence SEQ ID NO: 4, light chain CDR2 consisting of amino acid sequence SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence SEQ ID NO: 6. 2. The antibody of claim 1, wherein the antibody comprises a heavy chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 7, and a light chain variable region consisting of an amino acid sequence a sequence that is at least 80% or greater identical in sequence to the amino acid sequence of SEQ ID NO: 8. 3. An antibody according to claim 1 or 2, wherein the antibody is a single chain antibody. 4. The antibody of claim 3, wherein the single chain antibody comprises an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 165. 5. The antibody of claim 3, wherein the single chain antibody comprises an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 178; or contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 179; or contains an amino acid sequence that is at least 80% or greater identical in sequence to the amino acid sequence of SEQ ID NO: 180. 6. The antibody of claim 1 or 2, wherein the antibody comprises a heavy chain consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 9, and a light chain consisting of an amino acid sequence, which is at least 80% or greater identical in sequence to the amino acid sequence of SEQ ID NO: 10. 7. An antibody that specifically binds to a human GPC3 (glypican-3)-derived polypeptide consisting of the amino acid sequence SEQ ID NO: 155, wherein the antibody contains Heavy chain CDR1 consisting of the amino acid sequence SEQ ID NO: 11, heavy chain CDR2 consisting of the amino acid sequence SEQ ID NO: 12, and heavy chain CDR3 consisting of the amino acid sequence SEQ ID NO: 13, and light chain CDR1 consisting of amino acid sequence SEQ ID NO: 14, a light chain CDR2 consisting of the amino acid sequence SEQ ID NO: 15, and a light chain CDR3 consisting of the amino acid sequence SEQ ID NO: 16. 8. The antibody of claim 7, wherein the antibody comprises a heavy chain variable region consisting of an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 17, and a light chain variable region consisting of an amino acid sequence a sequence that is at least 80% or greater identical in sequence to the amino acid sequence of SEQ ID NO: 18. 9. An antibody according to claim 7 or 8, wherein the antibody is a single chain antibody. 10. The antibody of claim 9, wherein the single chain antibody comprises an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 166. 11. The antibody of claim 9, wherein the single chain antibody comprises an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 181; or contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 182; or contains an amino acid sequence that is at least 80% or more identical in sequence to the amino acid sequence of SEQ ID NO: 183; or contains an amino acid sequence that is at least 80% or greater identical in sequence to the amino acid sequence of SEQ ID NO: 184. -