Novel antibody specifically binding to GPC3 and use thereof

Abstract

The present invention relates to: an antibody specifically binding to GPC3; a CAR-T using same; a humanized antibody of the antibody; and a use of a CAR-T using the humanized antibody. A novel antibody specifically binding to GPC3 protein and a fragment or humanized antibody thereof having immunological activity according to the present invention specifically binds to GPC3, involved in signaling (YAP and Wnt / b-catenin signaling) playing an important role in cancer formation / progression, and exhibits an anticancer immune response, and thus can be used as an antibody therapeutic agent for cancer treatment and as a cell therapeutic agent using cancer cell-killing mechanisms via immune cell activation, and can also be used for cancer diagnosis.

Description

Novel antibodies specifically binding to GPC3 and uses thereof

[0001] The present invention relates to a novel antibody that specifically binds to GPC3, a chimeric antigen receptor T cell (CAR-T) using the same, and uses thereof.

[0002] Glypican 3 (GPC3) is known as an oncofetal antigen belonging to the glypican family of glycosyl-phosphatidylinositol-anchored heparin sulfate proteoglycans, and the cell membrane-bound glypican-3 is known to be composed of two subunits linked by one or more disulfide bonds.

[0003] Although GPC3 is not expressed in adult tissues other than the placenta, it is expressed in various cancer tissues such as hepatocellular carcinoma (HCC), hepatoblastoma, melanoma, ovarian clear cell carcinoma, Wilm's tumor, and lung squamous cell carcinoma. Thus, GPC3, like proteins such as α-fetoprotein (AFP) and carcinoembryonic antigen (CEA), is a protein expressed in fetal tissues, and is therefore classified as a fetal cancer antigen. In other words, although GPC3 is not expressed in normal tissue cells, it exhibits the characteristic of being specifically expressed in cancer cells, and therefore can be utilized as a target molecule for cancer treatment, a tumor marker, and a diagnostic marker.

[0004] In addition, among the currently developed drugs for treatment, they are largely divided into low-molecule drugs and high-molecule drugs, and high-molecule drugs with specificity are receiving attention as treatment compared to low-molecule drugs that have relatively large side effects due to lack of specificity.

[0005] Among these, protein therapeutics are rapidly replacing non-specific small-molecule compound therapeutics due to their high specificity for disease targets and low side effects and toxicity, and are widely used in clinical practice. In particular, antibody therapeutics are the main type of protein therapeutics currently used in clinical practice. Therapeutic antibodies are considered one of the most effective cancer treatments because they show much higher target specificity than existing small-molecule drugs, have low biotoxicity and side effects, and have an excellent blood half-life of approximately three weeks. In fact, major pharmaceutical companies and research institutes around the world are accelerating the research and development of therapeutic antibodies that specifically bind to cancer cells, including carcinogens, and effectively eliminate them. Therapeutic antibody drug development companies include pharmaceutical companies such as Roche, Amgen, Johnson & Johnson, Abbott, and BMS. Roche, in particular, has generated significant profits with its representative products such as Herceptin, Avastin, and Rituxan for anticancer treatment. These three therapeutic antibodies achieved global sales of approximately $19.5 billion in 2012, and are not only generating significant profits but also leading the global antibody drug market. Johnson & Johnson, which developed Remicade, is also rapidly growing in the global antibody market due to increasing sales. Pharmaceutical companies such as Abbott and BMS are also known to have a number of therapeutic antibodies in the final stages of development. As a result, the global pharmaceutical market, previously dominated by small molecule drugs, is quickly being replaced by biopharmaceuticals that include therapeutic antibodies that are specific to disease targets and have fewer side effects.

[0006] Meanwhile, immune cell-based immunotherapy has been developed, which, unlike conventional immunotherapy, specifically binds to cancer cells and eliminates them through the body's immune cells. These cancer treatments include T cells expressing chimeric antigen receptors (CARs) that can directly recognize tumor-associated antigens (CAR T cells), or T cells expressing antibody sequences specific to immune checkpoint receptors on cancer cells. These cancer treatments enable immune cells to specifically attack cancer cells. Human T cell therapy relies on enriched or modified human T cells to target and kill cancer cells in a patient. To enhance the ability of T cells to target and kill specific cancer cells, methods have been developed to engineer T cells to express constructs that direct them to specific target cancer cells. Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs) containing binding domains capable of interacting with specific tumor antigens enable T cells to target and kill cancer cells expressing specific tumor antigens. CAR(Chimeric Antigen Receptor)-T cell is a cell therapy that combines the sequence of an antibody that binds to tumor antigens (e.g. CD19) with a domain required for T cell signaling such as CD3 / 4-1BB / CD28 and inserts it into T cells. There are various methods for inserting these CAR genes into T cells, but most use a lentivirus delivery system. The structure of currently developed chimeric antigen receptors is divided into the scFv(single chain variable fragment) part that recognizes the antigen, the transmembrane domain, and the signaling domain that transmits signals into the cell.Currently, pharmaceutical companies such as Novartis, Gilead, Celgene, and Abclon, as well as clinicians, have developed CAR-T cells using scFv that specifically bind to the CD19 antigen of blood cancer cells as a treatment for blood cancer. However, to completely conquer cancer, the development of CAR-T cells that show more certain anti-cancer effects is required.

[0007] The purpose of the present invention is to provide an antibody specific for GPC3 or a fragment thereof having immunological activity.

[0008] Another object of the present invention is to provide a humanized antibody or antigen-binding fragment thereof that specifically binds to GPC3.

[0009] Another object of the present invention is to provide a hybridoma cell line.

[0010] Another object of the present invention is to provide a nucleic acid molecule.

[0011] Another object of the present invention is to provide a vector.

[0012] Another object of the present invention is to provide a transformed host cell.

[0013] Another object of the present invention is to provide a composition for detecting cancer antigens.

[0014] Another object of the present invention is to provide a kit for detecting cancer antigens.

[0015] Another object of the present invention is to provide an antibody-drug conjugate (ADC).

[0016] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer.

[0017] Another object of the present invention is to provide a composition for diagnosing cancer.

[0018] Another object of the present invention is to provide a kit for diagnosing cancer.

[0019] Another object of the present invention is to provide a method for providing information for diagnosing cancer.

[0020] Another object of the present invention is to provide a GPC3-specific chimeric antigen receptor (CAR).

[0021] Another object of the present invention is to provide a GPC3-specific humanized antibody-derived chimeric antigen receptor (CAR).

[0022] Another object of the present invention is to provide a polynucleotide.

[0023] Another object of the present invention is to provide a vector.

[0024] Another object of the present invention is to provide a method for producing a GPC3-specific CAR or a GPC3-specific humanized antibody-derived CAR.

[0025] Another object of the present invention is to provide a chimeric antigen receptor expressing cell.

[0026] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer.

[0027] Another object of the present invention is to provide a cell therapy agent for preventing or treating cancer.

[0028] Another object of the present invention is to provide an anticancer adjuvant.

[0029] Another object of the present invention is to provide a composition for diagnosing cancer.

[0030] Another object of the present invention is to provide a method for providing information for diagnosing cancer.

[0031] Another object of the present invention is to provide a method for detecting a cancer antigen.

[0032] Another object of the present invention is to provide a method for preventing or treating cancer.

[0033] Another object of the present invention is to provide a method for enhancing the anticancer treatment effect of an individual.

[0034] To solve the above problem, the present invention provides an isolated antibody that specifically binds to GPC3 (Glypican-3) protein or a fragment thereof having immunological activity.

[0035] In addition, the present invention provides a humanized antibody or antigen-binding fragment thereof that specifically binds to GPC3, comprising a human VH domain comprising at least one CDRH selected from the group consisting of CDRH (Complementarity determining regions Heavy chain) 1 comprising an amino acid sequence represented by SEQ ID NO: 1, CDRH2 comprising an amino acid sequence represented by SEQ ID NO: 2, and CDRH3 comprising an amino acid sequence represented by SEQ ID NO: 3; and a human VL domain comprising at least one CDRL selected from the group consisting of CDRL1 comprising an amino acid sequence represented by SEQ ID NO: 4, CDRL2 comprising an amino acid sequence represented by SEQ ID NO: 5, and CDRL3 comprising an amino acid sequence represented by SEQ ID NO: 6.

[0036] In addition, the present invention provides a hybridoma cell line producing an isolated antibody according to the present invention or a fragment thereof having immunological activity, or a humanized antibody according to the present invention or an antigen-binding fragment thereof.

[0037] The present invention also provides an isolated nucleic acid molecule encoding an isolated antibody according to the present invention or a fragment thereof having immunological activity, or a humanized antibody according to the present invention or an antigen-binding fragment thereof.

[0038] The present invention also provides a vector comprising an isolated nucleic acid molecule encoding an isolated antibody according to the present invention or a fragment thereof having immunological activity, or a humanized antibody according to the present invention or an antigen-binding fragment thereof.

[0039] The present invention also provides a host cell transformed with a vector comprising an isolated nucleic acid molecule encoding an isolated antibody according to the present invention or a fragment having immunological activity thereof, or a humanized antibody according to the present invention or an antigen-binding fragment thereof.

[0040] In addition, the present invention provides a composition for detecting a cancer antigen comprising an isolated antibody according to the present invention or a fragment thereof having immunological activity, or a humanized antibody according to the present invention or an antigen-binding fragment thereof.

[0041] In addition, the present invention provides a kit for detecting a cancer antigen comprising an isolated antibody according to the present invention or a fragment thereof having immunological activity, or a humanized antibody according to the present invention or an antigen-binding fragment thereof.

[0042] In addition, the present invention provides an antibody-drug conjugate (ADC) comprising an isolated antibody according to the present invention or a fragment thereof having immunological activity, or a humanized antibody according to the present invention or an antigen-binding fragment thereof; and a drug.

[0043] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an antibody-drug conjugate according to the present invention.

[0044] In addition, the present invention provides a composition for diagnosing cancer comprising an isolated antibody according to the present invention or a fragment thereof having immunological activity, a humanized antibody according to the present invention or an antigen-binding fragment thereof, or an antibody-drug conjugate according to the present invention.

[0045] In addition, the present invention provides a cancer diagnostic kit comprising an isolated antibody according to the present invention or a fragment thereof having immunological activity, a humanized antibody according to the present invention or an antigen-binding fragment thereof, or an antibody-drug conjugate according to the present invention.

[0046] In addition, the present invention provides a method for providing information for diagnosing cancer, comprising the steps of treating a sample with an isolated antibody or a fragment having immunological activity thereof according to the present invention, a humanized antibody or an antigen-binding fragment thereof according to the present invention, or an antibody-drug conjugate according to the present invention, labeled with a fluorescent substance; and measuring the expression of the isolated antibody or a fragment having immunological activity thereof, the humanized antibody or an antigen-binding fragment thereof, or the antibody-drug conjugate.

[0047] In order to solve the above problem, the present invention provides a GPC3-specific chimeric antigen receptor (CAR) comprising a single chain variable fragment (scFv) as an antigen binding domain, the single chain variable fragment comprising a heavy chain variable region comprising CDRH (Complementarity determining regions Heavy chain) 1 comprising an amino acid sequence represented by SEQ ID NO: 1, CDRH2 comprising an amino acid sequence represented by SEQ ID NO: 2, and CDRH3 comprising an amino acid sequence represented by SEQ ID NO: 3; and a light chain variable region comprising CDRL1 comprising an amino acid sequence represented by SEQ ID NO: 4, CDRL2 comprising an amino acid sequence represented by SEQ ID NO: 5, and CDRL3 comprising an amino acid sequence represented by SEQ ID NO: 6.

[0048] In addition, the present invention provides a GPC3-specific humanized antibody-derived chimeric antigen receptor (CAR) comprising a single chain variable fragment (scFv) as an antigen-binding domain, wherein the single chain variable fragment comprises: a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11; a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12; a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 13 and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 14; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 15; a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 16; a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 17 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 18.

[0049] In addition, the present invention provides a polynucleotide comprising a nucleic acid sequence encoding a GPC3-specific CAR according to the present invention or a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0050] In addition, the present invention provides a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a GPC3-specific CAR according to the present invention or a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0051] In addition, the present invention provides a method for producing a GPC3-specific CAR or a GPC3-specific humanized antibody-derived CAR, comprising a step of infecting a host cell with a vector comprising a polynucleotide, the vector comprising a nucleic acid sequence encoding a GPC3-specific CAR or a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0052] Additionally, the present invention provides a chimeric antigen receptor expressing cell transformed with a GPC3-specific CAR according to the present invention.

[0053] In addition, the present invention provides a chimeric antigen receptor expressing cell transformed with a CAR derived from a GPC3-specific humanized antibody according to the present invention.

[0054] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a GPC3-specific CAR according to the present invention, a CAR derived from a GPC3-specific humanized antibody according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a chimeric antigen receptor-expressing cell transformed with a CAR derived from a GPC3-specific humanized antibody according to the present invention.

[0055] In addition, the present invention provides a cell therapeutic agent for preventing or treating cancer, comprising a chimeric antigen receptor expressing cell transformed with a GPC3-specific CAR according to the present invention or a chimeric antigen receptor expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0056] In addition, the present invention provides an anticancer adjuvant comprising a GPC3-specific CAR according to the present invention, a GPC3-specific humanized antibody-derived CAR according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0057] In addition, the present invention provides a composition for diagnosing cancer, comprising a GPC3-specific CAR according to the present invention, a GPC3-specific humanized antibody-derived CAR according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0058] In addition, the present invention provides a method for providing information for diagnosing cancer, comprising the steps of treating a sample with a GPC3-specific CAR according to the present invention labeled with a fluorescent substance, a GPC3-specific humanized antibody-derived CAR according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention; and measuring the expression of the GPC3-specific CAR, the GPC3-specific humanized antibody-derived CAR, the chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR, or the chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR.

[0059] In addition, the present invention provides a method for detecting a cancer antigen, comprising the step of administering to a subject an isolated antibody according to the present invention or a fragment having immunological activity thereof, a humanized antibody according to the present invention or an antigen-binding fragment thereof, a GPC3-specific CAR according to the present invention, a CAR derived from a GPC3-specific humanized antibody according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a chimeric antigen receptor-expressing cell transformed with a CAR derived from a GPC3-specific humanized antibody according to the present invention.

[0060] In addition, the present invention provides a method for preventing or treating cancer, comprising the step of administering to a subject an antibody-drug conjugate according to the present invention, a GPC3-specific CAR according to the present invention, a GPC3-specific humanized antibody-derived CAR according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention, a cell therapeutic agent comprising a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a cell therapeutic agent comprising a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0061] In addition, the present invention provides a method for enhancing the anticancer treatment effect in a subject, comprising the step of administering to the subject a GPC3-specific CAR according to the present invention, a GPC3-specific humanized antibody-derived CAR according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0062] The present invention relates to an antibody specifically binding to GPC3, CAR-T using the same, a humanized antibody of the antibody, and a use of CAR-T using the humanized antibody. The novel antibody specifically binding to GPC3 protein of the present invention, a fragment thereof having immunological activity, or a humanized antibody thereof specifically binds to GPC3 involved in signal transduction (Yap and Wnt / b-catenin signaling) that plays an important role in cancer formation / progression, and exhibits an anticancer immune response, and therefore has the effect of being used as an antibody therapeutic agent for cancer treatment, a cell therapeutic agent utilizing a cancer cell death mechanism by immune cell activation, and has the effect of being used for the diagnosis of cancer.

[0063] Figure 1 is a diagram showing the process of producing a GPC3 (Glypican-3) specific antibody:

[0064] Left: Immune-induced GPC3 protein; and

[0065] Right: Process of deriving hybridoma antibody clones specific for GPC3.

[0066] Figure 2 is a diagram showing the results of a specificity ELISA analysis for GPC3 using the supernatant of each hybridoma single-cell clone.

[0067] Figure 3 shows the specific binding of the selected 10-1.4 antibody clone to GPC3 confirmed by flow cytometry:

[0068] Unstained: Unstained group;

[0069] Anti-GPC3 Ab: Positive control stained with anti-human GPC3:APC antibody;

[0070] Isolated Ab clone + 2' Ab: A group that was first stained with the culture supernatant of selected hybridoma antibody clone 10-1.4 and then secondarily stained with anti-mouse IgG:PE antibody; and

[0071] 2' Ab only: Group secondary stained with anti-mouse IgG:PE antibody without primary staining.

[0072] Figure 4 shows the sequence analysis results of the VH region (Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) and VL region (Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) of the hybridoma antibody clone 10-1.4 specific for GPC3 of the present invention.

[0073] Figure 5 is a diagram showing the specific binding of the scFv form antibody of the hybridoma antibody clone 10-1.4 of the present invention to GPC3 confirmed by flow cytometry:

[0074] Left: scFv form of hybridoma antibody clone 10-1.4;

[0075] Right: Flow cytometry results;

[0076] Unstained: Unstained group;

[0077] Commercial Ab: Positive control group stained with anti-human GPC3:APC antibody; and 10-1.4 (scFv): Group stained primarily with the FLAG tag-scFv form of the selected hybridoma clone 10-1.4, followed by secondary staining with anti-FLAG tag:APC antibody.

[0078] Figure 6 is a diagram showing the process of producing GPC3 CAR-T or CD19 (negative control) CAR-T of the present invention.

[0079] Figure 7 is a diagram showing the antigen specificity (a) and binding to the GPC3 antigen (b) of GPC3 CAR-T confirmed by flow cytometry:

[0080] NT-T: non-transduced T cell group (group without GPC3 CAR);

[0081] GPC3 CAR-T: T cell population introduced with GPC3 CAR;

[0082] CD19 CAR-T: CAR-T with scFv of FMC63 antibody clone that recognizes CD19 (negative control);

[0083] GPC3 CAR-T: T cell population introduced with GPC3 CAR; and

[0084] Left figure: Schematic diagram of a T cell introduced with a CAR comprising the GPC3 scFv of the present invention as an antigen-binding domain.

[0085] Figure 8 is a diagram confirming the GPC3-specific anti-cancer immune response of GPC3 CAR-T through cytokine release:

[0086] CD19 CAR-T (- HepG2): CD19 CAR-T cultured alone;

[0087] CD19 CAR-T (+ HepG2): CD19 CAR-T co-cultured with HepG2;

[0088] GPC3 CAR-T (- HepG2): GPC3 CAR-T cultured alone;

[0089] GPC3 CAR-T (+ HepG2): GPC3 CAR-T co-cultured with HepG2; and

[0090] Upper figure: Schematic diagram of cytokine release after binding of GPC3 CAR-T of the present invention to GPC3.

[0091] Figure 9 is a diagram showing CAR-T cells containing the GPC3 scFv of the present invention as an antigen binding domain and having CD28 or 4-1BB as a co-stimulatory domain, and confirming whether the cells are expressed on the surface thereof:

[0092] CD19 BBz: CAR-T with scFv of FMC63 antibody clone and 4-1BB costimulatory domain used as control;

[0093] CD19 28z: CAR-T with scFv of FMC63 antibody clone used as control and CD28 costimulatory domain;

[0094] GPC3 BBz: CAR-T with scFv sequence of clone 10-1.4 and 4-1BB costimulatory domain; and

[0095] GPC3 28z: CAR-T with scFv sequence of clone 10-1.4 and CD28 costimulatory domain.

[0096] Figure 10 is a diagram confirming the specific anticancer immune response to GPC3 of CAR-T cells containing the GPC3 scFv of the present invention as an antigen binding domain and having CD28 or 4-1BB as a co-stimulatory domain:

[0097] CD19 BBz: CAR-T with scFv of FMC63 antibody clone and 4-1BB costimulatory domain used as control;

[0098] CD19 28z: CAR-T with scFv of FMC63 antibody clone used as control and CD28 costimulatory domain;

[0099] GPC3 BBz: CAR-T with scFv sequence of clone 10-1.4 and 4-1BB costimulatory domain; and

[0100] GPC3 28z: CAR-T with scFv sequence of clone 10-1.4 and CD28 costimulatory domain.

[0101] Figure 11 is a diagram showing the anticancer efficacy of CAR-T cells containing the GPC3 scFv of the present invention as an antigen binding domain and having CD28 or 4-1BB as a co-stimulatory domain after administration to an animal model that induced liver cancer:

[0102] CD19 BBz: CAR-T with scFv of FMC63 antibody clone and 4-1BB costimulatory domain used as control;

[0103] CD19 28z: CAR-T with scFv of FMC63 antibody clone used as control and CD28 costimulatory domain; and

[0104] GPC3 28z: CAR-T with scFv sequence of clone 10-1.4 and CD28 costimulatory domain.

[0105] Figure 12 is a schematic diagram of the production of a humanized antibody by grafting the CDR sequence of the hybridoma antibody clone 10-1.4 specific to GPC3 of the present invention.

[0106] Figure 13 is a diagram confirming the GPC3 antigen specificity of a humanized antibody grafted with the CDR sequence of the GPC3-specific hybridoma antibody clone 10-1.4 of the present invention.

[0107] Figure 14 is a schematic diagram of a T cell introduced with a CAR that includes an antigen-binding domain of a scFv of a humanized antibody grafted with the CDR sequence of the hybridoma antibody clone 10-1.4 specific for GPC3 of the present invention, and a diagram confirming the GPC3 antigen specificity thereof.

[0108] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are provided as examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0109] Additionally, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customs of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout this specification, when a part is said to "include" a certain component, unless specifically stated otherwise, this does not mean that other components are excluded, but rather that other components may be included.

[0110] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited herein as references are incorporated herein by reference.

[0111] Throughout this specification, the conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as generally accepted three-letter codes for other amino acids, such as Aib (α-aminoisobutyric acid) and Sar (N-methylglycine). Furthermore, amino acids referred to herein by abbreviations are described according to the IUPAC-IUB nomenclature as follows:

[0112] Alanine: A, arginine: R, asparagine: N, aspartic acid: D, cysteine: C, glutamic acid: E, glutamine: Q, glycine: G, histidine: H, isoleucine: I, leucine: L, lysine: K, methionine: M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y, and valine: V.

[0113]

[0114] In one aspect, the present invention relates to an isolated antibody or a fragment thereof having immunological activity that specifically binds to GPC3 (Glypican-3) protein.

[0115] In one embodiment, the isolated antibody or immunologically active fragment thereof may comprise a VH domain comprising complementarity determining regions heavy chain (CDRH) 1 comprising the amino acid sequence of SEQ ID NO: 1, CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 3.

[0116] In one embodiment, the isolated antibody or immunologically active fragment thereof may comprise a VL domain comprising CDRL1 comprising an amino acid sequence represented by SEQ ID NO: 4, CDRL2 comprising an amino acid sequence represented by SEQ ID NO: 5, and CDRL3 comprising an amino acid sequence represented by SEQ ID NO: 6.

[0117] In one embodiment, the isolated antibody or immunologically active fragment thereof may comprise a VH domain comprising an amino acid sequence represented by SEQ ID NO: 7.

[0118] In one embodiment, the isolated antibody or immunologically active fragment thereof may comprise a VL domain comprising an amino acid sequence represented by SEQ ID NO: 8.

[0119] In one embodiment, the isolated antibody may be a chimeric antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a triabody, a tetrabody, and the immunologically active fragment thereof may be any one selected from the group consisting of, but not limited to, a Fab, Fd, Fd', Fab', dAb, F(ab'), F(ab')2, a single chain fragment variable (scFv), Fv, a single chain antibody, an Fv dimer (dsFv), or a complementarity determining region (CDR) fragment. Preferably, it may be a scFv.

[0120] In one embodiment, the antibody of the present invention may be a monoclonal antibody.

[0121] The above antibody is not only in the form of a whole antibody, but also includes functional fragments of an antibody molecule. A whole antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. A functional fragment of an antibody molecule means a fragment that possesses an antigen-binding function, and examples of antibody fragments include (i) a Fab fragment consisting of a variable region (VL) of a light chain and a variable region (VH) of a heavy chain and a constant region (CL) of a light chain and a first constant region (CH1) of a heavy chain; (ii) a Fd fragment consisting of the VH and CH1 domains; (iii) a Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain; (v) separate CDR regions; (vi) a F(ab')2 fragment, which is a bivalent fragment containing two linked Fab fragments; (vii) single-chain Fv molecules (scFv) joined by a peptide linker that joins the VH domain and the VL domain to form an antigen-binding site; (viii) bispecific single-chain Fv dimers; and (ix) diabodies, which are multivalent or multispecific fragments produced by genetic fusion.

[0122] The antibody of the present invention or a fragment thereof having immunological activity may be selected from the group consisting of, but is not limited to, animal-derived antibodies, chimeric antibodies, human antibodies, and fragments thereof having immunological activity. In addition, the antibody may be, but is not limited to, recombinantly or synthetically produced.

[0123] The above antibody or fragment having immunological activity may be isolated from a living organism (not existing in a living organism) or non-naturally occurring, for example, may be synthetically or recombinantly produced.

[0124] In the present invention, "antibody" refers to a substance produced by antigen stimulation within the immune system, and its type is not particularly limited, and can be obtained naturally or non-naturally (e.g., synthetically or recombinantly). Antibodies are highly stable both in vitro and in vivo and have a long half-life, making them advantageous for mass expression and production. In addition, antibodies inherently have a dimer structure, and thus have very high avidity. A complete antibody has a structure with two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. The constant region of antibodies is divided into the heavy chain constant region and the light chain constant region. The heavy chain constant region has the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and the subclasses are gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain has the kappa (κ) and lambda (λ) types.

[0125] In the present invention, the term "heavy chain" is interpreted to mean a full-length heavy chain and fragments thereof, including a variable region domain VH comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and three constant region domains CH1, CH2 and CH3 and a hinge. In addition, the term "light chain" is interpreted to mean a full-length light chain and fragments thereof, including a variable region domain VL comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and a constant region domain CL.

[0126] In the present invention, the term "variable region or variable domain" refers to a portion of an antibody molecule that exhibits many sequence variations while performing the function of specifically binding to an antigen, and the variable region includes complementarity determining regions (CDR1, CDR2, and CDR3). Between the CDRs, a framework region (FR) exists, which serves to support the CDR ring. The "complementarity determining region" is a ring-shaped region involved in antigen recognition, and the specificity of the antibody for the antigen is determined as the sequence of this region changes.

[0127] In the present invention, the term "scFv (single chain fragment variable)" refers to a single-chain antibody produced by expressing only the variable region of an antibody through genetic recombination, and refers to an antibody in the form of a single chain in which the VH region and VL region of an antibody are linked by a short peptide chain. The term "scFv" is intended to include scFv fragments, including antigen-binding fragments, unless otherwise specified or otherwise understood from the context. This will be apparent to those skilled in the art.

[0128] In the present invention, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of the heavy and light chains of an immunoglobulin. The heavy and light chains may each include three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for antibody binding to an antigen or epitope.

[0129] In the present invention, the terms “specifically bind” or “specifically recognize” have the same meaning as commonly known to those skilled in the art, and mean that an antigen and an antibody specifically interact to cause an immunological reaction.

[0130] In the present invention, the term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure, and a part of a polypeptide that includes a portion capable of binding an antigen. For example, it may be scFv, (scFv) 2, scFv-Fc, Fab, Fab', or F(ab') 2, but is not limited thereto. Among the antigen-binding fragments, Fab has a structure having variable regions of the light chain and heavy chain, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain.

[0131] F(ab') 2 antibodies are produced by disulfide bonds between cysteine ​​residues in the hinge region of Fab'. Fv is the minimum antibody fragment that has only a heavy chain variable region and a light chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art. A two-chain Fv has a heavy chain variable region and a light chain variable region linked non-covalently, and a single-chain Fv generally has a heavy chain variable region and a single chain variable region covalently linked via a peptide linker or directly linked at the C-terminus, so that they can form a dimer-like structure like a two-chain Fv. The linker may be a peptide linker composed of any 1 to 100 or 2 to 50 amino acids, and an appropriate sequence is known in the art. The above antigen-binding fragment can be obtained using a protein hydrolytic enzyme (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab') 2 fragment can be obtained by digestion with pepsin), and can be produced through genetic recombination technology.

[0132] The term "hinge region" as used herein refers to a region contained in the heavy chain of an antibody, which exists between the CH1 and CH2 regions and functions to provide flexibility to the antigen-binding site within the antibody. For example, the hinge may be derived from a human antibody, and specifically, may be derived from IgA, IgE, or IgG, such as IgG1, IgG2, IgG3, or IgG4.

[0133] In one aspect, the present invention relates to a humanized antibody or antigen-binding fragment thereof that specifically binds to GPC3, comprising a human VH domain comprising at least one CDRH selected from the group consisting of CDRH (Complementarity determining regions Heavy chain) 1 comprising an amino acid sequence represented by SEQ ID NO: 1, CDRH2 comprising an amino acid sequence represented by SEQ ID NO: 2, and CDRH3 comprising an amino acid sequence represented by SEQ ID NO: 3; and a human VL domain comprising at least one CDRL selected from the group consisting of CDRL1 comprising an amino acid sequence represented by SEQ ID NO: 4, CDRL2 comprising an amino acid sequence represented by SEQ ID NO: 5, and CDRL3 comprising an amino acid sequence represented by SEQ ID NO: 6.

[0134] In one embodiment, the human VH domain may be at least one selected from the group consisting of VH1 comprising an amino acid sequence represented by SEQ ID NO: 11; VH2 comprising an amino acid sequence represented by SEQ ID NO: 12; VH3 comprising an amino acid sequence represented by SEQ ID NO: 13; and VH4 comprising an amino acid sequence represented by SEQ ID NO: 14.

[0135] In one embodiment, the human VL domain may be at least one selected from the group consisting of VL1 comprising an amino acid sequence represented by SEQ ID NO: 15; VL2 comprising an amino acid sequence represented by SEQ ID NO: 16; VL3 comprising an amino acid sequence represented by SEQ ID NO: 17; and VL4 comprising an amino acid sequence represented by SEQ ID NO: 18.

[0136] In one embodiment, preferably, the humanized antibody comprises a VH1 comprising an amino acid sequence represented by SEQ ID NO: 11 and a VL1 comprising an amino acid sequence represented by SEQ ID NO: 15; a VH1 comprising an amino acid sequence represented by SEQ ID NO: 11 and a VL2 comprising an amino acid sequence represented by SEQ ID NO: 16; a VH1 comprising an amino acid sequence represented by SEQ ID NO: 11 and a VL3 comprising an amino acid sequence represented by SEQ ID NO: 17; a VH1 comprising an amino acid sequence represented by SEQ ID NO: 11 and a VL4 comprising an amino acid sequence represented by SEQ ID NO: 18; a VH2 comprising an amino acid sequence represented by SEQ ID NO: 12 and a VL1 comprising an amino acid sequence represented by SEQ ID NO: 15; a VH2 comprising an amino acid sequence represented by SEQ ID NO: 12 and a VL2 comprising an amino acid sequence represented by SEQ ID NO: 16; a VH2 comprising an amino acid sequence represented by SEQ ID NO: 12 and a VL3 comprising an amino acid sequence represented by SEQ ID NO: 17; It may include VH2 comprising an amino acid sequence represented by SEQ ID NO: 12 and VL4 comprising an amino acid sequence represented by SEQ ID NO: 18; VH4 comprising an amino acid sequence represented by SEQ ID NO: 14 and VL3 comprising an amino acid sequence represented by SEQ ID NO: 17; or VH4 comprising an amino acid sequence represented by SEQ ID NO: 14 and VL4 comprising an amino acid sequence represented by SEQ ID NO: 18.

[0137] Animal-derived antibodies, produced by immunizing an animal with a desired antigen, can generally elicit immune rejection when administered to humans for therapeutic purposes. Chimeric antibodies have been developed to suppress this immune rejection. Chimeric antibodies utilize genetic engineering to replace the constant region of an animal-derived antibody, which is responsible for the anti-isotype response, with the constant region of a human antibody. While chimeric antibodies offer significant improvements in anti-isotype response compared to animal-derived antibodies, they still contain animal-derived amino acids in the variable region, posing the risk of potential anti-idiotypic responses. Humanized antibodies were developed to improve this side effect. These antibodies are produced by grafting the complementarity determining regions (CDRs), which play a crucial role in antigen binding, from the variable region of a chimeric antibody onto a human antibody framework.

[0138] The most important aspect of CDR grafting technology for producing humanized antibodies is selecting an optimized human antibody that can best accommodate the CDR region of an animal-derived antibody. To this end, antibody databases, crystal structure analysis, and molecular modeling techniques are utilized. However, even when the CDR region of an animal-derived antibody is grafted onto an optimized human antibody framework, there are cases where amino acids located in the animal-derived antibody framework affect antigen binding, and thus antigen-binding ability is not preserved in many cases. Therefore, the application of additional antibody engineering techniques to restore antigen-binding ability can be said to be essential.

[0139] The antibody of the present invention or a fragment thereof having immunological activity, or a humanized antibody or antigen-binding fragment thereof, can be produced by any method known in the art. After encoding the polypeptide sequence of the antibody of the present invention or a fragment thereof having immunological activity, or a humanized antibody or antigen-binding fragment thereof, if desired, it is cloned into a host cell and used to form a nucleic acid that is expressed and assayed.

[0140] The nucleic acid encoding the antibody of the present invention or a fragment thereof having immunological activity, or a humanized antibody or an antigen-binding fragment thereof, can be inserted into an expression vector for protein expression. The expression vector typically includes the protein operably linked, i.e., in a functional relationship, with regulatory sequences, a selectable marker, an optional fusion partner, and / or additional elements. Under appropriate conditions, the antibody or a fragment thereof having immunological activity, or a humanized antibody or an antigen-binding fragment thereof, according to the present invention, can be produced by a method of inducing protein expression by culturing a host cell transformed with the nucleic acid, preferably an expression vector containing a nucleic acid encoding the antibody of the present invention or a fragment thereof having immunological activity, or a humanized antibody or an antigen-binding fragment thereof. Various suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are well known in the art and will vary depending on the host cell used. Preferably, the antibody or a fragment thereof having immunological activity, or a humanized antibody or an antigen-binding fragment thereof according to the present invention is produced using E. coli, which has a low production cost and high industrial utility value, as a host cell.

[0141] Accordingly, the scope of the present invention includes a method for producing an isolated antibody or a fragment thereof having immunological activity, or a humanized antibody or an antigen-binding fragment thereof that specifically binds to a GPC3 protein, comprising the steps of: culturing a host cell into which a nucleic acid encoding the antibody or a fragment thereof having immunological activity, or a humanized antibody or an antigen-binding fragment thereof, has been introduced under conditions suitable for protein expression; and purifying or separating the antibody or a fragment thereof having immunological activity expressed from the host cell.

[0142] Antibodies can be isolated or purified using a variety of methods known in the art. Standard purification methods include chromatography, electrophoresis, immunoassays, precipitation, dialysis, filtration, concentration, and chromatofocusing. As is known in the art, various natural proteins, such as bacterial proteins A, G, and L, bind to antibodies and can be used for purification. Often, purification using specific fusion partners may be possible.

[0143] In one aspect, the present invention relates to an isolated cell producing an antibody of the present invention or a fragment thereof having immunological activity, or a humanized antibody or an antigen-binding fragment thereof.

[0144] In one embodiment, the cell may be a bacterial cell, a yeast cell, a mammalian cell, an insect cell or a hybridoma cell, most preferably a hybridoma cell line.

[0145] In one aspect, the present invention relates to an isolated nucleic acid molecule encoding an antibody of the present invention or a fragment thereof having immunological activity, or a humanized antibody or an antigen-binding fragment thereof, a vector comprising the same, and a host cell transformed therewith.

[0146] In one embodiment, the nucleic acid molecule encoding the antibody or a fragment thereof having immunological activity may comprise a base sequence represented by any one or more sequence numbers selected from the group consisting of SEQ ID NOs: 9 and 10.

[0147] The nucleic acid molecules of the present invention may be isolated or recombinant, and include DNA and RNA in single-stranded and double-stranded forms, as well as corresponding complementary sequences. An isolated nucleic acid is a nucleic acid that has been separated from the surrounding genetic sequence present in the genome of the organism from which the nucleic acid was isolated, in the case of a nucleic acid isolated from a naturally occurring source. In the case of a nucleic acid synthesized enzymatically or chemically from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, the nucleic acid resulting from such a procedure may be understood as an isolated nucleic acid molecule. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, the DNA of a presequence or secretory leader is operably linked to the DNA of a polypeptide if the polypeptide is expressed as a preprotein, i.e., the form in which the polypeptide is secreted; a promoter or enhancer is operably linked to a coding sequence if it influences the transcription of the polypeptide sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Operably linked DNA sequences are generally contiguous, and in the case of a secretory leader, contiguous and in the same reading frame. However, enhancers need not be contiguous. Linkage is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in a conventional manner.

[0148] The isolated nucleic acid molecule encoding the antibody of the present invention or a fragment having immunological activity thereof, or a humanized antibody or an antigen-binding fragment thereof, may have various modifications in the coding region within a range that does not change the amino acid sequence of the antibody expressed from the coding region due to codon degeneracy or in consideration of the codons preferred in the organism to which the antibody is to be expressed, and various modifications or alterations may also be made in a portion excluding the coding region within a range that does not affect the expression of the gene, and it will be well understood by those skilled in the art that such modified genes are also included in the scope of the present invention. That is, the nucleic acid molecule of the present invention may have one or more nucleic acid bases mutated by substitution, deletion, insertion, or a combination thereof, as long as it encodes a protein having an activity equivalent thereto, and these are also included in the scope of the present invention. The sequence of such a nucleic acid molecule may be single-stranded or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.

[0149] An isolated nucleic acid molecule encoding an antibody of the present invention or a fragment thereof having immunological activity, or a humanized antibody or an antigen-binding fragment thereof, according to the present invention, can be inserted into an expression vector for protein expression. The expression vector typically comprises the protein operably linked, i.e., in a functional relationship, with regulatory sequences, a selectable marker, an optional fusion partner, and / or additional elements. Under appropriate conditions, a host cell transformed with the nucleic acid, preferably an expression vector containing an isolated nucleic acid molecule encoding an antibody of the present invention or a fragment thereof having immunological activity, or a humanized antibody or an antigen-binding fragment thereof, can be produced by inducing protein expression by culturing the expression vector. Various suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are well known in the art and will vary depending on the host cell used. Preferably, E. coli, which has a low production cost and high industrial utility value, can be produced as a host cell.

[0150] The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, a viral vector, and the like. A suitable vector may include, in addition to expression control elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer, a signal sequence or a leader sequence for membrane targeting or secretion, and may be manufactured in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The signal sequence may include, but is not limited to, a PhoA signal sequence, an OmpA signal sequence, etc. when the host is an Escherichia sp. fungus; an α-amylase signal sequence, a subtilisin signal sequence, etc. when the host is a Bacillus sp. fungus; an MFα signal sequence, a SUC2 signal sequence, etc. when the host is a yeast; and an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, etc. when the host is an animal cell. Additionally, the vector may include a selection marker for selecting host cells containing the vector, and, if it is a replicable expression vector, an origin of replication.

[0151] As used herein, the term "vector" refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence. The nucleic acid sequence may be exogenous or heterologous. Examples of vectors include, but are not limited to, plasmids, cosmids, and viruses (e.g., bacteriophages).

[0152] A person skilled in the art can construct the vector by standard recombinant techniques.

[0153] In one embodiment, when producing the vector, expression control sequences such as promoters, terminators, enhancers, etc., sequences for membrane targeting or secretion, etc. may be appropriately selected and combined in various ways according to the purpose, depending on the type of host cell to be used to produce the antibody.

[0154] As used herein, the term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a transcribed gene product. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors may contain various regulatory sequences. In addition to regulatory sequences that regulate transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that provide additional functions.

[0155] In the present invention, the term "host cell" includes eukaryotes and prokaryotes, and refers to any transformable organism capable of replicating the vector or expressing a gene encoded by the vector. The host cell may be transfected or transformed by the vector, which refers to the process by which an exogenous nucleic acid molecule is transferred or introduced into the host cell.

[0156] In one embodiment, the host cell can be a bacterial cell, a yeast cell, a mammalian cell, an insect cell or a hybridoma cell, and can be a CHO cell, a HEK 293 cell, a CAP cell, a Per.C6 cell, a BHK cell, a Vero cell, an MDCK cell, a hybridoma cell or a fibroblast cell.

[0157] In one aspect, the present invention relates to a composition for detecting a cancer antigen comprising an antibody according to the present invention or a fragment thereof having immunological activity, or a humanized antibody according to the present invention or an antigen-binding fragment thereof.

[0158] In one embodiment, the cancer antigen may be GPC3.

[0159] The term "detection" in the present invention refers to the detection of an antigen-antibody complex, and can be performed using various labels. Specific examples of such labels include, but are not limited to, enzymes, fluorescent substances, ligands, luminescent substances, microparticles, or radioactive isotopes.

[0160] Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, and β-ratamase; fluorescent substances include fluorescein, Eu3+, Eu3+ chelate, or cryptate; ligands include biotin derivatives; luminescent substances include acridinium ester and isoluminol derivatives; microparticles include colloidal gold and colored latex; and radioactive isotopes include 57Co, 3H, 125I, and 125I-Bonton Hunter reagent.

[0161] The detection composition of the present invention can detect antigen-antibody complexes using an enzyme-linked immunosorbent assay (ELISA). ELISA includes various ELISA methods, such as a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of antibodies that recognize the antigen attached to the solid support, a direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of antibodies and antigens attached to the solid support, and an indirect sandwich ELISA using a labeled secondary antibody that recognizes the antibody after reacting it with another antibody that recognizes the antigen in a complex of antibodies and antigens attached to the solid support. The antibodies according to the present invention may have a detection label, and when they do not have a detection label, these monoclonal antibodies can be captured and identified by treating them with another antibody that has a detection label.

[0162] Furthermore, the "detection" described herein can also be achieved through label-free methods that do not use a label. Specifically, methods selected include surface plasmon resonance, isothermal titration calorimetry, and bio-layer interferometry. However, the present invention is not limited to these methods.

[0163] In one aspect, the present invention relates to a kit for detecting a cancer antigen comprising an antibody according to the present invention or a fragment thereof having immunological activity, or a humanized antibody according to the present invention or an antigen-binding fragment thereof.

[0164] In one embodiment, the cancer antigen may be GPC3.

[0165] The cancer antigen detection kit of the present invention may include an antibody or a fragment thereof having immunological activity according to the present invention, or a humanized antibody or an antigen-binding fragment thereof according to the present invention, and tools and reagents used for immunological analysis. Tools / reagents used for immunological analysis include a suitable carrier, a labeling substance capable of generating a detectable signal, a solubilizing agent, a detergent, and the like. Furthermore, if the labeling substance is an enzyme, a substrate capable of measuring enzyme activity and a reaction terminator may be included.

[0166] Suitable carriers include, but are not limited to, soluble carriers, such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers, such as polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesins, cross-linked dextrans, polysaccharides, latex-plated magnetic microparticles, other paper, glass, metal, agarose, and combinations thereof.

[0167] Assay systems for use in the detection composition or kit of the present invention include, but are not limited to, ELISA plates, dip-stick devices, immunochromatographic test strips, radial fractionation immunoassay devices, and flow-through devices.

[0168] In one aspect, the present invention relates to an antibody-drug conjugate (ADC) comprising an isolated antibody of the present invention or a fragment thereof having immunological activity, or a humanized antibody of the present invention or an antigen-binding fragment thereof; and a drug.

[0169] In one embodiment, the drug may be an immunogenic apoptosis inducer, a microtubulin structure formation inhibitor, a meiosis inhibitor, a topoisomerase inhibitor, a DNA intercalator, a toxin, or a radionuclide, and the immunogenic apoptosis inducer may be at least one selected from the group consisting of anthracycline-based anticancer agents, taxane-based anticancer agents, anti-EGFR antibodies, BK channel agonists, bortezomib, cardiac glycosides, cyclophosphamide-based anticancer agents, GADD34 / PP1 inhibitors, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, or oxaliplatin, but is not limited thereto.

[0170] In one embodiment, the drug is SN-38 (7-Ethyl-10-hydroxy-camptothecin), daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), Streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C, vincristine, vinblastine, teniposide, topotecan, iridotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, duocarmycin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine,Capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, izabepilone, vindesine, vinorelbine, estramustine, maytansine, DM1 (mertansine), DM4, dolastatin, auristatin E, It may be selected from the group consisting of auristatin F, monomethyl auristatin E (MMAE), monomethyl auristatin F, and derivatives thereof, but is not limited thereto.

[0171] In one embodiment, the antibody-drug conjugate may further comprise an ADC linker, which may be, but is not limited to, 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC), valine-citrulline-p-aminobenzyloxycarbonyl (val-cit-PAB), or N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB).

[0172] In one embodiment, the antibody-drug conjugate can form a complex with the drug via an ADC linker, the antibody of the present invention or a fragment thereof having immunological activity.

[0173] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, comprising an antibody-drug conjugate comprising an isolated antibody of the present invention or a fragment thereof having immunological activity, or a humanized antibody of the present invention or an antigen-binding fragment thereof; and a drug.

[0174] In one embodiment, the cancer may be a cancer refractory to an immune checkpoint inhibitor.

[0175] In one embodiment, the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer (TNBC), lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem It may be one or more selected from the group consisting of, but is not limited to, glioma and pituitary adenoma.

[0176] The pharmaceutical composition of the present invention for preventing or treating cancer can be administered in combination with a chemotherapy drug (anticancer agent) to enhance the cancer treatment effect of conventional anticancer agents through its cancer cell killing effect. Co-administration may occur simultaneously with or sequentially with the anticancer agent. The anticancer agent may be one or more selected from the group consisting of cytotoxic anticancer agents, targeted anticancer agents, and immunotherapy agents, but is not limited thereto.The cytotoxic anticancer agent may be doxorubicin, cyclophosphamide, temozolomide, irinotecan, cisplatin, vinblastine, vincristine, actinomycin-D, 5-fluorouracil, docetaxel, cabazitaxel, paclitaxel, or pembrolizumab; Targeted anticancer drugs include trametinib, vemurafenib, alpelisib, dactolisib, gefitinib, erlotinib, lapatinib, sunitinib, sorafenib, crizotinib, dabrafenib, trastuzumab, cetuximab, bevacizumab, panitumumab, ipilimumab, pertuzumab, tofacitinib, imatinib, bortezomib, ofatumumab, or It may be Alemtuzumab; and the immunotherapy agent may be anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-LAG-3, anti-TIGIT, anti-TIM-3, anti-GITR, CAR-T or NK cell.

[0177] In addition, DNA alkylating agents include mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, and carboplatin; anti-cancer antibiotics include dactinomycin (actinomycin D), plicamycin, and mitomycin C; and plant alkaloids such as vincristine, vinblastine, etoposide, teniposide, topotecan, and iridotecan, but are not limited thereto.

[0178] In the present invention, the term “prevention” means any act of inhibiting or delaying the occurrence, spread, and recurrence of cancer by administering a pharmaceutical composition according to the present invention.

[0179] The term "treatment" as used herein refers to any action that kills cancer cells or improves or beneficially alters the symptoms of cancer through administration of the composition of the present invention. Those skilled in the art to which the present invention pertains will be able to reference materials provided by the Korean Medical Association and other sources to determine the precise criteria for diseases for which the composition of the present invention is effective, and to determine the degree of improvement, enhancement, and treatment achieved.

[0180] The term "therapeutically effective amount" in the present invention refers to the amount of a pharmaceutically acceptable salt of a composition effective in preventing or treating a target disease. The therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the administration method, target site, and patient condition. Therefore, when used in the human body, the dosage should be determined as an appropriate amount while taking both safety and efficacy into consideration.

[0181] It is also possible to estimate the amount to be used in humans from the effective dose determined through animal testing.

[0182] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. The effective dosage level may be determined based on factors including the patient's health condition, cancer type and severity, drug activity, water sensitivity, administration method, administration time, administration route and excretion rate, treatment period, combination or concurrent use of drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to pharmaceutically composition an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0183] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0184] The composition of the present invention may also include a carrier, diluent, excipient, or a combination of two or more thereof commonly used in biological preparations. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and for example, a compound, saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. If necessary, other common additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into a main use form such as an aqueous solution, suspension, emulsion, pills, capsules, granules, or tablets. Furthermore, the composition may be preferably formulated according to each disease or component using an appropriate method in the art.

[0185] The composition of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally as an injection formulation) or orally, depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and it is more preferable to administer the pharmaceutical composition once or several times a day.

[0186] Liquid preparations for oral administration of the composition of the present invention include suspensions, solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc.

[0187] In one aspect, the present invention relates to a composition for diagnosing cancer comprising an isolated antibody of the present invention or a fragment thereof having immunological activity, a humanized antibody of the present invention or an antigen-binding fragment thereof, or an antibody-drug conjugate of the present invention.

[0188] In one aspect, the present invention relates to a kit for diagnosing cancer comprising an isolated antibody of the present invention or a fragment thereof having immunological activity, a humanized antibody of the present invention or an antigen-binding fragment thereof, or an antibody-drug conjugate of the present invention.

[0189] In the present invention, the specific description of the cancer diagnosis composition and cancer diagnosis kit is the same as that described in the above cancer antigen detection composition and cancer antigen detection kit.

[0190] In one aspect, the present invention provides a method for providing information for diagnosing cancer, comprising the steps of treating a sample with an isolated antibody of the present invention or a fragment having immunological activity thereof, a humanized antibody of the present invention or an antigen-binding fragment thereof, or an antibody-drug conjugate of the present invention, labeled with a fluorescent substance; and measuring the expression of the isolated antibody or a fragment having immunological activity thereof, a humanized antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate.

[0191] The above fluorescent material has the same meaning as the “label” of the above cancer antigen detection composition, and the description thereof is also the same.

[0192] In one embodiment, when expression of the isolated antibody or a fragment thereof having immunological activity, a humanized antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate is measured, a diagnosis of cancer can be made.

[0193] In one embodiment, the term "sample" is used in the broadest sense. On the one hand, it encompasses a specimen or culture (e.g., a microbial culture). On the other hand, it encompasses both biological and environmental samples. Furthermore, the sample may include a sample of synthetic origin. The above biological samples include whole blood, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, leukocytes, peripheral blood mononuclear cells, buffy coat, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, trachea. It may be any one or more selected from the group consisting of, but is not limited to, organ secretions, cells, cell extracts, and cerebrospinal fluid.

[0194] In one aspect, the present invention relates to a method for producing an isolated antibody or a fragment having immunological activity thereof, or a humanized antibody or an antigen-binding fragment thereof, which specifically binds to a GPC3 protein, comprising the step of culturing a hybridoma cell line producing the isolated antibody or a fragment having immunological activity thereof, or a humanized antibody or an antigen-binding fragment thereof, of the present invention.

[0195] In one aspect, the present invention relates to a method for producing an isolated antibody or a fragment having immunological activity thereof, or a humanized antibody or an antigen-binding fragment thereof, which specifically binds to a GPC3 protein, comprising the steps of: culturing a host cell transformed with a vector comprising an isolated nucleic acid molecule encoding an isolated antibody or a fragment having immunological activity thereof, or a humanized antibody or an antigen-binding fragment thereof; and recovering the antibody or the fragment having immunological activity thereof from the host cell culture.

[0196] In one aspect, the present invention relates to a GPC3-specific chimeric antigen receptor (CAR) comprising a single chain variable fragment (scFv) as an antigen-binding domain, wherein the single chain variable fragment comprises a heavy chain variable region comprising CDRH (Complementarity determining regions Heavy chain) 1 comprising an amino acid sequence represented by SEQ ID NO: 1, CDRH2 comprising an amino acid sequence represented by SEQ ID NO: 2, and CDRH3 comprising an amino acid sequence represented by SEQ ID NO: 3; and a light chain variable region comprising CDRL1 comprising an amino acid sequence represented by SEQ ID NO: 4, CDRL2 comprising an amino acid sequence represented by SEQ ID NO: 5, and CDRL3 comprising an amino acid sequence represented by SEQ ID NO: 6.

[0197] In one embodiment, the light chain variable region may comprise an amino acid sequence represented by SEQ ID NO: 19.

[0198] In one embodiment, the heavy chain variable region may comprise an amino acid sequence represented by SEQ ID NO: 20.

[0199] In one embodiment, the heavy chain variable region and the light chain variable region may be connected by a GS linker. The GS linker may comprise an amino acid sequence represented by SEQ ID NO: 21.

[0200] Therefore, in one embodiment, the GPC3-specific chimeric antigen receptor of the present invention, in which a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 19 and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 20 are linked by a GS linker comprising an amino acid sequence represented by SEQ ID NO: 21, may include an scFv comprising an amino acid sequence represented by SEQ ID NO: 22.

[0201] In one aspect, the present invention relates to a GPC3-specific humanized antibody-derived chimeric antigen receptor (CAR), comprising a single chain variable fragment (scFv) as an antigen-binding domain, wherein the single chain variable fragment comprises: a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11; a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12; a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 13 and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 14; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 15; a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 16; a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 17 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 18.

[0202] In one embodiment, the scFv may include any one selected from the group consisting of a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 15; a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 17; a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 16; and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 18.

[0203] In one embodiment, the heavy chain variable region and the light chain variable region may be connected by a GS linker. The GS linker may include an amino acid sequence represented by SEQ ID NO: 21.

[0204] Therefore, in one embodiment, the GPC3-specific humanized antibody-derived chimeric antigen receptor of the present invention, in which a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 15 are linked by a GS linker comprising an amino acid sequence represented by SEQ ID NO: 21, may include an amino acid sequence represented by SEQ ID NO: 23.

[0205] In another embodiment, the GPC3-specific humanized antibody-derived chimeric antigen receptor of the present invention, wherein a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 17 are linked by a GS linker comprising an amino acid sequence represented by SEQ ID NO: 21, may include an amino acid sequence represented by SEQ ID NO: 24.

[0206] In addition, in one embodiment, the GPC3-specific humanized antibody-derived chimeric antigen receptor of the present invention, wherein a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 16 are linked by a GS linker comprising an amino acid sequence represented by SEQ ID NO: 21, may include an amino acid sequence represented by SEQ ID NO: 25.

[0207] In addition, in one embodiment, the GPC3-specific humanized antibody-derived chimeric antigen receptor of the present invention, wherein a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 18 are linked by a GS linker comprising an amino acid sequence represented by SEQ ID NO: 21, may include an amino acid sequence represented by SEQ ID NO: 26.

[0208] The antibodies, humanized antibodies, GPC3-specific chimeric antigen receptors, or GPC3-specific humanized antibody-derived CARs of the present invention may include variants of the amino acid sequences set forth in the attached sequence listing within the scope of specifically recognizing GPC3. For example, the amino acid sequence of the antibody may be changed to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody.

[0209] The above amino acid mutations are based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0210] When introducing mutations, the hydrophobicity index of an amino acid can be considered. Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0211] The hydrophobic amino acid index is crucial for imparting interactive biological functions to proteins. It is well known that amino acids with similar hydrophobic indices must be substituted to retain similar biological activity. When introducing mutations based on hydrophobic indices, substitutions are preferably made between amino acids with a difference in hydrophobicity index of within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0212] Meanwhile, it is also well known that substitutions between amino acids with similar hydrophilicity values ​​result in proteins with equivalent biological activity. The following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

[0213] When introducing mutations with reference to hydrophilicity values, substitutions are made between amino acids that exhibit a difference in hydrophilicity values ​​of preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0214] The most common exchanges in proteins that do not alter the overall activity of the molecule are those between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

[0215] As used herein, the term "CAR (chimeric antigen receptor)" refers to a non-naturally occurring receptor capable of imparting specificity for a specific antigen to immune effector cells. Typically, the CAR refers to a receptor used to transplant the specificity of a monoclonal antibody into T cells. In addition, the GPC3-specific chimeric antigen receptor or GPC3-specific humanized antibody-derived chimeric antigen receptor of the present invention may further include at least one selected from the group consisting of a signal peptide, a stalk domain, a membrane-penetrating domain, and an intracellular signaling domain.

[0216] In the present invention, the signal peptide serves to send the initial protein to the endoplasmic reticulum, and any polypeptide known to have the same or similar function as the signal peptide may be used without limitation, regardless of whether derived from a natural or synthetic source. This may include, but is not limited to, one or more selected from the group consisting of CD8, CD28, GM-CSF, CD4, and CD137.

[0217] In the present invention, the term “stem domain” means any polypeptide that functions to link a membrane-penetrating domain to the scFv of the present invention. Any polypeptide known to have the same or similar function as the above may be used without limitation, regardless of whether it is derived from a natural or synthetic source. It may be, but is not limited to, one selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, and CD8 hinge.

[0218] In the present invention, the term “transmembrane domain” means any polypeptide that allows the CAR according to the present invention to be expressed on the surface membrane of a cell. A transmembrane domain suitable for the CAR disclosed in the present invention has (a) the ability to be expressed on the surface of a cell, for example, an immune cell, such as, but not limited to, a lymphocyte cell or a natural killer (NK) cell, and (b) the ability to interact with the scFv and an intracellular signaling domain according to the present invention to direct a cellular response of the immune cell to a predefined target cell. The transmembrane domain may also be used without limitation, regardless of whether it is derived from a natural or synthetic source, as long as it is a polypeptide known to have the same or similar function as described above. but are not limited to, FcγR, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2Rβ, CD40, DAP10, MHC class I molecule, TNF receptor protein, Immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activating NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1,CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83-specific ligand, CD247, CD3δ, CD3ε, CD3γ, CD3ζ, CD8, Ig alpha (CD79a), IL-2Rγ, IL-7Rα, PD-1, TNFSF14, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD154, alpha chain of T cell receptor, beta chain of T cell receptor and may be at least one selected from the group consisting of the zeta chain of the T cell receptor.

[0219] In the present invention, the term “intracellular signaling domain” refers to a portion of a protein that transmits an effector signaling function signal and instructs a cell to perform a specialized function. This is responsible for intracellular signaling after the scFv according to the present invention binds to a target, thereby causing T cell activation. In addition, the intracellular signaling domain may be used without limitation regardless of whether it is derived from a natural or synthetic source, as long as it is a polypeptide known to have the same or similar function as the above.

[0220] Although not limited thereto, the intracellular transduction domain may be selected from the group consisting of CD3ζ, FcγR, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2Rβ, IL-15R-α, MyD88, DAP10, DAP12, MHC class I molecule, TNF receptor protein, Immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activating NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CD40, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, The immunoglobulin may be at least one selected from the group consisting of IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 specific ligand, CD247, CD3δ, CD3ε, CD3γ, CD8, Ig alpha (CD79a), IL-2Rγ, IL-7Rα, PD-1, and TNFSF14.

[0221] The GPC3-specific chimeric antigen receptor or GPC3-specific humanized antibody-derived chimeric antigen receptor of the present invention may further include a tag peptide that can be used to determine whether it is expressed. Any known tag peptide, such as a flag peptide, may be used without limitation.

[0222] In one embodiment, a GPC3-specific CAR comprising a CD8 signal peptide, a FLAG tag, a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 19, a GS linker represented by SEQ ID NO: 21, a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 20, CD28 and CD3ζ sequentially linked comprises an amino acid sequence represented by SEQ ID NO: 28.

[0223] In one embodiment, a GPC3-specific CAR comprises a CD8 signal peptide, a FLAG tag, a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 19, a GS linker represented by SEQ ID NO: 21, a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 20, a CD8 transmembrane domain, 4-1BB and CD3ζ sequentially linked to the amino acid sequence represented by SEQ ID NO: 29.

[0224] In one embodiment, a GPC3-specific humanized antibody-derived CAR comprises a CD8 signal peptide, a FLAG tag, a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 15, a GS linker represented by SEQ ID NO: 21, a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11, CD28 and CD3ζ sequentially linked, and comprises an amino acid sequence represented by SEQ ID NO: 30.

[0225] In one embodiment, a GPC3-specific humanized antibody-derived CAR comprises a CD8 signal peptide, a FLAG tag, a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 17, a GS linker represented by SEQ ID NO: 21, a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11, CD28 and CD3ζ sequentially linked, and comprises an amino acid sequence represented by SEQ ID NO: 31.

[0226] In one embodiment, a GPC3-specific humanized antibody-derived CAR comprises a CD8 signal peptide, a FLAG tag, a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 16, a GS linker represented by SEQ ID NO: 21, a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12, CD28 and CD3ζ sequentially linked, and comprises an amino acid sequence represented by SEQ ID NO: 32.

[0227] In one embodiment, a GPC3-specific humanized antibody-derived CAR comprises a CD8 signal peptide, a FLAG tag, a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 18, a GS linker represented by SEQ ID NO: 21, a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12, CD28 and CD3ζ sequentially linked, and comprises an amino acid sequence represented by SEQ ID NO: 33.

[0228] In one aspect, the present invention relates to a polynucleotide comprising a nucleic acid sequence encoding a GPC3-specific CAR according to the present invention or a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0229] The nucleic acid molecules of the present invention may be isolated or recombinant, and include DNA and RNA in single-stranded and double-stranded forms, as well as corresponding complementary sequences. An isolated nucleic acid is a nucleic acid that has been separated from the surrounding genetic sequence present in the genome of the organism from which the nucleic acid was isolated, in the case of a nucleic acid isolated from a naturally occurring source. In the case of a nucleic acid synthesized enzymatically or chemically from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, the nucleic acid resulting from such a procedure may be understood as an isolated nucleic acid molecule. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, the DNA of a presequence or secretory leader is operably linked to the DNA of a polypeptide if the polypeptide is expressed as a preprotein, i.e., the form in which the polypeptide is secreted; a promoter or enhancer is operably linked to a coding sequence if it influences the transcription of the polypeptide sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Operably linked DNA sequences are generally contiguous, and in the case of a secretory leader, contiguous and in the same reading frame. However, enhancers need not be contiguous. Linkage is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in a conventional manner.

[0230] The polynucleotide encoding the GPC3-specific CAR of the present invention or the GPC3-specific humanized antibody-derived CAR of the present invention may have various modifications in the coding region within a range that does not change the amino acid sequence of the antibody expressed from the coding region due to the degeneracy of the codon or in consideration of the codon preferred in the organism to which the antibody is to be expressed, and various modifications or alterations may be made in a portion excluding the coding region within a range that does not affect the expression of the gene, and it will be well understood by those skilled in the art that such modified genes are also included in the scope of the present invention. That is, the nucleic acid molecule of the present invention may have one or more nucleic acid bases mutated by substitution, deletion, insertion, or a combination thereof, as long as it encodes a protein having an activity equivalent thereto, and these are also included in the scope of the present invention. The sequence of such a nucleic acid molecule may be single-stranded or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.

[0231] The GPC3-specific CAR of the present invention or the CAR derived from the GPC3-specific humanized antibody of the present invention can be produced by any method known in the art. After encoding the polypeptide sequence of the GPC3-specific CAR of the present invention or the CAR derived from the GPC3-specific humanized antibody of the present invention, if desired, it is cloned into a host cell and used to form a nucleic acid that is expressed and assayed.

[0232] A nucleic acid encoding the GPC3-specific CAR of the present invention or the GPC3-specific humanized antibody-derived CAR of the present invention can be inserted into an expression vector for protein expression.

[0233] In one aspect, the present invention relates to a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a GPC3-specific CAR according to the present invention or a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0234] In one aspect, the present invention provides a method for producing a GPC3-specific CAR or a GPC3-specific humanized antibody-derived CAR, comprising the step of infecting a host cell with a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a GPC3-specific CAR according to the present invention or a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0235] The vector of the present invention and the method for producing a GPC3-specific CAR or a GPC3-specific humanized antibody-derived CAR using the same can be equally applied to the descriptions given above for the antibody of the present invention or a fragment thereof having immunological activity, or the humanized antibody or an antigen-binding fragment thereof.

[0236] In one aspect, the present invention relates to a chimeric antigen receptor expressing cell transformed with a GPC3-specific CAR of the present invention.

[0237] In one aspect, the present invention relates to a chimeric antigen receptor expressing cell transformed with a GPC3-specific humanized antibody-derived CAR of the present invention.

[0238] In one embodiment, the chimeric antigen receptor expressing cell can be a chimeric antigen receptor expressing macrophage (CAR-macrophage), a chimeric antigen receptor expressing T (CAR-T) cell, a chimeric antigen receptor expressing-gamma-delta T (CAR-Gamma-delta T) cell, or a natural killer (CAR-NK) cell.

[0239] In the present invention, the term "chimeric antigen receptor-expressing T (CAR-T) cell" refers to a T cell expressing a CAR. The chimeric antigen receptor-expressing T (CAR-T) cell has the advantage of: i) recognizing a cancer antigen in a manner independent of HLA (human leukocyte antigen), and thus being able to treat cancers that evade the action of anticancer drugs by reducing HLA expression on the cell surface; ii) being independent of HLA type, and thus being able to be used for treatment regardless of a patient's HLA type; and iii) being able to produce a large amount of cancer-specific T cells in a short period of time, and thus being able to exhibit an excellent anticancer effect.

[0240] The above T cells are CD4 + T cells (helper T cells, TH cells), CD8 + There are T cells (cytotoxic T cells, CTL), memory T cells, regulatory T cells (Treg cells), natural killer T cells, etc., and in the present invention, the T cells into which the CAR is introduced are preferably CD8 + T cells, but not limited to these.

[0241] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising a GPC3-specific CAR according to the present invention, a GPC3-specific humanized antibody-derived CAR according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0242] The pharmaceutical composition of the present invention can be equally applied to the descriptions given above for the antibody of the present invention or a fragment thereof having immunological activity, or a humanized antibody or an antigen-binding fragment thereof.

[0243] In one aspect, the present invention relates to a cell therapy for preventing or treating cancer, comprising a chimeric antigen receptor expressing cell transformed with a GPC3-specific CAR according to the present invention or a chimeric antigen receptor expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0244] The term "cell therapy agent" used in the present invention refers to a drug (as defined by the US FDA) used for the purposes of treatment, diagnosis, and prevention by separating, culturing, and manufacturing cells and tissues from an individual through special manipulation, and by proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro to restore the function of cells or tissues, or by changing the biological characteristics of cells through other methods.

[0245] In one aspect, the present invention relates to an anticancer adjuvant comprising a GPC3-specific CAR according to the present invention, a GPC3-specific humanized antibody-derived CAR according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0246] In one embodiment, the anticancer adjuvant of the present invention may be administered concurrently, separately, or sequentially with the immunotherapy agent.

[0247] In one embodiment, the anticancer adjuvant can improve refractoriness to immunotherapy.

[0248] In one embodiment, the immunotherapy agent may be an immune checkpoint inhibitor, an immunosuppressant controlling drug, a cancer vaccine, an immunoadjuvant, an immune cell for cancer treatment, an immune cell activation cofactor, an antibody for cancer treatment, or a cytokine required for maintaining the activity of an immune cell for cancer treatment.

[0249] In one embodiment, the immune checkpoint inhibitor may be an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA or A2aR.

[0250] In one aspect, the present invention relates to a composition for diagnosing cancer, comprising a GPC3-specific CAR according to the present invention, a CAR derived from a GPC3-specific humanized antibody according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a chimeric antigen receptor-expressing cell transformed with a CAR derived from a GPC3-specific humanized antibody according to the present invention.

[0251] In one aspect, the present invention relates to a cancer diagnostic kit comprising a GPC3-specific CAR according to the present invention, a GPC3-specific humanized antibody-derived CAR according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0252] In one embodiment, the cancer diagnostic composition and cancer diagnostic kit of the present invention can diagnose cancer by detecting GPC3, a cancer antigen.

[0253] The cancer diagnostic composition and cancer diagnostic kit of the present invention can be equally applied to the descriptions given above for the antibody of the present invention or a fragment thereof having immunological activity, or a humanized antibody or an antigen-binding fragment thereof.

[0254] In one aspect, the present invention provides a method for providing information for diagnosing cancer, comprising the steps of treating a sample with a GPC3-specific CAR according to the present invention labeled with a fluorescent substance, a GPC3-specific humanized antibody-derived CAR according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention; and measuring the expression of the GPC3-specific CAR, the GPC3-specific humanized antibody-derived CAR, the chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR, or the chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR.

[0255] In one embodiment, when expression of the GPC3-specific CAR, the GPC3-specific humanized antibody-derived CAR, the chimeric antigen receptor-expressing cell transformed with the GPC3-specific CAR, or the chimeric antigen receptor-expressing cell transformed with the GPC3-specific humanized antibody-derived CAR is measured, cancer can be diagnosed as having developed.

[0256] The information providing method of the present invention can be equally applied to the descriptions given above regarding the antibody of the present invention or a fragment thereof having immunological activity, or a humanized antibody or an antigen-binding fragment thereof.

[0257] In one aspect, the present invention provides a method for detecting a cancer antigen, comprising the step of administering to a subject an isolated antibody according to the present invention or a fragment having immunological activity thereof, a humanized antibody according to the present invention or an antigen-binding fragment thereof, a GPC3-specific CAR according to the present invention, a CAR derived from a GPC3-specific humanized antibody according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a chimeric antigen receptor-expressing cell transformed with a CAR derived from a GPC3-specific humanized antibody according to the present invention.

[0258] In one embodiment, the cancer antigen may be GPC3.

[0259] In addition, in one aspect, the present invention provides a method for preventing or treating cancer, comprising the step of administering to a subject an antibody-drug conjugate according to the present invention, a GPC3-specific CAR according to the present invention, a GPC3-specific humanized antibody-derived CAR according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention, a cell therapeutic agent comprising a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a cell therapeutic agent comprising a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0260] In one embodiment, the cancer may be a cancer refractory to an immune checkpoint inhibitor.

[0261] In one embodiment, the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer (TNBC), lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem It may be any one or more selected from the group consisting of, but is not limited to, gliomas and pituitary adenomas.

[0262] In one aspect, the present invention provides a method for enhancing the anticancer treatment effect in a subject, comprising the step of administering to the subject a GPC3-specific CAR according to the present invention, a GPC3-specific humanized antibody-derived CAR according to the present invention, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR according to the present invention, or a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR according to the present invention.

[0263] In one embodiment, enhancing the anticancer therapeutic effect of the subject may be improving refractoriness to immunotherapy.

[0264] The above-mentioned individual is preferably a mammal, including a human, and is a patient who needs to be confirmed whether or not he or she has a cancer antigen, or a patient who needs treatment for a cancer disease, including all patients who are undergoing treatment, patients who have received treatment, and patients who need to receive treatment, and may also include patients who have undergone surgical operation for the treatment of a cancer disease.

[0265]

[0266] The present invention is described in more detail through the following examples. However, the following examples are intended only to concretize the content of the present invention and are not intended to limit the present invention.

[0267]

[0268] Example 1. Production of GPC3 (Glypican-3)-specific antibodies

[0269] 1-1. Antibody production

[0270] Balb / c wild-type mice were immunized twice with GPC3 protein, and after the second immunization, immune cells in the lymph nodes were isolated and fused with the SP2 / 0 cell line to produce hybridoma cells. The produced hybridoma cells were cultured as single-cell clones, and to confirm the specificity of each clone for GPC3, the supernatant of each clone was used for ELISA analysis to derive a GPC3-specific hybridoma clone 10-1.4 (Figs. 1 and 2). Thereafter, HepG2 cell lines and A549 cells (negative control group) overexpressing GPC3 were divided into the following groups, stained with each antibody, and flow cytometry analysis was performed to confirm the specific binding of the 10-1.4 antibody clone to GPC3 (Fig. 3): Unstained group; Positive control group stained with anti-human GPC3:APC antibody (Anti-GPC3 Ab); A group stained with anti-mouse IgG:PE antibody after primary staining with the culture supernatant of selected hybridoma clone 10-1.4 (Isolated Ab clone + 2' Ab); and a group stained with anti-mouse IgG:PE antibody without primary staining (2' Ab only).

[0271] Through this, a hybridoma antibody clone 10-1.4 specific for GPC3 was derived.

[0272]

[0273] 1-2. Antibody sequence analysis

[0274] RT-PCR and cDNA sequencing analyses were performed on the GPC3-specific hybridoma antibody clone 10-1.4 derived in Example 1-1 above, to analyze the VH region (amino acid sequence: SEQ ID NO: 7; and base sequence: SEQ ID NO: 9) and VL region (amino acid sequence: SEQ ID NO: 8; and base sequence: SEQ ID NO: 10) sequences of the GPC3-specific antibody, and the signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 sequences of each VH and VL were confirmed (Fig. 4).

[0275]

[0276] 1-3. Production of scFv

[0277] After expressing the hybridoma antibody clone 10-1.4 sequenced above as a FLAG tag-VL-(linker)-VH scFv format protein, to confirm the binding ability to GPC3, HepG2 cell lines overexpressing GPC3 were divided into the following groups and stained with each antibody and flow cytometry analysis was performed: unstained group; positive control group stained with anti-human GPC3:APC antibody; and group stained first with FLAG tag-scFv format protein of the selected hybridoma clone 10-1.4 and then secondarily stained with anti-FLAG tag:APC antibody.

[0278] As a result, the scFv of hybridoma clone 10-1.4 was found to bind specifically to GPC3 (Fig. 5).

[0279]

[0280] Example 2. Production of GPC3 CAR-T and confirmation of antigen specificity.

[0281] To produce GPC3 CAR-T, a chimeric antigen receptor (CAR) containing GPC3 scFv as an antigen-binding domain was transduced into T cells (Fig. 6). Specifically, a CAR vector was constructed by linking a FLAG-tag sequence (DYKDDDDK) to the N-terminal portion of the scFv sequence of antibody clone 10-1.4 produced in Example 1 and linking a chimeric antigen receptor (CAR) sequence (CD28-CD3z) to the C-terminal portion, and a lentivirus was constructed using this vector. In addition, CD4 / CD8-positive T cells were isolated from peripheral blood derived from normal donors, and T cells were stimulated by contacting the cells with microbeads coated with anti-human CD2 / 3 / 28 antibodies. One day after T cell stimulation, the T cells were transduced with the gene by contacting them with a lentivirus containing the CAR produced as described above. After this, the cell number was checked at 2-3 day intervals and CAR-T was proliferated through the process of re-culturing, and on the 11th day, CAR-T was collected and stained with anti-human CD8:FITC and anti-FLAG tag:APC to confirm the stable CAR expression pattern on the cell surface by flow cytometry. In addition, to confirm whether the GPC3 CAR expressed on the T cell recognizes and binds to the GPC3 antigen, the GPC3 CAR-T of the present invention manufactured by the method of FIG. 5 was stained with soluble GPC3:APC protein and anti-human CD8:FITC and then flow cytometry was performed. As a negative control, CAR-T having scFv of the FMC63 antibody clone that recognizes CD19 was stained and compared.

[0282] As a result, it was shown that the GPC3 CAR-T of the present invention stably expressed CAR on the cell surface (Fig. 7a), and the GPC3 CAR expressed on T cells was shown to recognize and bind to the GPC3 antigen (Fig. 7b).

[0283]

[0284] Example 3. Confirmation of GPC3-specific anticancer immune response of GPC3 CAR-T

[0285] To confirm the GPC3-specific anti-tumor immune response of GPC3 CAR-T, CD19 CAR-T (negative control) and GPC3 CR-T were co-cultured with HepG2 cell lines to determine whether CAR-T exhibited an antigen-specific immune response. Specifically, each CAR-T was added to a vessel culturing HepG2 cells, and a protein transporter inhibitor cocktail (eBioscience) was added to the culture medium to inhibit the extracellular release of cytokines. After 6 hours of co-culture, cytokine expression was confirmed by flow cytometry using extracellular anti-CD8:FITC staining and intracellular cytokine staining (anti-human IL2:BV421, anti-human TNF:PerCP-Cy5.5, and anti-human IFN-γ:PE-Cy7).

[0286] As a result, it was confirmed that the GPC3 CAR-T of the present invention reacted with GPC3 of the HepG2 cell line and increased the expression of cytokines IL-2, TNF, and IFN-γ (Fig. 8).

[0287]

[0288] Example 4. Production of GPC3 CAR-T cells with CD28 or 4-1BB as co-stimulatory domains and confirmation of antigen specificity.

[0289] GPC3 BBz and GPC3 28z CAR-T having CD28 or 4-1BB as a costimulatory domain were produced in the same manner as in Example 2. Specifically, a CAR vector was produced by linking a FLAG-tag sequence (DYKDDDDK) to the N-terminal portion of the scFv sequence of antibody clone 10-1.4 produced in Example 1 and linking a CAR sequence (4-1BB-CD3z or CD28-CD3z) to the C-terminal portion, and a lentivirus was produced using this vector. This was introduced into T cells to produce GPC3 CAR-T having CD28 or 4-1BB as a costimulatory domain, and flow cytometry analysis was performed by staining with anti-human CD8:FITC and anti-FLAG tag:APC, and the results are shown in Fig. 9. In the above Figure 9, CD19 BBz refers to a CAR-T having the scFv of the FMC63 antibody clone used as a control and the 4-1BB costimulatory domain, CD19 28z refers to a CAR-T having the scFv of the FMC63 antibody clone used as a control and the CD28 costimulatory domain, GPC3 BBz refers to a CAR-T having the scFv sequence of clone 10-1.4 and the 4-1BB costimulatory domain, and GPC3 28z refers to a CAR-T having the scFv sequence of clone 10-1.4 and the CD28 costimulatory domain.

[0290] As a result, as shown in Fig. 9, it was confirmed that GPC3 BBz and GPC3 28z CAR-T having CD28 or 4-1BB as a co-stimulatory domain according to the present invention were stably expressed on the cell surface, respectively.

[0291] In addition, whether the GPC3 BBz and GPC3 28z CAR-T exhibited a specific anticancer immune response against GPC3 was confirmed in the same manner as in Example 3, and the results are shown in FIG. 10. In FIG. 10, CD19 BBz refers to a CAR-T having the scFv of the FMC63 antibody clone used as a control and the 4-1BB costimulatory domain, CD19 28z refers to a CAR-T having the scFv of the FMC63 antibody clone used as a control and the CD28 costimulatory domain, GPC3 BBz refers to a CAR-T having the scFv sequence of clone 10-1.4 and the 4-1BB costimulatory domain, and GPC3 28z refers to a CAR-T having the scFv sequence of clone 10-1.4 and the CD28 costimulatory domain.

[0292] As a result, as shown in Fig. 10, it was confirmed that GPC3 BBz and GPC3 28z CAR-T having CD28 or 4-1BB as a co-stimulatory domain according to the present invention exhibited a specific anti-cancer immune response against GPC3.

[0293]

[0294] Example 5. In vivo anticancer effect of GPC3 CAR-T cells with CD28 or 4-1BB as co-stimulatory domains.

[0295] In order to confirm the in vivo effect of GPC3 CAR-T according to the present invention, a HepG2 cell line overexpressing human CD19 protein and Firefly luciferase was first inoculated into NOG (NOD.Cg-Prkdcscid II2rgtm1Sug / Jic), a mouse with severe immunodeficiency, to induce cancer growth.

[0296] In addition, CD19 CAR-T (CAR having 4-1BB or CD28 as a co-stimulatory domain) and GPC3 CAR-T (CAR having CD28 as a co-stimulatory domain) produced in the same manner as in Example 2 were produced and injected into mice inoculated with HepG2-hCD19-luc2, and the cancer suppression ability was confirmed by measuring luciferase activity.

[0297] As a result, as shown in Figure 11, it was confirmed that cancer growth was suppressed in mice injected with GPC3 CAR-T. In particular, when compared with mice administered with previously approved CD19 CAR-T, it was confirmed that they showed equivalent or superior anticancer efficacy.

[0298]

[0299] Example 6. Production of humanized antibodies using hybridoma antibodies specific for GPC3 and confirmation of antigen specificity.

[0300] According to the present invention, a humanized antibody was produced through a humanization process using the hybridoma clone 10-1.4 antibody sequence specific for GPC3. Specifically, as shown in Fig. 12, the VH and VL region CDR1~3 sequences of clone 10-1.4 were converted to human V H 4 types of frames and V L A total of 16 types of humanized antibody sequences, v1 to v16, were designed by grafting them onto four types of frames and expressing them in combination. When each antibody sequence was expressed in HEK293T cells, it was confirmed that 10 types of humanized antibodies, excluding v9 to 14, were stably secreted into the cell culture medium in the form of antibodies (Fig. 12 bottom (yield obtained by purifying 80 mL of supernatant by affinity chromatography)).

[0301] In addition, HepG2 and Huh-7 cell lines overexpressing GPC3, Nalm6 as a negative control, and Nalm6-GPC3 cells (overexpressing GPC3) as a positive control were stained with the 10 humanized antibodies, followed by secondary staining with anti-hIgG-Fc:PE and flow cytometry analysis was performed, which is shown in Fig. 13. In Fig. 13, 2nd only refers to a group stained only with the secondary antibody anti-hIgG-Fc:PE, Mouse refers to a group stained with a chimeric antibody produced by linking the VH, VL sequences of the original mouse antibody clone 10-1.4 to human CH, CL sequences, and v1 to v16 refer to groups stained with each humanized antibody. As a result, it was confirmed that all humanized antibodies according to the present invention exhibited GPC3 antigen specificity.

[0302]

[0303] Example 7. Production of CAR-T cells using humanized antibodies and confirmation of antigen specificity.

[0304] CAR-T was produced using humanized antibodies v1, v3, v6, and v8 in the same manner as in Example 2. More specifically, a FLAG-tag sequence (DYKDDDDK (SEQ ID NO: 34)) was linked to the N-terminal part of the scFv sequence of humanized antibodies v1, v3, v6, and v8, and a CAR sequence (CD28-CD3z) was linked to the C-terminal part to produce a vector in the form of a second-generation CAR. After producing a lentivirus with the vector, it was introduced into T cells to produce GPC3 CAR-T. This was stained with anti-human CD8:FITC and anti-FLAG tag:APC, and at the same time, the GPC3 CAR expressed on the T cells was analyzed by flow cytometry to determine whether it recognized and bound to the GPC3 antigen by interacting with the soluble GPC3:APC protein. At this time, the concentration of the soluble GPC3:APC protein was gradually diluted, and the stained samples were simultaneously analyzed. As a result, as shown in Fig. 14, it was confirmed that the GPC3 antibody according to the present invention was stably expressed in T cells in the form of a humanized CAR sequence. In addition, it was confirmed that V1 and V3 had excellent binding affinity to the GPC3 antigen, while V6 and V8 had somewhat lower binding affinity. In Fig. 14, Original is an experimental group for CAR-T having the mouse form (before humanization) scFv sequence of clone 10-1.4 used as a positive control.

[0305]

[0306] The amino acid sequence and base sequence of the novel antibody specifically binding to GPC3 produced in the present invention and the CAR-T using the same are as shown in Table 1 below.

[0307] <h2 style=";text-align:left;direction:ltr">VH domainMLLGLKWVFF VVFYQGVHCE VQLVESGGGL VQPKGSLKLS CAASGFTFNT YAMNWVRQAP GKGLEWVARI RSKSNNYATY YADSVKDRFT ISRDDSQSML YLQMNNLKTE DTAMYYCVRL GYRYDGYAMD YWGQGTSVTV SS7GPC3 antibody VL domainMDFQVQIFSF LLISASVIIS RGQIVLTQSP AIMSASPGEK VTTMTCSASSS VSYMYWYQQK SGTSPKRWIY DTSKLASGVP ARFSGSGSGT SYSLTISSME AEDAATYYCQ QWSSNPPTFG GGTKLEIK8GPC3 antibody VH domain sequenceatgctgttgg ggctgaagtg ggttttcttt gttgtttttt atcaaggtgt gcattgtgag gtgcagcttg ttgagtctgg tggaggattg gtgcagccta aagggtcatt gaaactctca tgtgcagcct ctggattcac cttcaatacc tacgccatga actgggtccg ccaggctcca ggaaagggtt tggaatgggt tgctcgcata agaagtaaaa gtaataatta tgcaacatat tatgccgatt cagtgaaaga caggttcacc atctccagag atgattcaca aagcatgctc tatctgcaaa tgaacaactt gaaaactgag gacacagcca tgtattactg tgtgagactt ggctataggt acgacggcta tgctatggac tactggggtc aaggaacctc agtcaccgtc tcctca9GPC3 antibody VL domain sequenceatggattttcaagtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatatcc agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatctcc aggggagaag gtcaccatga cctgcagtgc cagctcaagt gtaagttata tgtattggta ccagcagaag tcaggcacct cccccaaaag atggatttat gacacatcca aactggcttc tggagtccct gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagcatggag gctgaagatg ctgccactta ttactgccag cagtggagta gtaacccacc cacgttcgga ggggggacca agctggaaat aaaa10Humanized VH1 domainEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYADSVKGRFTISRDDSQSMLYLQMNSLKTEDTAvyYCVRLGYRYDGYAMDYWGQGTTVTVSS11Humanized VH2 domainEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYCVRLGYRYDGYAMDYWGQGTTVTVSS12Humanized VH3 domainQVQLQESGPGLVKPSETLSLTCTASGFTFNTYAMNWVRQPPGKGLEWVARIRSKSNNYATYADSLKSRFTISRDDSQSMLSLKLSSVTAADTAVYYCVRLGYRYDGYAMDYWGQGTTVTVSS13Humanized VH4domainQVQLQESGPGLVKPSETLSLTCTASGFTFNTYAMNWIRQPPGKGLEWIARIRSKSNNYATYYADSLKSRVTISRDDSKNQLSLKLSSVTAADTAVYYCVRLGYRYDGYAMDYWGQGTTVTVSS14Humanized VL1 domainQIVLTQSPATLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTSYSLTISSLEPEDFATYYCQQWSSNPPTFGGGTKLEIK15Humanized VL2 domainEIVLTQSPATLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPTFGGGTKLEIK16Humanized VL3 domainQIQLTQSPSSLSASVGDRVTITCRASSSVSYMYWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTSYSLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIK17Humanized VL4 domainDIQLTQSPSSLSASVGDRVTITCRASSSVSYMYWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIK18CAR-T_GPC antibody VLQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIK19CAR-T_GPC antibody VHEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRLGYRYDGYAMDYWGQGTSVTVSS20GS linkerGGGGSGGGGSGGGGS21GPC antibodyCARQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRLGYRYDGYAMDYWGQGTSVTVSS22v1 CAR scFv_VH1VL1QIVLTQSPATLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTSYSLTISSLEPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKGRFTISRDDSQSMLYLQMNSLKTEDTAVYYCVRLGYRYDGYAMDYWGQGTTVTVSS23v3 CAR scFv_VH1VL3QIQLTQSPSSLSASVGDRVTITCRASSSVSYMYWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTSYSLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKGRFTISRDDSQSMLYLQMNSLKTEDTAVYYCVRLGYRYDGYAMDYWGQGTTVTVSS24v6 CARscFv_VH2VL2EIVLTQSPATLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRLGYRYDGYAMDYWGQGTTVTVSS25v8 CAR scFv_VH2VL4DIQLTQSPSSLSASVGDRVTITCRASSSVSYMYWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRLGYRYDGYAMDYWGQGTTVTVSS26GPC3 CAR (10-1.4 original mouse CD8a signal-FLAG tag-VL-GSlinker-VH-CD28-CD3z)MALPVTALLLPLALLLHAARPDYKDDDDKQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRLGYRYDGYAMDYWGQGTSVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR27GPC3 CAR (10-1.4 original mouse CD8a signal-FLAG tag-VL-GSlinker-VH-CD28-CD3z)MALPVTALLLPLALLLHAARPDYKDDDDKQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRLGYRYDGYAMDYWGQGTSVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR28GPC3 CAR (10-1.4 original mouse CD8a signal-FLAG tag-VL-GS linker-VH-CD8transmembrane-4-1BB-CD3z)MALPVTALLLPLALLLHAARPDYKDDDDKQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRLGYRYDGYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR29Humanized v1 GPC3 CAR (CD8a signal-FLAG tag-VL-GSlinker-VH-CD28-CD3z)MALPVTALLLPLALLLHAARPDYKDDDDKQIVLTQSPATLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTSYSLTISSLEPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKGRFTISRDDSQSMLYLQMNSLKTEDTAVYYCVRLGYRYDGYAMDYWGQGTTVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR30Humanized v3 GPC3 CAR (CD8a signal-FLAG tag-VL-GSlinker-VH-CD28-CD3z)MALPVTALLLPLALLLHAARPDYKDDDDKQIQLTQSPSSLSASVGDRVTITCRASSSVSYMYWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTSYSLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKGRFTISRDDSQSMLYLQMNSLKTEDTAVYYCVRLGYRYDGYAMDYWGQGTTVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR31Humanized v6 GPC3 CAR (CD8a signal-FLAG tag-VL-GSlinker-VH-CD28-CD3z)MALPVTALLLPLALLLHAARPDYKDDDDKEIVLTQSPATLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRLGYRYDGYAMDYWGQGTTVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR32Humanized v8 GPC3 CAR (CD8a signal-FLAG tag-VL-GSlinker-VH-CD28-CD3z)MALPVTALLLPLALLLHAARPDYKDDDDKDIQLTQSPSSLSASVGDRVTITCRASSSVSYMYWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRLGYRYDGYAMDYWGQGTTVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR33FLAG-tagDYKDDDDK34

Claims

1. An isolated antibody or a fragment thereof having immunological activity that specifically binds to GPC3 (Glypican-3) protein.

2. In paragraph 1, An isolated antibody or a fragment thereof having immunological activity, wherein the antibody comprises a VH domain comprising CDRH (Complementarity determining regions Heavy chain) 1 comprising an amino acid sequence represented by SEQ ID NO: 1, CDRH2 comprising an amino acid sequence represented by SEQ ID NO: 2, and CDRH3 comprising an amino acid sequence represented by SEQ ID NO:

3.

3. In paragraph 1, An isolated antibody or a fragment thereof having immunological activity, wherein the antibody comprises a VL domain comprising CDRL1 comprising an amino acid sequence represented by SEQ ID NO: 4, CDRL2 comprising an amino acid sequence represented by SEQ ID NO: 5, and CDRL3 comprising an amino acid sequence represented by SEQ ID NO:

6.

4. In paragraph 1, The above antibody is an isolated antibody or an immunologically active fragment thereof, comprising a VH domain comprising an amino acid sequence represented by SEQ ID NO:

7.

5. In paragraph 1, The above antibody is an isolated antibody or an immunologically active fragment thereof, comprising a VL domain comprising an amino acid sequence represented by SEQ ID NO:

8.

6. In paragraph 1, An isolated antibody or a fragment thereof having immunological activity, wherein the antibody is a chimeric antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a triabody or a tetrabody.

7. In paragraph 1, An isolated antibody or a fragment thereof having immunological activity, wherein the fragment having immunological activity is any one selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, single-chain antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody, and diabody.

8. A human VH domain comprising at least one CDRH selected from the group consisting of CDRH (Complementarity determining regions Heavy chain) 1 comprising an amino acid sequence represented by SEQ ID NO: 1, CDRH2 comprising an amino acid sequence represented by SEQ ID NO: 2, and CDRH3 comprising an amino acid sequence represented by SEQ ID NO: 3; and A humanized antibody or antigen-binding fragment thereof that specifically binds to GPC3, comprising a human VL domain comprising at least one CDRL selected from the group consisting of CDRL1 comprising an amino acid sequence represented by SEQ ID NO: 4, CDRL2 comprising an amino acid sequence represented by SEQ ID NO: 5, and CDRL3 comprising an amino acid sequence represented by SEQ ID NO:

6.

9. In paragraph 8, A humanized antibody or antigen-binding fragment thereof, wherein the human VH domain is at least one selected from the group consisting of VH1 comprising an amino acid sequence represented by SEQ ID NO: 11; VH2 comprising an amino acid sequence represented by SEQ ID NO: 12; VH3 comprising an amino acid sequence represented by SEQ ID NO: 13; and VH4 comprising an amino acid sequence represented by SEQ ID NO:

14.

10. In paragraph 8, A humanized antibody or antigen-binding fragment thereof, wherein the human VL domain is at least one selected from the group consisting of VL1 comprising an amino acid sequence represented by SEQ ID NO: 15; VL2 comprising an amino acid sequence represented by SEQ ID NO: 16; VL3 comprising an amino acid sequence represented by SEQ ID NO: 17; and VL4 comprising an amino acid sequence represented by SEQ ID NO:

18.

11. In paragraph 8, The humanized antibody comprises: a VH1 comprising an amino acid sequence represented by SEQ ID NO: 11 and a VL1 comprising an amino acid sequence represented by SEQ ID NO: 15; a VH1 comprising an amino acid sequence represented by SEQ ID NO: 11 and a VL2 comprising an amino acid sequence represented by SEQ ID NO: 16; a VH1 comprising an amino acid sequence represented by SEQ ID NO: 11 and a VL3 comprising an amino acid sequence represented by SEQ ID NO: 17; a VH1 comprising an amino acid sequence represented by SEQ ID NO: 11 and a VL4 comprising an amino acid sequence represented by SEQ ID NO: 18; a VH2 comprising an amino acid sequence represented by SEQ ID NO: 12 and a VL1 comprising an amino acid sequence represented by SEQ ID NO: 15; a VH2 comprising an amino acid sequence represented by SEQ ID NO: 12 and a VL2 comprising an amino acid sequence represented by SEQ ID NO: 16; a VH2 comprising an amino acid sequence represented by SEQ ID NO: 12 and a VL3 comprising an amino acid sequence represented by SEQ ID NO: 17; A humanized antibody or antigen-binding fragment thereof, comprising: a VH2 comprising an amino acid sequence represented by SEQ ID NO: 12 and a VL4 comprising an amino acid sequence represented by SEQ ID NO: 18; a VH4 comprising an amino acid sequence represented by SEQ ID NO: 14 and a VL3 comprising an amino acid sequence represented by SEQ ID NO: 17; or a VH4 comprising an amino acid sequence represented by SEQ ID NO: 14 and a VL4 comprising an amino acid sequence represented by SEQ ID NO:

18.

12. A hybridoma cell line producing the isolated antibody of paragraph 1 or a fragment thereof having immunological activity or the humanized antibody of paragraph 8 or an antigen-binding fragment thereof.

13. An isolated nucleic acid molecule encoding the isolated antibody of paragraph 1 or a fragment thereof having immunological activity or the humanized antibody of paragraph 8 or an antigen-binding fragment thereof.

14. In paragraph 13, An isolated nucleic acid molecule encoding the isolated antibody of the first paragraph or a fragment thereof having immunological activity, wherein the nucleic acid molecule comprises a base sequence represented by one or more sequence numbers selected from the group consisting of SEQ ID NOs: 9 and 10.

15. A vector comprising an isolated nucleic acid molecule encoding the isolated antibody of paragraph 1 or a fragment thereof having immunological activity or the humanized antibody of paragraph 8 or an antigen-binding fragment thereof.

16. A host cell transformed with a vector comprising an isolated nucleic acid molecule encoding the isolated antibody of paragraph 1 or a fragment thereof having immunological activity or the humanized antibody of paragraph 8 or an antigen-binding fragment thereof.

17. A composition for detecting a cancer antigen comprising the isolated antibody of claim 1 or a fragment thereof having immunological activity, or the humanized antibody of claim 8 or an antigen-binding fragment thereof.

18. In paragraph 17, A composition for detecting a cancer antigen, wherein the cancer antigen is GPC3.

19. A kit for detecting a cancer antigen comprising the isolated antibody of paragraph 1 or a fragment thereof having immunological activity, or the humanized antibody of paragraph 8 or an antigen-binding fragment thereof.

20. The isolated antibody of paragraph 1 or a fragment thereof having immunological activity, or the humanized antibody of paragraph 8 or an antigen-binding fragment thereof; and Antibody-Drug Conjugate (ADC), containing a drug.

21. In paragraph 20, An antibody-drug conjugate, wherein the drug is an immunogenic apoptosis inducer, a microtubulin structure formation inhibitor, a meiosis inhibitor, a topoisomerase inhibitor, a DNA intercalator, a toxin, or a radionuclide.

22. A pharmaceutical composition for preventing or treating cancer comprising the antibody-drug conjugate of Article 20.

23. In paragraph 22, The above cancers include brain tumors, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer (TNBC), lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumors, primary central nervous system lymphoma, spinal cord tumors, brainstem glioma, and A pharmaceutical composition for the prevention or treatment of cancer, wherein the composition comprises at least one selected from the group consisting of pituitary adenomas.

24. A composition for diagnosing cancer comprising the isolated antibody of claim 1 or a fragment thereof having immunological activity, the humanized antibody of claim 8 or an antigen-binding fragment thereof, or the antibody-drug conjugate of claim 20.

25. A kit for diagnosing cancer comprising the isolated antibody of paragraph 1 or a fragment thereof having immunological activity, the humanized antibody of paragraph 8 or an antigen-binding fragment thereof, or the antibody-drug conjugate of paragraph 20.

26. A step of treating a sample with the isolated antibody of claim 1 or a fragment thereof having immunological activity labeled with a fluorescent material, the humanized antibody of claim 8 or an antigen-binding fragment thereof, or the antibody-drug conjugate of claim 20; and A method for providing information for diagnosing cancer, comprising: a step of measuring the expression of the isolated antibody or a fragment thereof having immunological activity, a humanized antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate.

27. A heavy chain variable region comprising CDRH (Complementarity determining regions Heavy chain) 1 comprising an amino acid sequence represented by SEQ ID NO: 1, CDRH2 comprising an amino acid sequence represented by SEQ ID NO: 2, and CDRH3 comprising an amino acid sequence represented by SEQ ID NO: 3; and A GPC3-specific chimeric antigen receptor (CAR) comprising a single chain variable fragment (scFv) as an antigen binding domain, wherein the single chain variable fragment comprises a light chain variable region comprising a CDRL1 comprising an amino acid sequence represented by SEQ ID NO: 4, a CDRL2 comprising an amino acid sequence represented by SEQ ID NO: 5, and a CDRL3 comprising an amino acid sequence represented by SEQ ID NO:

6.

28. In paragraph 27, A GPC3-specific CAR, wherein the heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO:

20.

29. In paragraph 27, A GPC3-specific CAR, wherein the light chain variable region comprises an amino acid sequence represented by SEQ ID NO:

19.

30. In paragraph 27, The above scFv is a GPC3-specific CAR comprising an amino acid sequence represented by SEQ ID NO:

22.

31. A heavy chain variable region selected from the group consisting of a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11; a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12; a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 13; and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 14; and A GPC3-specific humanized antibody-derived chimeric antigen receptor (CAR) comprising a single chain variable fragment (scFv) as an antigen-binding domain, wherein the single chain variable fragment comprises any one of a light chain variable region selected from the group consisting of a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 15; a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 16; a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 17; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO:

18.

32. In paragraph 31, A GPC-specific humanized antibody-derived CAR, wherein the scFv comprises any one selected from the group consisting of a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 15; a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 17; a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 16; and a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12 and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO:

18.

33. In paragraph 31, A GPC3-specific humanized antibody-derived CAR, wherein the scFv comprises an amino acid sequence represented by any one of the sequence numbers selected from the group consisting of sequence numbers 23, 24, 25, and 26.

34. A polynucleotide comprising a nucleic acid sequence encoding a GPC3-specific CAR of claim 27 or a GPC3-specific humanized antibody-derived CAR of claim 31.

35. A vector comprising a polynucleotide comprising a nucleic acid sequence encoding a GPC3-specific CAR of claim 27 or a GPC3-specific humanized antibody-derived CAR of claim 31.

36. A method for producing a GPC3-specific CAR or a GPC3-specific humanized antibody-derived CAR, comprising a step of infecting a host cell with a vector comprising a polynucleotide comprising a nucleic acid sequence encoding the GPC3-specific CAR of item 27 or the GPC3-specific humanized antibody-derived CAR of item 31.

37. A chimeric antigen receptor expressing cell transformed with the GPC3-specific CAR of item 27.

38. A chimeric antigen receptor-expressing cell transformed with a CAR derived from a GPC3-specific humanized antibody of item 31.

39. In paragraph 37 or 38, The above chimeric antigen receptor expressing cell is a chimeric antigen receptor expressing cell, which is a chimeric antigen receptor expressing macrophage (CAR-macrophage), a chimeric antigen receptor expressing T (CAR-T) cell, a chimeric antigen receptor expressing-gamma-delta T (CAR-Gamma-delta T) cell, or a natural killer (CAR-NK) cell.

40. A pharmaceutical composition for preventing or treating cancer, comprising a GPC3-specific CAR of claim 27, a GPC3-specific humanized antibody-derived CAR of claim 31, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR of claim 37, or a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR of claim 38.

41. In paragraph 40, A pharmaceutical composition for preventing or treating cancer, characterized in that the cancer is refractory to an immune checkpoint inhibitor.

42. In paragraph 40, The above cancers include brain tumors, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer (TNBC), lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumors, primary central nervous system lymphoma, spinal cord tumors, brainstem glioma, and A pharmaceutical composition for the prevention or treatment of cancer, wherein the composition comprises at least one selected from the group consisting of pituitary adenomas.

43. A cell therapy agent for preventing or treating cancer, comprising a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR of item 37 or a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR of item 38.

44. An anticancer adjuvant comprising a chimeric antigen receptor-expressing cell transformed with the GPC3-specific CAR of item 27, a GPC3-specific humanized antibody-derived CAR of item 31, a GPC3-specific CAR of item 37, or a chimeric antigen receptor-expressing cell transformed with the GPC3-specific humanized antibody-derived CAR of item 38.

45. In paragraph 44, The above anticancer adjuvant is an anticancer adjuvant that improves refractoriness to immunotherapy.

46. In paragraph 45, The above immunotherapy agents include immune checkpoint inhibitors, immunosuppressant control drugs, cancer vaccines, immunoadjuvants, cancer treatment immune cells, immune cell activation cofactors, cancer treatment antibodies, or cytokines necessary for maintaining the activity of cancer treatment immune cells, and anticancer adjuvants.

47. In paragraph 46, The above immune checkpoint inhibitor is an anticancer adjuvant that is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA or A2aR.

48. A composition for diagnosing cancer, comprising a chimeric antigen receptor-expressing cell transformed with the GPC3-specific CAR of claim 27, a GPC3-specific humanized antibody-derived CAR of claim 31, a GPC3-specific CAR of claim 37, or a chimeric antigen receptor-expressing cell transformed with the GPC3-specific humanized antibody-derived CAR of claim 38.

49. A method for providing information for diagnosing cancer, comprising: a step of treating a sample with a GPC3-specific CAR of claim 27, a GPC3-specific humanized antibody-derived CAR of claim 31, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR of claim 37, or a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR of claim 38, all labeled with a fluorescent substance; and a step of measuring the expression of the GPC3-specific CAR, the GPC3-specific humanized antibody-derived CAR, the chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR, or the chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR.

50. A method for detecting a cancer antigen, comprising the step of administering to a subject an isolated antibody of claim 1 or a fragment having immunological activity thereof, a humanized antibody of claim 8 or an antigen-binding fragment thereof, a GPC3-specific CAR of claim 27, a CAR derived from a GPC3-specific humanized antibody of claim 31, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR of claim 37, or a chimeric antigen receptor-expressing cell transformed with a CAR derived from a GPC3-specific humanized antibody of claim 38.

51. In paragraph 50, A method for detecting a cancer antigen, wherein the cancer antigen is GPC3.

52. A method for preventing or treating cancer, comprising the step of administering to a subject an antibody-drug conjugate of claim 20, a GPC3-specific CAR of claim 27, a GPC3-specific humanized antibody-derived CAR of claim 31, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR of claim 37, a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR of claim 38, a cell therapeutic agent comprising a chimeric antigen receptor-expressing cell transformed with a GPC3-specific CAR of claim 37, or a cell therapeutic agent comprising a chimeric antigen receptor-expressing cell transformed with a GPC3-specific humanized antibody-derived CAR of claim 38.

53. In paragraph 52, A method for preventing or treating cancer, characterized in that the cancer is refractory to an immune checkpoint inhibitor.

54. In paragraph 52, The above cancers include brain tumors, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer (TNBC), lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumors, primary central nervous system lymphoma, spinal cord tumors, brainstem glioma, and A method for preventing or treating cancer, wherein the cancer is at least one selected from the group consisting of pituitary adenomas.

55. A method for enhancing the anticancer treatment effect in a subject, comprising administering to the subject a chimeric antigen receptor-expressing cell transformed with the GPC3-specific CAR of claim 27, a GPC3-specific humanized antibody-derived CAR of claim 31, a GPC3-specific CAR of claim 37, or a chimeric antigen receptor-expressing cell transformed with the GPC3-specific humanized antibody-derived CAR of claim 38.

56. In paragraph 55, A method for enhancing the anticancer treatment effect of an individual, wherein enhancing the anticancer treatment effect of the individual improves the refractoriness to an immunotherapy agent.

Images