Immune effector cell expressing chimeric antigen receptor targeting GPC3 and chimeric receptor targeting CD276, and uses thereof

Abstract

The present invention relates to an immune effector cell expressing a chimeric antigen receptor targeting GPC3 and a chimeric antigen receptor targeting CD276, and uses thereof. In the present invention, immune effector cells (IBC301, IBC302, IBC302CA, or IBC303 inteliCAR-T) were developed to simultaneously express chimeric antigen receptors targeting GPC3 and CD276, respectively. It was confirmed that the immune effector cells developed in the present invention effectively recognize GPC3 S and CD276 S, thereby not only activating CAR-T cells, but also promoting the apoptosis of liver cancer cells, which are one of solid cancers, and effectively inhibiting the growth of tumors. In particular, IBC302CA inteliCAR-T, prepared to prevent CD276 signal leakage by blocking disulfide bonds, most effectively inhibits tumor growth and suppresses tumor recurrence through an excellent memory response, and thus can be effectively applied to the treatment of cancers or tumors expressing GPC3 and CD276.

Description

Immune effector cells expressing a chimeric antigen receptor targeting GPC3 and a chimeric antigen receptor targeting CD276, and uses thereof

[0001] The present invention relates to an immune effector cell expressing a chimeric antigen receptor targeting GPC3 and a chimeric antigen receptor targeting CD276, and uses thereof, and relates to an AND-gate chimeric antigen receptor composed of a chimeric antigen receptor targeting GPC3 comprising a GPC3-binding domain and a receptor targeting CD276 comprising a CD276-binding domain, a polynucleotide encoding the same, an immune effector cell expressing the same and targeting GPC3 and CD276, and a pharmaceutical composition for preventing or treating cancer expressing GPC3 and CD276, comprising the immune effector cell.

[0002]

[0003] GPC3 (Glypican 3) is known as a type of 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.

[0004] Although GPC3 is not expressed in adult tissues other than the placenta, it is expressed in various cancer tissues such as hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, and lung squamous cell carcinoma. Thus, GPC3, like 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.

[0005] Regarding the function and use of GPC3, it has been reported that it can function as a hepatocellular carcinoma marker. Recently, it has been reported that GPC3 is expressed in various cancers, especially hepatocellular carcinoma (HCC), melanoma, Wilm's tumor, and hepatoblastoma (Jakubovic and Jothy, Ex Mol Path, 82:184-189, 2007; Nakatsura and Nishimura, Biodrugs, 19(2):71-77 2005), and it has been reported that it is possible to treat tumors such as liver cancer using antibodies against GPC3 (International Patent Publication No. WO 2014 / 097648; International Patent Publication No. WO 2018-131586; Republic of Korea Patent Publication No. 10-2017-0049831, etc.).

[0006] Meanwhile, CAR-T cell therapy has emerged as a groundbreaking approach to tumor immunotherapy in recent years. CAR-T cells containing CD19-specific antibodies have been reported to be effective against benign acute lymphoblastic leukemia and other blood cancers. However, CAR-T cell activity against solid tumors has been shown to be limited, and research is underway to address this issue by incorporating various immune checkpoints.

[0007] Among them, CD276 (Cluster of Differentiation 276), an immune checkpoint protein of the CD28 family, has been reported to be expressed in solid cancers, and many studies are being conducted on CAR-T cell therapy targeting CD276 (International Patent Publication No. WO2021-099347; International Patent Publication No. WO2021-101991).

[0008] CD276 acts as an inhibitor of T-cell stimulation that simultaneously stimulates the production of effector cytokines such as IL-4 and IFN-γ, and is known to be involved in the stimulation of primary CD8 cytotoxic T-cells (CTLs), which have been shown to reduce tumor growth rates (Prasad, DV,et al.,J Immunol.,173(4):500-6, 2004).

[0009] CD276 protein expression is very limited in normal cells, but is highly upregulated in primary and metastatic tumors as well as tumor vasculature, and is found in multiple cell types including differentiated tumor cells, tumor initiating or cancer stem cells, and its expression has been reported to be associated with poor prognosis in some tumor types (Steven Seaman,et al.,cancer cell, 31(4):501-515, 2017).

[0010]

[0011] Accordingly, in the present invention, as a result of extensive efforts to develop CAR-T cells with increased therapeutic effects on solid cancers known to have low CAR-T cell activity, immune effector cells expressing a chimeric antigen receptor targeting GPC3 and a chimeric antigen receptor targeting CD276, which are known to be overexpressed in various types of solid cancers, were produced, and it was confirmed that the immune effector cells produced in the present invention effectively promote cell death of liver cancer, one of solid cancers, and inhibit tumor growth, thereby completing the present invention.

[0012]

[0013] Accordingly, an object of the present invention is to provide an AND-gate chimeric antigen receptor specific for GPC3 and CD276, comprising a chimeric antigen receptor targeting GPC3 and a chimeric antigen receptor targeting CD276, a polynucleotide encoding the same, a vector comprising the same, and an immune effector cell expressing the AND-gate chimeric antigen receptor specific for GPC3 and CD276.

[0014] Another object of the present invention is to provide an AND-gate chimeric antigen receptor specific for GPC3, CD276, and TGF-β, comprising a chimeric antigen receptor targeting GPC3, a chimeric antigen receptor targeting CD276, and TGFβDNR, a polynucleotide encoding the same, a vector comprising the same, and an immune effector cell expressing the AND-gate chimeric antigen receptor specific for GPC3, CD276, and TGF-β.

[0015] Another object of the present invention is to provide an AND-gate chimeric antigen receptor specific for GPC3, CD276, and 4-1BB, comprising a chimeric antigen receptor targeting GPC3, a chimeric antigen receptor targeting CD276, and a 4-1BB ligand, a polynucleotide encoding the same, a vector comprising the same, and an immune effector cell expressing the AND-gate chimeric antigen receptor specific for GPC3, CD276, and 4-1BB.

[0016]

[0017] To achieve the above-mentioned purpose,

[0018] The present invention relates to a chimeric antigen receptor (GPC3-CAR) targeting GPC3 comprising a signal peptide, a GPC3-binding domain, a hinge region, a transmembrane domain, and an intracellular signal transduction domain; and

[0019] It consists of a chimeric antigen receptor (CD276-CAR) targeting CD276, which comprises a signal peptide, a CD276-binding domain, a hinge region, a transmembrane domain, and an intracellular costimulatory domain.

[0020] The above GPC3-binding domain is a GPC3-specific humanized antibody or fragment thereof, which comprises a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 6.

[0021] The above CD276-binding domain is characterized in that it is a CD276-specific humanized antibody or fragment thereof, which comprises a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 11, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 12, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 14, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 15, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 16.

[0022] In a preferred embodiment of the present invention, the GPC3-binding domain may be represented by the amino acid sequence of SEQ ID NO: 22, and the CD276-binding domain may be represented by the amino acid sequence of SEQ ID NO: 28.

[0023] In another preferred embodiment of the present invention, the signal peptide is a protein derived from IgGκ or CD33, the hinge region is a protein derived from CD8α or CD28, the transmembrane domain is a protein derived from any one selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1, the intracellular signaling domain is a protein derived from CD3ζ, and the intracellular costimulatory domain may be a protein derived from a CD28-derived intracellular domain, a 4-1BB-derived intracellular domain and an OX40-derived intracellular domain.

[0024]

[0025] In another preferred embodiment of the present invention, in order to prevent disulfide bonds in the chimeric antigen receptor (CD276-CAR) targeting CD276, the 28th sequence of SEQ ID NO: 31 is a CD28 hinge region in which cysteine ​​(C) is substituted with alanine (A), and

[0026] The 13th sequence of SEQ ID NO: 33 may include a CD28-derived transmembrane domain in which cysteine ​​is substituted with alanine.

[0027]

[0028] In addition, the present invention provides a polynucleotide encoding a chimeric antigen receptor (GPC3-CAR) targeting GPC3 and a chimeric antigen receptor (CD276-CAR) targeting CD276.

[0029] In addition, the present invention provides a vector comprising a polynucleotide encoding a chimeric antigen receptor targeting GPC3 (GPC3-CAR) and a chimeric antigen receptor targeting CD276 (CD276-CAR).

[0030] In addition, the present invention provides an immune effector cell (IBC301 InteliCAR-T) targeting GPC3 and CD276, which expresses a chimeric antigen receptor (GPC3-CAR) targeting GPC3 and a chimeric antigen receptor (CD276-CAR) targeting CD276.

[0031] In a preferred embodiment of the present invention, the immune effector cell may be a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a myeloid cell, a monocyte, or a macrophage.

[0032] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer or tumors, comprising immune effector cells targeting the GPC3 and CD276.

[0033] In a preferred embodiment of the present invention, the cancer or tumor may be selected from the group consisting of prostate cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, lung cancer, liver cancer, pancreatic cancer, head and neck cancer, melanoma, glioblastoma, and neuroblastoma, and preferably may be a cancer or tumor expressing GPC3 and CD276.

[0034]

[0035] To achieve other purposes,

[0036] The present invention relates to a chimeric antigen receptor (GPC3-CAR) targeting GPC3 comprising a signal peptide, a GPC3-binding domain, a hinge region, a transmembrane domain, and an intracellular signal transduction domain; and

[0037] A chimeric antigen receptor (CD276-CAR) targeting CD276 comprising a signal peptide, a CD276-binding domain, a hinge region, a transmembrane domain, and an intracellular costimulatory domain; and

[0038] It is composed of TGF-β receptor II (TGFβDNR), which is composed of TGF-β receptor II extracellular domain (TGF-β RII ECD), TGF-β receptor II transmembrane domain (TGF-β RII TM), and truncated TGF-β receptor II intracellular domain (Truncated TGF-β RII ICD).

[0039] The above GPC3-binding domain is a GPC3-specific humanized antibody or fragment thereof, which comprises a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 6.

[0040] The present invention provides an AND-gate chimeric antigen receptor specific for GPC3, CD276 and TGF-β, characterized in that the CD276-binding domain is a CD276-specific humanized antibody or fragment thereof, comprising a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 11, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 12 and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 14, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 15 and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 16.

[0041] In a preferred embodiment of the present invention, the signal peptide is a protein derived from IgGκ or CD33, the hinge region is a protein derived from CD8α or CD28, the transmembrane domain is a protein derived from any one selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1, the intracellular signaling domain is a protein derived from CD3ζ, and the intracellular costimulatory domain may be a protein derived from a CD28-derived intracellular domain, a 4-1BB-derived intracellular domain and an OX40-derived intracellular domain.

[0042] In another preferred embodiment of the present invention, the TGFβDNR may be represented by the amino acid sequence of SEQ ID NO: 36.

[0043] In another preferred embodiment of the present invention, in order to prevent disulfide bonds in the chimeric antigen receptor (CD276-CAR) targeting CD276, the 28th sequence of SEQ ID NO: 31 is a CD28 hinge region in which cysteine ​​(C) is substituted with alanine (A), and

[0044] The 13th sequence of SEQ ID NO: 33 may include a CD28-derived transmembrane domain in which cysteine ​​is substituted with alanine.

[0045]

[0046] In addition, the present invention provides a chimeric antigen receptor targeting GPC3 (GPC3-CAR), a chimeric antigen receptor targeting CD276 (CD276-CAR), and a polynucleotide encoding TGF-β receptor II (TGFβDNR).

[0047] In addition, the present invention provides a vector comprising a polynucleotide encoding a chimeric antigen receptor targeting GPC3 (GPC3-CAR), a chimeric antigen receptor targeting CD276 (CD276-CAR), and TGF-β receptor II (TGFβDNR).

[0048] In addition, the present invention provides an immune effector cell (IBC302 InteliCAR-T) that targets GPC3, CD276, and TGF-β, which expresses a chimeric antigen receptor (GPC3-CAR) targeting GPC3, a chimeric antigen receptor (CD276-CAR) targeting CD276, and TGF-β receptor II (TGFβDNR).

[0049] In a preferred embodiment of the present invention, the immune effector cell may be a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a myeloid cell, a monocyte, or a macrophage.

[0050] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer or tumors, comprising immune effector cells targeting GPC3, CD276, and TGF-β.

[0051] In a preferred embodiment of the present invention, the cancer or tumor may be selected from the group consisting of prostate cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, lung cancer, liver cancer, pancreatic cancer, head and neck cancer, melanoma, glioblastoma, and neuroblastoma, and preferably may be a cancer or tumor expressing GPC3 and CD276.

[0052]

[0053] To achieve another purpose,

[0054] The present invention relates to a chimeric antigen receptor (GPC3-CAR) targeting GPC3 comprising a signal peptide, a GPC3-binding domain, a hinge region, a transmembrane domain, and an intracellular signal transduction domain; and

[0055] A chimeric antigen receptor (CD276-CAR) targeting CD276 comprising a signal peptide, a CD276-binding domain, a hinge region, a transmembrane domain, and an intracellular costimulatory domain; and

[0056] It consists of 4-1BB ligand (4-1BBL), which is composed of 4-1BB ligand extracellular domain (4-1BBL ECD), 4-1BB ligand transmembrane domain (4-1BBL TM), and 4-1BB ligand intracellular domain (4-1BBL ICD).

[0057] The above GPC3-binding domain is a GPC3-specific humanized antibody or fragment thereof, which comprises a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 6.

[0058] The present invention provides an AND-gate chimeric antigen receptor specific for GPC3, CD276, and 4-1BB, characterized in that the CD276-binding domain is a CD276-specific humanized antibody or fragment thereof, comprising a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 11, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 12, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 14, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 15, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 16.

[0059] In a preferred embodiment of the present invention, the signal peptide is a protein derived from IgGκ or CD33, the hinge region is a protein derived from CD8α or CD28, the transmembrane domain is a protein derived from any one selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1, the intracellular signaling domain is a protein derived from CD3ζ, and the intracellular costimulatory domain may be a protein derived from a CD28-derived intracellular domain, a 4-1BB-derived intracellular domain and an OX40-derived intracellular domain.

[0060] In another preferred embodiment of the present invention, the 4-1BB ligand may be represented by the amino acid sequence of SEQ ID NO: 37.

[0061] In another preferred embodiment of the present invention, in order to prevent disulfide bonds in the chimeric antigen receptor (CD276-CAR) targeting CD276, the 28th sequence of SEQ ID NO: 31 is a CD28 hinge region in which cysteine ​​(C) is substituted with alanine (A), and

[0062] The 13th sequence of SEQ ID NO: 33 may include a CD28-derived transmembrane domain in which cysteine ​​is substituted with alanine.

[0063]

[0064] In addition, the present invention provides a polynucleotide encoding a chimeric antigen receptor targeting GPC3 (GPC3-CAR), a chimeric antigen receptor targeting CD276 (CD276-CAR), and a 4-1BB ligand (4-1BBL).

[0065] In addition, the present invention provides a vector comprising a polynucleotide encoding a chimeric antigen receptor targeting GPC3 (GPC3-CAR), a chimeric antigen receptor targeting CD276 (CD276-CAR), and a 4-1BB ligand (4-1BBL).

[0066] In addition, the present invention provides an immune effector cell (IBC303 InteliCAR-T) targeting GPC3, CD276, and 4-1BB, which expresses a chimeric antigen receptor targeting GPC3 (GPC3-CAR), a chimeric antigen receptor targeting CD276 (CD276-CAR), and a 4-1BB ligand (4-1BBL).

[0067] In a preferred embodiment of the present invention, the immune effector cell may be a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a myeloid cell, a monocyte, or a macrophage.

[0068] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer or tumors, comprising immune effector cells targeting GPC3, CD276, and 4-1BB.

[0069] In a preferred embodiment of the present invention, the cancer or tumor may be selected from the group consisting of prostate cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, lung cancer, liver cancer, pancreatic cancer, head and neck cancer, melanoma, glioblastoma, and neuroblastoma, and preferably may be a cancer or tumor expressing GPC3 and CD276.

[0070]

[0071] In the present invention, immune effector cells (IBC301, IBC302, IBC302CA or IBC303 inteliCAR-T) that simultaneously express a chimeric antigen receptor targeting GPC3 and a chimeric antigen receptor targeting CD276 were produced.

[0072] It was confirmed that the immune effector cells manufactured in the present invention not only effectively recognize GPC3 and CD276 to activate CAR-T cells, but also promote apoptosis of liver cancer cells, a type of solid cancer, and effectively suppress tumor growth. In particular, the IBC302CA inteliCAR-T, manufactured to block CD276 signal leakage by blocking disulfide bonds, most effectively suppressed tumor growth and suppressed tumor recurrence through excellent memory response, and thus can be effectively applied to the treatment of cancers or tumors expressing GPC3 and CD276.

[0073]

[0074] Figure 1 shows data confirming the binding affinity of a humanized antibody (IBA501v5) specific for GPC3 to HepG2 cells derived from human liver cancer expressing GPC3, using flow cytometry.

[0075] Figure 2 is data confirming the binding affinity of a humanized antibody (IBA201v6) specific for CD276 to human ovarian cancer-derived cells (OVCAR3) expressing CD276 by flow cytometry.

[0076] Figure 3 is a schematic diagram showing the lentiviral vectors of IBC301, IBC302, and IBC303 inteliCAR-T (left) and the structure of IBC301, IBC302, and IBC303 inteliCAR-T targeting GPC3 and CD276 on the human T cell membrane (right).

[0077] Figure 4 is a schematic diagram showing (a) a method for manufacturing inteliCAR-T and (b) a method for confirming the expression levels of GPC3-CAR and CD276-CAR and binding ability to GPC3 / CD276 of the manufactured inteliCAR-T.

[0078] Figure 5 shows data confirming the expression levels of GPC3-CAR and CD276-CAR in IBC301, IBC302, and IBC303 inteliCAR-T cells. Figure 5a shows data on day 6 of T cell activation, Figure 5b shows data on day 9 of T cell activation, and Figure 5c shows data on day 13 of T cell activation.

[0079] Figure 6 shows data confirming the killing effect of (a) human lung adenocarcinoma-derived A549 cells and (b) human liver cancer-derived HepG2 cells by IBC301 and IBC302 inteliCAR-T on the 13th day of T cell activation.

[0080] Figure 7 shows the ex vivo culture profiles of IBC301, IBC302, and IBC303 inteliCAR-T, evaluated based on total viable cell count (left axis) and viability (right axis) for (a) the 13-day process and (b) the 9-day process.

[0081] Figure 8 is data confirming the killing effect of IBC301, IBC302, and IBC303 inteliCAR-T on human liver cancer-derived HepG2 (GPC3+CD276+) cells, and is data confirming the degree of HepG2 cell killing during (a) the 13-day process and (b) the 9-day process.

[0082] Figure 9 shows data showing the results of animal experiments to confirm the anti-tumor effects of IBC301 inteliCAR-T and IBC302 inteliCAR-T.

[0083] Figure 9a is a schematic diagram illustrating the animal experiment schedule to confirm the antitumor effects of IBC301 inteliCAR-T and IBC302 inteliCAR-T. Figure 9b shows data obtained by IVIS SpectrumCT of luminescence expressed in HepG2-Luc cells injected into mice. In the figure, UT represents the untreated group. Figure 9c shows numerical data representing the luminescence intensity of the IVIS image of Figure 9b.

[0084] Figure 10 is data for confirming the memory response according to the second tumor administration after the first tumor administration of Figure 9 in an animal experiment to confirm the anti-tumor effect of IBC302 inteliCAR-T.

[0085] Figure 10a shows data obtained by capturing the luminescence expressed in secondary HepG2-Luc cells injected into mice using IVIS SpectrumCT. Figure 10b shows data representing the luminescence intensity of the IVIS image of Figure 10a in numerical form.

[0086] Figure 11 is a schematic diagram illustrating a strategy to reduce the possibility of signal leakage through CD276 to the intracellular signaling domain (CD3ζ) by blocking disulfide bonds.

[0087] Figure 12 shows data comparing IFN-γ production through intracellular cytokine staining after co-culture of IBC302CA inteliCAR-T and BC302 inteliCAR-T with Cys replaced with Ala in the CD28 hinge and TM domains with hepatoma cell lines Hep1 (GPC3-CD276+) and HepG2 (GPC3+CD276+) for 6 hours.

[0088] Figure 13 shows data confirming the level of GPC3-CAR and CD276-CAR expression in IBC302 and IBC302CA inteliCAR-T cells. Figure 13a shows data on day 6 of T cell activation, Figure 13b shows data on day 9 of T cell activation, and Figure 13c shows data on day 13 of T cell activation.

[0089] Figure 14 shows the ex vivo culture profiles of IBC302 and IBC302CA inteliCAR-T, evaluated based on the total viable cell count (left axis) and viability (right axis) of (a) the 13-day process and (b) the 9-day process.

[0090] Figure 15 is data confirming the killing effect of IBC302 and IBC302CA inteliCAR-T on human liver cancer-derived HepG2 (GPC3+CD276+) cells, and is data confirming the degree of HepG2 cell killing during (a) a 13-day process and (b) a 9-day process.

[0091] Figure 16 shows data showing the results of an animal experiment to confirm the anti-tumor effect of IBC302 inteliCAR-T and IBC302CA inteliCAR-T.

[0092] Figure 16a is a schematic diagram illustrating the animal experiment schedule to confirm the antitumor effects of IBC302 inteliCAR-T and IBC302CA inteliCAR-T. Figure 16b shows data obtained by capturing luminescence expressed in HepG2-Luc cells injected into mice using IVIS SpectrumCT. Figure 16c shows numerical data representing the luminescence intensity of the IVIS image of Figure 16b.

[0093] Figure 17 shows data for confirming the memory response according to the second tumor administration after the first tumor administration of Figure 16 in an animal experiment to confirm the anti-tumor effect of IBC302CA inteliCAR-T. Figure 17a shows data for confirming the luminescence expressed in the second HepG2-Luc cells injected into the mouse, captured using IVIS SpectrumCT. Figure 17b shows data numerically representing the luminescence level of the IVIS image of Figure 17a.

[0094] Figure 18 is a schematic diagram showing the characteristics of IBC302CA inteliCAR-T.

[0095]

[0096] Hereinafter, the present invention will be described in detail.

[0097]

[0098] <GPC3-CAR 및 CD276-CAR로 구성된 inteliCAR-T 플랫폼>

[0099] AND-gated chimeric antigen receptor specific for GPC3 and CD276

[0100] The present invention relates to a chimeric antigen receptor (GPC3-CAR) targeting GPC3, comprising a signal peptide, a GPC3-binding domain, a hinge region, a transmembrane domain, and an intracellular signal transduction domain; and

[0101] It consists of a chimeric antigen receptor (CD276-CAR) targeting CD276, which comprises a signal peptide, a CD276-binding domain, a hinge region, a transmembrane domain, and an intracellular costimulatory domain.

[0102] The above GPC3-binding domain is a GPC3-specific humanized antibody or fragment thereof, which comprises a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 6.

[0103] The present invention relates to an AND-gate chimeric antigen receptor specific for GPC3 and CD27, characterized in that the CD276-binding domain is a CD276-specific humanized antibody or fragment thereof, comprising a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 11, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 12, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 14, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 15, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 16.

[0104] In the present invention, the GPC3-binding domain may be represented by the amino acid sequence of SEQ ID NO: 22, and the CD276-binding domain may be represented by the amino acid sequence of SEQ ID NO: 28.

[0105]

[0106] The "AND-gate chimeric antigen receptor" of the present invention is a protein that specifically binds to a specific antigen or molecule, and may be composed of an antibody, a ligand, a receptor, or a protein containing these.

[0107] In the present invention, the term "antibody" may be used not only for a complete form having two full-length light chains and two full-length heavy chains, but also for fragments of antibody molecules. A fragment of an antibody molecule means a fragment that possesses at least a peptide tag (epitope) binding function, and includes scFv, Fab, F(ab'), F(ab')2, single domain, etc.

[0108] In the present invention, the antibody may be a monoclonal antibody. In the present invention, the term "monoclonal antibody", also called a monoclonal antibody or monoclonal antibody, refers to an antibody produced by a single antibody-producing cell and characterized by a uniform primary structure (amino acid sequence). It recognizes only one antigenic determinant and is generally produced by culturing hybridoma cells, which are cancer cells and antibody-producing cells fused together. However, it can also be produced using other recombinant protein-expressing host cells utilizing the secured antibody gene sequence.

[0109]

[0110] In the present invention, the term "chimeric antigen receptor (CAR)" generally refers to a fusion protein containing an extracellular domain capable of binding an antigen and one or more intracellular domains. The CAR is the core component of a chimeric antigen receptor T cell (CAR-T) and may include an antigen (e.g., GPC3 or CD276) binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. The CAR may be combined with a T cell receptor-activating intracellular domain based on the antigen specificity of an antibody. Genetically modified CAR-expressing T cells can specifically identify and eliminate target antigen-expressing malignant cells.

[0111]

[0112] In the present invention, the "signal peptide" generally refers to a peptide chain that guides protein transport. The signal peptide may be a short peptide having a length of 5 to 30 amino acids. In the present invention, the IgGκ signal peptide represented by the amino acid sequence of SEQ ID NO: 21 or the CD33 signal peptide represented by SEQ ID NO: 27 is preferably used.

[0113] In the present invention, the "hinge region" generally refers to a connecting region between an antigen-binding region and an immune cell Fc receptor (FcR)-binding region. A "hinge region" is included between the C-terminus of the binding peptide and the N-terminus of the transmembrane domain, and the hinge region may be a CD8α-derived hinge region represented by the amino acid sequence of SEQ ID NO: 23, or a CD28-derived hinge region represented by the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31.

[0114] The CD28-derived hinge region represented by the amino acid sequence of SEQ ID NO: 29 is a short form, and the CD28-derived hinge region represented by the amino acid sequence of SEQ ID NO: 30 is a long form. In addition, the CD28-derived hinge region represented by the amino acid sequence of SEQ ID NO: 31 is a form in which cysteine ​​(C) in the CD28 hinge (28th sequence of SEQ ID NO: 31) is substituted to prevent disulfide bonds.

[0115] In the present invention, the "transmembrane domain" generally refers to a domain of a CAR that passes through a cell membrane and is connected to an intracellular signaling domain to play a role in signal transduction. The transmembrane domain may be derived from a protein selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1, and may preferably be a CD8α-derived transmembrane domain represented by the amino acid sequence of SEQ ID NO: 24, or a CD28-derived transmembrane domain represented by the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33.

[0116] The CD28-derived transmembrane domain represented by the amino acid sequence of the above sequence number 33 is in the form in which cysteine ​​(C) in the CD28 hinge (the 13th sequence of the sequence number 33) is substituted to prevent disulfide bonds.

[0117] In the present invention, the term "intracellular signal transduction domain" generally refers to a domain located within a cell and capable of transmitting a signal. In the present invention, the intracellular signal transduction domain is an intracellular signal transduction domain of a chimeric antigen receptor. For example, the intracellular signal transduction domain may be selected from a CD3ζ-derived intracellular domain, and preferably, it may be a CD3ζ-derived intracellular domain represented by the amino acid sequence of SEQ ID NO: 25.

[0118] In the present invention, the "intracellular costimulatory domain" generally refers to a domain capable of transmitting a costimulatory signal. In the present invention, the intracellular costimulatory domain is a costimulatory domain of a chimeric antigen receptor. For example, the intracellular costimulatory domain may be selected from a CD28-derived intracellular domain, a 4-1BB-derived intracellular domain, and an OX40-derived intracellular domain, and preferably, a CD28-derived intracellular domain represented by the amino acid sequence of SEQ ID NO: 34.

[0119]

[0120] Polynucleotides and recombinant vectors

[0121] In another aspect, the present invention relates to a polynucleotide encoding a chimeric antigen receptor targeting GPC3 (GPC3-CAR) and a chimeric antigen receptor targeting CD276 (CD276-CAR).

[0122] In another aspect, the present invention relates to a vector comprising a polynucleotide encoding a chimeric antigen receptor targeting GPC3 (GPC3-CAR) and a chimeric antigen receptor targeting CD276 (CD276-CAR).

[0123]

[0124] In the present invention, the term "polynucleotide" generally refers to a nucleic acid molecule, deoxyribonucleotide or ribonucleotide, or an analog thereof, separated into any length. In some embodiments, the polynucleotide of the present invention can be prepared by (1) in vitro amplification such as polymerase chain reaction (PCR) amplification; (2) cloning and recombination; (3) purification such as digestion and gel electrophoresis separation; and (4) synthetically such as chemical synthesis, and preferably, the isolated polynucleotide is prepared by recombinant DNA technology.

[0125]

[0126] In the present invention, the polynucleotide may be composed of [polynucleotide encoding a chimeric antigen receptor targeting GPC3 - linker - polynucleotide encoding a chimeric antigen receptor targeting CD276].

[0127] Preferably, the polynucleotide encoding the chimeric antigen receptor targeting GPC3 may include a polynucleotide encoding an IgGκ signal peptide (SEQ ID NO: 39); a polynucleotide encoding a GPC3-binding domain (SEQ ID NO: 40); a polynucleotide encoding a CD8α hinge region (SEQ ID NO: 41); a polynucleotide encoding a CD8α transmembrane domain (SEQ ID NO: 42); and a polynucleotide encoding a CD3ζ intracellular signal transduction domain (SEQ ID NO: 43).

[0128] The chimeric antigen receptor targeting CD276 may include a polynucleotide encoding a CD33 signal peptide (SEQ ID NO: 45); a polynucleotide encoding a CD276-binding domain (SEQ ID NO: 46); a polynucleotide encoding a CD28 hinge region (SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49); a polynucleotide encoding a CD28 transmembrane domain (SEQ ID NO: 50 or SEQ ID NO: 51); and a polynucleotide encoding a CD28 intracellular costimulatory domain (SEQ ID NO: 52).

[0129] The above linker may be composed of any sequence that does not inhibit the expression of each chimeric antigen receptor, and in the present invention, T2A represented by the base sequence of SEQ ID NO: 44 was used as shown in FIG. 3.

[0130]

[0131] In the present invention, the vector is a recombinant viral vector, preferably a lentiviral vector, and comprises an operably linked EF1α promoter; a polynucleotide encoding a chimeric antigen receptor targeting GPC3; a linker; and a polynucleotide encoding a chimeric antigen receptor targeting CD276, and may additionally include a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to increase protein expression (Fig. 3).

[0132] The above EF1α promoter may be represented by the base sequence of SEQ ID NO: 38, and may include a sequence that is 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the base sequence, as needed. In addition, the promoter is operably linked to induce the expression of a chimeric antigen receptor targeting GPC3 and a chimeric antigen receptor targeting CD276.

[0133]

[0134] Biological methods for introducing polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, and particularly retroviral vectors, are the most widely used methods for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses, and adeno-associated viruses.

[0135] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). Other methods for targeted delivery of nucleic acids, such as targeted nanoparticles or other suitable submicron-sized delivery systems, are available.

[0136] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, entrapped within a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension within a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. The lipid, lipid / DNA, or lipid / expression vector associated composition is not limited to any particular structure in solution.

[0137]

[0138] Immune effector cells (IBC301 InteliCAR-T)

[0139] In another aspect, the present invention relates to immune effector cells targeting GPC3 and CD276, which express a chimeric antigen receptor targeting GPC3 (GPC3-CAR) and a chimeric antigen receptor targeting CD276 (CD276-CAR).

[0140] In the present invention, the immune effector cell may include a polynucleotide encoding a chimeric antigen receptor targeting GPC3 (GPC3-CAR) and a chimeric antigen receptor targeting CD276 (CD276-CAR) of the present invention, or a vector including the polynucleotide.

[0141] In the present invention, the immune effector cell may be an isolated cell derived from a mammal, preferably a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a myeloid cell, a monocyte, or a macrophage, more preferably a T cell.

[0142] In the present invention, the [immune effector cells expressing GPC3-CAR and CD276-CAR] can be produced by introducing the [GPC3-CAR and CD276-CAR coding polynucleotide] or the [GPC3-CAR and CD276-CAR expression vector] of the present invention into an immune effector cell, for example, a T cell or an NK cell.

[0143] Specifically, [GPC3-CAR and CD276-CAR expression vectors] can be introduced into cells by methods known in the art, such as electroporation, lipofectamine (lipofectamine 2000, Invitrogen), etc. For example, immune effector cells can be transfected with a lentiviral vector to integrate the viral genome carrying the CAR molecule into the host genome, thereby ensuring long-term and stable expression of the target gene. In another example, a transposon can be used to introduce the CAR carrying plasmid (transposon) and the transposase carrying plasmid into the target cell. In another example, the CAR molecule can be added to the genome by a gene editing method (e.g., CRISPRCas9).

[0144]

[0145] <GPC3-CAR, CD276-CAR 및 TGFβDNR로 구성된 inteliCAR-T 플랫폼>

[0146] AND-gated chimeric antigen receptor specific for GPC3, CD276, and TGF-β

[0147] In another aspect, the present invention relates to a chimeric antigen receptor (GPC3-CAR) targeting GPC3 comprising a signal peptide, a GPC3-binding domain, a hinge region, a transmembrane domain, and an intracellular signal transduction domain; and

[0148] A chimeric antigen receptor (CD276-CAR) targeting CD276 comprising a signal peptide, a CD276-binding domain, a hinge region, a transmembrane domain, and an intracellular costimulatory domain; and

[0149] It is composed of TGF-β receptor II (TGFβDNR), which is composed of TGF-β receptor II extracellular domain (TGF-β RII ECD), TGF-β receptor II transmembrane domain (TGF-β RII TM), and truncated TGF-β receptor II intracellular domain (Truncated TGF-β RII ICD).

[0150] The above GPC3-binding domain is a GPC3-specific humanized antibody or fragment thereof, which comprises a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 6.

[0151] The present invention relates to an AND-gate chimeric antigen receptor specific for GPC3, CD276 and TGF-β, characterized in that the CD276-binding domain is a CD276-specific humanized antibody or fragment thereof, comprising a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 11, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 12 and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 14, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 15 and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 16.

[0152]

[0153] For specific details on AND-gated chimeric antigen receptors specific for GPC3, CD276 and TGF-β,<GPC3-CAR 및 CD276-CAR로 구성된 inteliCAR-T 플랫폼> As described above, the TGF-β receptor Ⅱ (TGFβDNR) can be represented by the amino acid sequence of sequence number 36.

[0154]

[0155] Polynucleotides and recombinant vectors

[0156] In another aspect, the present invention relates to a chimeric antigen receptor targeting GPC3 (GPC3-CAR), a chimeric antigen receptor targeting CD276 (CD276-CAR), and a polynucleotide encoding TGF-β receptor II (TGFβDNR).

[0157] In another aspect, the present invention relates to a vector comprising a chimeric antigen receptor targeting GPC3 (GPC3-CAR), a chimeric antigen receptor targeting CD276 (CD276-CAR), and a polynucleotide encoding TGF-β receptor II (TGFβDNR).

[0158] In the present invention, the polynucleotide may be composed of [polynucleotide encoding a chimeric antigen receptor targeting GPC3 - linker - polynucleotide encoding a chimeric antigen receptor targeting CD276 - linker - polynucleotide encoding TGFβDNR].

[0159] Specific details regarding polynucleotides and vectors containing them are as follows:<GPC3-CAR 및 CD276-CAR로 구성된 inteliCAR-T 플랫폼> As described above, the polynucleotide encoding the TGFβDNR can be represented by the base sequence of SEQ ID NO: 54.

[0160] The above linker may be composed of any sequence that does not inhibit the expression of each chimeric antigen receptor, and in the present invention, T2A represented by the base sequence of SEQ ID NO: 44 and P2A represented by the base sequence of SEQ ID NO: 53 were used as shown in FIG. 3.

[0161]

[0162] Immune effector cells (IBC302 InteliCAR-T)

[0163] In another aspect, the present invention relates to an immune effector cell targeting GPC3, CD276, and TGF-β, which expresses a chimeric antigen receptor targeting GPC3 (GPC3-CAR), a chimeric antigen receptor targeting CD276 (CD276-CAR), and TGF-β receptor II (TGFβDNR).

[0164]

[0165] In the present invention, the [immune effector cell expressing GPC3-CAR, CD276-CAR and TGFβDNR] can be produced by introducing the [GPC3-CAR, CD276-CAR and TGFβDNR coding polynucleotide] or the [GPC3-CAR, CD276-CAR and TGFβDNR expression vector] of the present invention into an immune effector cell, for example, a T cell or an NK cell, and specific details about the immune effector cell are as follows.<GPC3-CAR 및 CD276-CAR로 구성된 inteliCAR-T 플랫폼> It is the same as described above.

[0166]

[0167] <GPC3-CAR, CD276-CAR 및 4-1BBL로 구성된 inteliCAR-T 플랫폼>

[0168] chimeric antigen receptor

[0169] In another aspect, the present invention relates to a chimeric antigen receptor (GPC3-CAR) targeting GPC3 comprising a signal peptide, a GPC3-binding domain, a hinge region, a transmembrane domain, and an intracellular signal transduction domain; and

[0170] A chimeric antigen receptor (CD276-CAR) targeting CD276 comprising a signal peptide, a CD276-binding domain, a hinge region, a transmembrane domain, and an intracellular costimulatory domain; and

[0171] It consists of 4-1BB ligand (4-1BBL), which is composed of 4-1BB ligand extracellular domain (4-1BBL ECD), 4-1BB ligand transmembrane domain (4-1BBL TM), and 4-1BB ligand intracellular domain (4-1BBL ICD).

[0172] The above GPC3-binding domain is a GPC3-specific humanized antibody or fragment thereof, which comprises a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 6.

[0173] The present invention relates to an AND-gate chimeric antigen receptor specific for GPC3, CD276 and 4-1BB, characterized in that the CD276-binding domain is a CD276-specific humanized antibody or fragment thereof, comprising a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 11, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 12 and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 14, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 15 and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 16.

[0174]

[0175] For specific details on AND-gated chimeric antigen receptors specific for GPC3, CD276 and 4-1BB,<GPC3-CAR 및 CD276-CAR로 구성된 inteliCAR-T 플랫폼> As described above, the 4-1BB ligand (4-1BBL) can be represented by the amino acid sequence of SEQ ID NO: 37.

[0176]

[0177] Polynucleotides and recombinant vectors

[0178] In another aspect, the present invention relates to a polynucleotide encoding a chimeric antigen receptor targeting GPC3 (GPC3-CAR), a chimeric antigen receptor targeting CD276 (CD276-CAR), and a 4-1BB ligand (4-1BBL).

[0179] In another aspect, the present invention relates to a vector comprising a polynucleotide encoding a chimeric antigen receptor targeting GPC3 (GPC3-CAR), a chimeric antigen receptor targeting CD276 (CD276-CAR), and a 4-1BB ligand (4-1BBL).

[0180] In the present invention, the polynucleotide may be composed of [polynucleotide encoding a chimeric antigen receptor targeting GPC3 - linker - polynucleotide encoding a chimeric antigen receptor targeting CD276 - linker - polynucleotide encoding 4-1BBL].

[0181] Specific details regarding polynucleotides and vectors containing them are as follows:<GPC3-CAR 및 CD276-CAR로 구성된 inteliCAR-T 플랫폼> As described above, the polynucleotide encoding the above 4-1BBL can be represented by the base sequence of SEQ ID NO: 55.

[0182] The above linker may be composed of any sequence that does not inhibit the expression of each chimeric antigen receptor, and in the present invention, T2A represented by the base sequence of SEQ ID NO: 44 and P2A represented by the base sequence of SEQ ID NO: 53 were used as shown in FIG. 3.

[0183]

[0184] Immune effector cells (IBC303 InteliCAR-T)

[0185] In another aspect, the present invention relates to an immune effector cell targeting GPC3, CD276, and 4-1BB, which expresses a chimeric antigen receptor targeting GPC3 (GPC3-CAR), a chimeric antigen receptor targeting CD276 (CD276-CAR), and a 4-1BB ligand (4-1BBL).

[0186] In the present invention, the [immune effector cell expressing GPC3-CAR, CD276-CAR and 4-1BBL] can be produced by introducing the [GPC3-CAR, CD276-CAR and 4-1BBL coding polynucleotide] or the [GPC3-CAR, CD276-CAR and 4-1BBL expression vector] of the present invention into an immune effector cell, for example, a T cell or an NK cell, and specific details about the immune effector cell are as follows.<GPC3-CAR 및 CD276-CAR로 구성된 inteliCAR-T 플랫폼> It is the same as described above.

[0187]

[0188] In a specific embodiment of the present invention, in order to produce an antibody that specifically binds to GPC3, a hybridoma producing GPC3 protein was produced and screened, and an antibody (scFv) that specifically binds to GPC3 was selected, and a humanized antibody, IBA501v5, was produced using this.

[0189] It was confirmed that the above IBA501v5 antibody comprises a heavy chain variable region including a CDR1 region (GYTFSRYW) represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region (ILPGSGST) represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region (ARSARATYYFDY) represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region including a CDR1 region (QDISNY) represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region (YTS) represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region (QQGNALPYT) represented by an amino acid sequence of SEQ ID NO: 6.

[0190] Specifically, the IBA501v5 antibody is composed of a heavy chain variable region represented by an amino acid sequence of SEQ ID NO: 7 and a light chain variable region represented by an amino acid sequence of SEQ ID NO: 8, and it was confirmed that the heavy chain variable region is encoded by a base sequence of SEQ ID NO: 9, and the light chain variable region is encoded by a base sequence of SEQ ID NO: 10. Preferably, in the present invention, inteliCAR-T was manufactured using the IBA501v5 antibody represented by an amino acid sequence of SEQ ID NO: 22.

[0191]

[0192] In addition, in order to produce an antibody that specifically binds to CD276, hybridomas producing CD276 protein were produced and screened, and an antibody (scFv) that specifically binds to CD276 was selected, and a humanized antibody, IBA201v6, was produced using this.

[0193] The above IBA201v6 antibody was confirmed to be composed of a heavy chain variable region including a CDR1 region (DYAMH) represented by an amino acid sequence of SEQ ID NO: 11, a CDR2 region (VINTYSGNTNYNQKFQG) represented by an amino acid sequence of SEQ ID NO: 12, and a CDR3 region (GLGPYWYFDV) represented by an amino acid sequence of SEQ ID NO: 13, and a light chain variable region including a CDR1 region (RASSSVIYMH) represented by an amino acid sequence of SEQ ID NO: 14, a CDR2 region (ATSNRAT) represented by an amino acid sequence of SEQ ID NO: 15, and a CDR3 region (QQWSSNPYT) represented by an amino acid sequence of SEQ ID NO: 16.

[0194] Specifically, the IBA201v6 antibody is composed of a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 17 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 18, and it was confirmed that the heavy chain variable region is encoded by the base sequence of SEQ ID NO: 19, and the light chain variable region is encoded by the base sequence of SEQ ID NO: 20. Preferably, in the present invention, inteliCAR-T was manufactured using the IBA201v6 antibody represented by the amino acid sequence of SEQ ID NO: 28.

[0195]

[0196] In particular, it was confirmed that the IBA501v5 antibody specifically recognizes HepG2 cells derived from human liver cancer expressing GPC3, and the IBA201v6 antibody specifically recognizes OVCAR3 cells derived from human ovarian cancer expressing CD276.

[0197] In another specific embodiment of the present invention, as shown in the schematic diagram of FIG. 3, [a lentiviral vector having polynucleotides encoding GPC3-CAR and CD276-CAR inserted] was prepared, and this was transduced into T cells to produce IBC301 inteliCAR-T, which is an immune effector cell.

[0198] In addition, [a lentiviral vector containing polynucleotides encoding GPC3-CAR, CD276-CAR, and TGFβDNR] was produced, and this was transduced into T cells to produce IBC302 inteliCAR-T, an immune effector cell.

[0199] In addition, [a lentiviral vector containing polynucleotides encoding GPC3-CAR, CD276-CAR, and 4-1BBL] was produced, and this was transduced into T cells to produce IBC303 inteliCAR-T, an immune effector cell.

[0200] It was confirmed that all manufactured IBC301, IBC302, and IBC303 inteliCAR-T stably expressed GPC3-CAR and CD276-CAR (Fig. 5). It was confirmed that there was no cell killing effect on A549 cells expressing only CD276, but there was an excellent cell killing effect on HepG2 cells overexpressing both GPC3 and CD276 (Fig. 6).

[0201] In addition, to measure the in vitro culture profiles of IBC301, IBC302, and IBC303 inteliCAR-T, cultured for 14 and 9 days, it was confirmed that both IBC301, IBC302, and IBC303 inteliCAR-T of the 14-day process and the 9-day process had excellent cell killing effects on HepG2 cells overexpressing both GPC3 and CD276 (Figs. 7 and 8).

[0202]

[0203] <GPC3 및 CD276를 발현하는 암 또는 종양의 예방 또는 치료용 조성물>

[0204] In another aspect of the present invention, immune effector cells targeting GPC3 and CD276;

[0205] Immune effector cells targeting GPC3, CD276, and TGF-β; or

[0206] The present invention relates to a pharmaceutical composition for preventing or treating cancer or tumors, comprising immune effector cells targeting GPC3, CD276 and 4-1BB.

[0207] In the present invention, the cancer or tumor may be selected from the group consisting of prostate cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, lung cancer, liver cancer, pancreatic cancer, head and neck cancer, melanoma, glioblastoma, and neuroblastoma, and preferably may be a cancer or tumor expressing GPC3 and CD276.

[0208] In the present invention, the composition may include a therapeutic agent for cancer or a tumor, and the therapeutic agent may be administered in combination with the immune effector cells of the present invention.

[0209] The above therapeutic agents include small molecule drugs, peptide drugs, toxins (e.g., cytotoxins), etc.

[0210] The above small molecule drug exhibits the pharmaceutical activity of interest and may be a compound having a molecular weight of about 800 Da or less or 2000 Da or less. Inorganic small molecules refer to molecules that do not contain any carbon atoms, whereas organic small molecules refer to compounds that contain at least one carbon atom.

[0211] The above peptide drugs refer to amino acid-containing polymeric compounds, including naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides, and proteins, as well as peptide mimetics. The peptide drugs can be obtained by chemical synthesis or produced from genetically encoded sources (e.g., recombinant sources). The molecular weight of the peptide drugs can range from 200 Da to 10 kDa or more.

[0212] The toxin is preferably a cytotoxin, which includes, but is not limited to, ricin, abrin, diphtheria toxin, Pseudomonas exotoxins (e.g., PE35, PE37, PE38, PE40, etc.), saporin, gelonin, pore antiviral protein (PAP), botulinum toxin, bryodin, momordin, and buganin.

[0213] Additionally, the therapeutic agent may be an anticancer agent. Anticancer agents include non-peptide (i.e., non-protein-based) compounds that reduce the proliferation of cancer cells and include cytotoxic agents and cytostatic agents. Non-limiting examples of anticancer agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptide compounds may also be used.

[0214]

[0215] In a specific embodiment of the present invention, an animal experiment was performed to confirm the anti-tumor effect of IBC301 inteliCAR-T and IBC302 inteliCAR-T (Fig. 9a), and it was confirmed that the growth of HepG2-Luc cells xenografted into mice was inhibited by IBC301 inteliCAR-T and IBC302 inteliCAR-T. In particular, it was confirmed that most cancer cells were killed 14 days after inoculation by IBC302 inteliCAR-T (Figs. 9b and 9c).

[0216] In addition, to confirm the memory response by IBC301 inteliCAR-T and IBC302 inteliCAR-T, HepG2-Luc cells were administered for the second time to mice that completed the animal experiment in Fig. 9, and it was confirmed that tumor growth was suppressed even without additional administration of IBC301 inteliCAR-T and IBC302 inteliCAR-T (Fig. 10).

[0217]

[0218] In another specific embodiment of the present invention, in order to block CD276 signal leakage, IBC302CA inteliCAR-T was prepared by substituting cysteine ​​(C) in the hinge and TM domains included in the CD276-CAR portion of IBC302 inteliCAR-T with alanine (A). The IBC302CA inteliCAR-T blocks signal leakage to CD3ζ due to CD276 binding by reducing the possibility of disulfide bonds, thereby increasing stability (Figs. 11 and 12).

[0219] The manufactured IBC302CA inteliCAR-T was confirmed to stably express GPC3-CAR and CD276-CAR on the surface of T cells (Figs. 13a to 13c), and it was confirmed to have an excellent cell killing effect on HepG2 cells overexpressing both GPC3 and CD276 (Figs. 14 and 15).

[0220] In addition, as a result of conducting an animal experiment to confirm the anti-tumor effect of IBC302CA inteliCAR-T (Fig. 16a), it was confirmed that most cancer cells were killed 14 days after inoculation by IBC302CA inteliCAR-T (Figs. 16b and 16c).

[0221] In addition, to confirm the memory response by IBC302CA inteliCAR-T, HepG2-Luc cells were administered for the second time to mice that completed the animal experiment in Fig. 9, and it was confirmed that tumor growth was effectively suppressed without recurrence even without additional administration of IBC302CA inteliCAR-T (Figs. 17a and 17b).

[0222]

[0223] That is, the IBC302 inteliCAR-T and IBC302CA inteliCAR-T of the present invention can be usefully utilized as a pharmaceutical composition for the prevention or treatment of diseases associated with GPC3 and CD276 overexpression, preferably cancer or tumor.

[0224]

[0225] In the pharmaceutical composition, the immune effector cells may be the sole active ingredient in the composition, or may be used together with other active ingredients, including, for example, other antibody components such as anti-T cells, anti-IFNγ or anti-LPS antibodies, or non-antibody components such as xanthines.

[0226] The pharmaceutical composition preferably comprises a therapeutically effective amount of the antibody of the present invention. As used herein, the term "therapeutically effective amount" refers to the amount of a therapeutic agent necessary to treat, improve, or prevent a target disease or condition, or to produce a detectable therapeutic or preventive effect. For certain antibodies, the therapeutically effective dose can be initially determined using cell culture assays or animal models, typically rodents, rabbits, dogs, pigs, or primates. Animal models can also be used to determine appropriate concentration ranges and administration routes. This information can be used to determine useful dosages and routes for human administration.

[0227] The precise effective dose for a human patient may vary depending on the severity of the disease, the patient's general health, the patient's age, weight, and sex, diet, administration time and frequency, the formulation of the drug, sensitivity, and tolerance / response to treatment. The amount can be determined through routine experiments and is within the clinician's discretion. Typically, the effective dosage is 0.01 to 50 mg / kg, preferably 0.1 to 20 mg / kg, and more preferably about 15 mg / kg. The composition may be administered individually to a patient or in combination with other agents, drugs, or hormones.

[0228] The dosage at which the antibody of the present invention is administered will vary depending on the nature of the condition to be treated, the grade of the malignant lymphoma or leukemia, and whether the antibody is used for disease prevention or to treat an existing condition.

[0229] The frequency of administration depends on the half-life of the antibody molecule and the duration of the drug effect. If the antibody molecule has a short half-life (e.g., 2 to 10 hours), it may need to be administered once or more times daily. Alternatively, if the antibody molecule has a long half-life (e.g., 2 to 15 days), it may need to be administered once daily, once weekly, or once every month or two months.

[0230] Additionally, the pharmaceutical composition may contain a pharmaceutically acceptable carrier for the administration of antibodies. The carrier must not induce the production of harmful antibodies in the subject receiving the composition and must be nontoxic. Suitable carriers include slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers, and inactivated virus particles.

[0231] Pharmaceutically acceptable salts may be used, for example, mineral salts such as hydrochlorides, hydrobromates, phosphates and sulfates, or salts of organic acids such as acetic, propionic, malonic and benzoic acids.

[0232] Pharmaceutically acceptable carriers within the therapeutic composition may additionally include liquids such as water, saline, glycerol, and ethanol. Additionally, auxiliary substances such as wetting agents, emulsifiers, or pH buffering agents may be present within the composition. The carriers may be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, and suspensions for patient ingestion of the pharmaceutical composition.

[0233] Preferred forms for administration include those suitable for parenteral administration, for example, by injection or infusion (e.g., bolus injection or continuous infusion). When the product is for injection or infusion, it may take the form of a suspension, solution, or emulsion in an oil or water-soluble vehicle, which may contain preservatives, stabilizers, and / or dispersing agents. Alternatively, the antibody molecule may be in anhydrous form and reconstituted with a suitable sterile solution before use.

[0234] Once formulated, the composition of the present invention can be administered directly to a patient. The patients to be treated may be animals. However, it is preferred that the composition be tailored for administration to human patients.

[0235] The pharmaceutical composition of the present invention may be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (see, e.g., WO 98 / 20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal, or rectal routes. A hypospray may be used to administer the pharmaceutical composition of the present invention. Typically, the therapeutic composition may be prepared as an injectable material, either as a liquid solution or suspension. In addition, a solid form suitable for solution or suspension in a liquid excipient may be prepared prior to injection.

[0236] Direct delivery of the composition can generally be achieved by injection, subcutaneous injection, intraperitoneal injection, intravenous injection, or intramuscular injection, or it can be delivered into the interstitial space of a tissue. Additionally, the composition can be administered to a wound site. Dosage regimens can be single-dose or multiple-dose.

[0237] The active ingredient in the composition may be an antibody molecule. This molecule itself may be susceptible to degradation in the gastrointestinal tract. Therefore, if the composition is administered via the gastrointestinal route, it will need to contain an agent that protects the antibody from degradation but releases it from the gastrointestinal tract once absorbed.

[0238]

[0239] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0240]

[0241] Example 1: Preparation of humanized antibodies that specifically bind to GPC3

[0242] To select GPC3 peptide-specific antibodies, hybridomas producing antibodies that bind to GPC3 were prepared and antibodies were selected.

[0243] First, splenocytes were isolated by immunization with GPC3 protein (Acrobiosystems, cat#GP3-H52H4) and hybridoma cells were generated through cell fusion with mouse bone marrow progenitor cells.

[0244] Mouse myeloma cells used for cell fusion do not possess HGPRT (Hypoxanthine Guanidine-Phosphoribosyl-Transferase) and therefore cannot survive in HAT medium. However, hybridomas can survive in HAT medium by fusing with spleen cells. This allows for the proliferation of only hybridomas, so they are usually grown in HAT medium until hybridomas are established.

[0245] To select hybridomas that produce antibodies that bind to GPC3 among the expanded hybridomas, the limiting dilution method was used. First, the number of cells per 96-well was reduced to less than 1. Then, the antibody obtained from a clone expanded from a single cell was confirmed to bind to GPC3 using ELISA, and clones that did bind to GPC3 were selected. This process was repeated three times to select hybridomas that produce antibodies that bind to GPC3.

[0246] An antibody binding to GPC3 was obtained in this manner, and then a humanized antibody was prepared by changing the selected antibody into a structure corresponding to a human.

[0247] Specifically, a humanized antibody was produced by replacing the CDR of a mouse antibody that binds to GPC3 with the CDR of a human antibody in frame with the germline sequence of the human antibody using the CDR grafting method, and this was named IBA501v5.

[0248]

[0249] IBA501v5 antibody sequence information IBA501v5 sequence information Sequence number Heavy chain variable region CDR1 GYTFSRYW Sequence number 1 Heavy chain variable region CDR2 ILPGSGST Sequence number 2 Heavy chain variable region CDR3 ARSARATYYFDY Sequence number 3 Light chain variable region CDR1 QDISNY Sequence number 4 Light chain variable region CDR2 YTS Sequence number 5 Light chain variable region CDR3 QQGNALPYT Sequence number 6 Heavy chain variable region amino acid sequence QVQLVQSGAEVKKPGASVKVSCKATGYTFSRYWMHWVRQAPGQGLEWMGEILPGSGSTSYNEKFQGRVTMTADTSTSTAYMELSSLRSEDTAVYYCARSARATYYFDYWGQGTTVTVSS Sequence number 7 Light chain variable region amino acid sequence DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAVKLLIYYTSTLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNALPYTFGGGTKLEIKSequence number 8 Heavy chain variable region base sequence CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTCAAGAAGCCCGGCGCTTCCGTAAAGGTGTCATGCAAGGCAACTGGCTACACCTTTTCACGTTATTGGATGCACTGGGTCCGCCAGGCCCCGGGACAGGGCCTGGGAGTGGATGGGCGAGATCCTGCCTGGTTCTGGCTCCACTA GCTACAACGAGAAATTTCAGGGTCGGGTTACGATGACGGCGGACACTTCCACATCGACCGCCTACATGGAGCTGAGCTCGCTAAGGTCCGAGGACACCGCAGTGTACTACTGTGCCCGCAGCGCGCGAGCTACCTACTACTTCGACTATTGGGGCCAGGGAACTACCGTCACCGTGTCCTCCSEQ ID NO.9Light chain variable region nucleotide sequence GACATCCAGATGACCCAGAGCCCCTCTAGTCTTTCCGCCTCCGTGGGTGATCGCGTGACCATCACCTGCCAGGCTTCTCAGGACATCAGCAACTACCTCAATTGGTACCAGCAGAAGCCTGGCAAGGCCGTGAAACTGCTGATTTACTACACCTCCA CCTTGCATAGTGGGGTCCCGTCCCGCTTCTCTGGTTCGGGGTCCGGCACCGACTACACGTTCACCATTTCTTCTCTGCAGCCAGAAGATATCGCCACTTACTTCTGTCAACAGGGCAACGCGCTGCCCTATACATTCGGCGGTGGGACCAAGCTGGAGATCAAGSSEQ ID NO. 10

[0250] In the present invention, a chimeric antigen receptor was produced using an IBA501v5 antibody in the form of scFv (single chain variable fragment), and the antibody is composed of an amino acid sequence of SEQ ID NO: 22 and encoded by a base sequence of SEQ ID NO: 40.

[0251]

[0252] Example 2: Preparation of humanized antibodies that specifically bind to CD276

[0253] To select CD276 peptide-specific antibodies, hybridomas producing antibodies that bind to CD276 were prepared and the antibodies were selected.

[0254] First, splenocytes were extracted by immunizing with CD276 protein, and hybridoma cells were produced through cell fusion with mouse myeloma cells.

[0255] Mouse myeloma cells used for cell fusion do not possess HGPRT (Hypoxanthine Guanidine-Phosphoribosyl-Transferase) and therefore cannot survive in HAT medium. However, hybridomas can survive in HAT medium by fusing with spleen cells. This allows for the proliferation of only hybridomas, so they are usually grown in HAT medium until hybridomas are established.

[0256] To select hybridomas that produce antibodies that bind to CD276 among the expanded hybridomas, the limiting dilution method was used. First, no more than one cell was seeded per 96-well. Then, antibodies obtained from clones grown from a single cell were confirmed to bind to CD276 using ELISA, and clones that did bind to CD276 were selected. This process was repeated three times to select hybridomas that produce antibodies that bind to CD276.

[0257] An antibody binding to CD276 was obtained in this manner, and then a humanized antibody was prepared by changing the selected antibody into a structure corresponding to a human.

[0258] Specifically, a humanized antibody was produced by the CDR grafting method, which replaces the CDR of a mouse antibody that binds to CD276 with the CDR of a human antibody in frame with the germline sequence of the human antibody, and this was named IBA201v6.

[0259]

[0260] Sequence information of IBA201v6 antibody IBA201v6 Sequence information Sequence number Heavy chain variable region CDR1 DYAMH SEQ ID NO: 11 Heavy chain variable region CDR2 VINTYSGNTNYNQKFQG SEQ ID NO: 12 Heavy chain variable region CDR3 GLGPYWYFDV SEQ ID NO: 13 Light chain variable region CDR1 RASSSVIYMH SEQ ID NO: 14 Light chain variable region CDR2 ATSNRAT SEQ ID NO: 15 Light chain variable region CDR3 QQWSSNPYT SEQ ID NO: 16 Heavy chain variable region amino acid sequence QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAMHWVRQAPGQRLEWMGVINTYSGNTNYNQKFQGRVTITVDKSASTAYMELSSLRSEDTAVYYCARGLGPYWYFDVWGQGTTVTVSS SEQ ID NO: 17 Light chain variable region amino acid sequence EIVLTQSPATLSLSPGERATLSCRASSSVIYMHWYQQKPGQAPRPLIYATSNRATGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPYTFGGGTKLEIK sequence number 18 Heavy chain variable region nucleotide sequence CAAGTGCAGCTGGTGCAGAGCGGGGCGGAGGTGAAGAAGCCGGGTGCTTCCGTAAAGGTGTCATGCAAAGGCTCCGGCTACACGTTCACCGACTATGCCATGCACTGGGTCCGCCAGGCCCCTGGACAGCGCCTGGAGTGGATGGGCGTGATTAATACCTACTCCGGCAACACG AACTACAACCAGAAGTTCCAGGGCCGGGTTACCATCACCGTGGACAAGAGTGCATCTACTGCCTACATGGAGCTGAGCTCTTTGCGCTCGGAAGACACCGCGGTGTACTACTGTGCTCGTGGTCTCGGGCCCTATTGGTACTTTGACGTTGGGGCCAGGGAACAACTGTCACGTGTCGTCCSEQ ID NO.19Light chain variable region base sequenceGAGATTGTCCTGACCCAGAGCCCCGCTACACTGAGCTTGAGTCCTGGGGAGCGGGCAACTCTTTCGTGCCGTGCGTCGTCCTCCGTGATCTACATGCACTGGTACCAGCAGAAGCCCGGCCAGGCCCCGCGCCCGCTCATTTACGCGACTAGCAA CCGCGCCACCGGCATCCCTGCTCGCTTTTCAGGTTCCGGGCTCCGGAACCGACTACACCCTGACCATCTCTTCTCTGGAGCCCGAAGATTTCGCCGTGTACTACTGTCAACAGTGGTCTTCCAACCCATATACCTTCGGCGGCGGCACCAAGCTGGAGATCAAASEQ ID NO. 20

[0261] In the present invention, a chimeric antigen receptor was produced using an IBA201v6 antibody in the form of scFv (single chain variable fragment), and the antibody is composed of an amino acid sequence of SEQ ID NO: 28 and encoded by a base sequence of SEQ ID NO: 46.

[0262]

[0263] Example 3: Confirmation of the specificity of selected antibodies for GPC3 and CD276

[0264] 3-1: Confirmation of specificity for GPC3

[0265] In the present invention, to confirm the specificity of the IBA501v5 antibody established in Example 1 for GPC3, flow cytometry analysis was performed.

[0266] First, HepG2 cells (4 x 10) derived from human liver cancer overexpressing GPC3 5 After reacting with each of the dog) and IBA501v5 antibodies (1 μg) for 30 minutes, the surface was stained with a secondary antibody and then measured using a flow cytometer.

[0267] GPC3 antibody (APC anti-human GPC3; Sino biology cat#100393-R024-A, 10 μl) was used as a positive control, and PE-conjugated anti-mouse IgG antibody (PE-conjugated goat anti-mouse IgG; Biolegend Inc., cat# 405307, USA, 3 μl) was used as a secondary antibody.

[0268]

[0269] MFI (mean fluorescence intensity) value Antibody type Count IBA501v5 Untreated (none) 79.22 Secondary antibody treatment (2nd Ab alone) 157 IBA501v5 550 97 Positive control (positive) Untreated (none) 7.22 Commercial anti-GPC3 antibody treatment 2953

[0270] As a result, as shown in Fig. 1, it was confirmed that the IBA501v5 antibody specifically binds to cells expressing GPC3.

[0271] 3-2: Confirmation of specificity for CD276

[0272] In the present invention, to confirm the specificity of the IBA201v6 antibody established in Example 2 for CD276, flow cytometry analysis was performed.

[0273] First, OVCAR3 (4 x 10), a human ovarian cancer cell line expressing CD276 5 After reacting with each of the primary antibodies (dog) and IBA201v6 antibody (2 μg) for 30 minutes, the surface was stained with a secondary antibody and then measured using a flow cytometer.

[0274] A commercially available APC-anti-CD276 antibody (APC-HuCD276 (CD276), Invitrogen, cat#17-2769-42, USA, 5 μl) was used as a positive control, and a PE-conjugated anti-mouse IgG antibody (PE-conjugated goat anti-mouse IgG; Biolegend Inc., cat# 405307, USA, 3 μl) was used as a secondary antibody.

[0275]

[0276] MFI (mean fluorescence intensity) value Antibody type Count IBA201v6 Untreated (none) 23.72 Secondary antibody treatment (2nd Ab alone) 425 IBA201v6 2962 Positive control (positive) Untreated (none) 18 Commercial anti-CD276 antibody treatment 7325

[0277] As a result, as shown in Fig. 2, it was confirmed that the IBA201v6 antibody specifically binds to cells expressing CD276.

[0278]

[0279] Example 4: Production of expression vectors for a chimeric antigen receptor targeting GPC3 (GPC3-CAR) and a chimeric antigen receptor targeting CD276 (CD276-CAR).

[0280] In the present invention, a lentiviral vector in which GPC3-CAR and CD276-CAR are simultaneously expressed was produced using the IBA501v5 antibody and the IBA201v6 antibody.

[0281] As shown in the schematic diagram of Fig. 3, an IBC301 lentiviral vector comprising [polynucleotide encoding a chimeric antigen receptor targeting GPC3 - linker - polynucleotide encoding a chimeric antigen receptor targeting CD276],

[0282] IBC302 lentiviral vector comprising [polynucleotide encoding a chimeric antigen receptor targeting GPC3 - linker - polynucleotide encoding a chimeric antigen receptor targeting CD276 - linker - polynucleotide encoding TGFβDNR], and

[0283] An IBC303 lentiviral vector was prepared comprising [polynucleotide encoding a chimeric antigen receptor targeting GPC3 - linker - polynucleotide encoding a chimeric antigen receptor targeting CD276 - linker - polynucleotide encoding 4-1BBL].

[0284]

[0285] Specifically, the IBC301 lentiviral vector

[0286] EF1α promoter (SEQ ID NO: 38)

[0287] A polynucleotide encoding an IgGκ signal peptide (SEQ ID NO: 39);

[0288] A polynucleotide encoding a GPC3-binding domain (SEQ ID NO: 40);

[0289] A polynucleotide encoding a CD8α hinge region (SEQ ID NO: 41);

[0290] A polynucleotide encoding a CD8α transmembrane domain (SEQ ID NO: 42);

[0291] A polynucleotide encoding the CD3ζ intracellular signal transduction domain (SEQ ID NO: 43);

[0292] T2A linker (SEQ ID NO: 44);

[0293] A polynucleotide encoding a CD33 signal peptide (SEQ ID NO: 45);

[0294] A polynucleotide encoding a CD276-binding domain (SEQ ID NO: 46);

[0295] A polynucleotide encoding the CD28 hinge region (SEQ ID NO: 47);

[0296] A polynucleotide encoding the CD28 transmembrane domain (SEQ ID NO: 50);

[0297] A polynucleotide encoding the CD28 intracellular costimulatory domain (SEQ ID NO: 52) and

[0298] IBC301 inteli-CAR DNA, consisting of a polynucleotide encoding WPRE (SEQ ID NO: 56), was synthesized in vitro and inserted into a third-generation lentiviral vector.

[0299]

[0300] In addition, the IBC302 lentiviral vector, between the polynucleotide encoding the CD28 intracellular costimulatory domain and WPRE of the IBC301 inteli-CAR DNA,

[0301] P2A linker (SEQ ID NO: 53); and

[0302] IBC302 inteli-CAR DNA, additionally containing a polynucleotide encoding TGFβDNR (SEQ ID NO: 54), was synthesized in vitro and inserted into a third-generation lentiviral vector.

[0303] The above TGFβDNR is specifically composed of [TGF-β RⅡ extracellular domain (TGF-β RⅡ ECD) - TGF-β RⅡ transmembrane domain (TGF-β RⅡ TM) - TGF-β RⅡ intracellular domain (Truncated TGF-β RⅡ ICD)].

[0304]

[0305] In addition, the IBC303 lentiviral vector comprises a polynucleotide encoding the CD28 intracellular costimulatory domain and WPRE of the IBC301 inteli-CAR DNA.

[0306] P2A linker (SEQ ID NO: 53); and

[0307] IBC303 inteli-CAR DNA, which additionally includes a polynucleotide (SEQ ID NO: 55) encoding 4-1BB ligand (4-1BBL), was synthesized in vitro and inserted into a third-generation lentiviral vector.

[0308] The above 4-1BBL is specifically composed of [4-1BB ligand extracellular domain (4-1BBL ECD) - 4-1BB ligand transmembrane domain (4-1BBL TM) - 4-1BB ligand intracellular domain (4-1BBL ICD)].

[0309]

[0310] Example 5: Manufacturing of IBC301, IBC302, and IBC303 inteliCAR-T

[0311] In the present invention, peripheral blood mononuclear cells (PBMC) were separated from blood, and then T cells were activated using T cell activation beads (Miltenyi Biotec, cat# 130-091-441). Activated T cells were transfected with the IBC301, IBC302, and IBC303 lentiviral vectors prepared in <Example 4> to produce IBC301 inteliCAR-T, which is an [immune effector cell expressing GPC3-CAR and CD276-CAR] in which GPC3-CAR and CD276-CAR are simultaneously expressed, IBC302 inteliCAR-T, which is an [immune effector cell expressing GPC3-CAR, CD276-CAR, and TGFβ], and IBC303 inteliCAR-T, which is an [immune effector cell expressing GPC3-CAR, CD276-CAR, and 4-1BBL].

[0312]

[0313] The expression levels of GPC3-CAR and CD276-CAR in IBC301, IBC302, and IBC303 inteliCAR-T were confirmed using flow cytometry.

[0314] IBC301, IBC302, and IBC303 inteliCAR-T cells at 6, 9, and 13 days of T cell activation were sorted into CD3-activated cells using anti-CD3 antibody, and then reacted with B7H3-PE peptide and GPC3-FITC peptide, and fluorescence intensity was measured using a flow cytometer. hPBMCs not transduced with lentiviral vectors were used as a control.

[0315]

[0316] As a result, as shown in FIGS. 5A to 5C, it was confirmed that all CD3-activated IBC301, IBC302, and IBC303 inteliCAR-T cells similarly expressed GPC3-CAR and CD276-CAR as well as TGFbR2 on the surface of T cells.

[0317]

[0318] Example 6: Confirmation of the apoptotic effect of IBC301 and IBC302 inteliCAR-T on cancer cells overexpressing GPC3 and CD276.

[0319] In the present invention, the target cell killing effect by IBC301 and IBC302 inteliCAR-T prepared in the above <Example 5> was confirmed.

[0320] A549 cells derived from human lung adenocarcinoma and HepG2 cells derived from human liver cancer were used as target cells. IBC301 and IBC302 inteliCAR-T cells with 13 days of T cell activation and target cells were mixed at ratios of 4:1, 2:1, 1:1, 0.5:1, and 0.25:1, respectively, and cultured for 12 hours, and then luminescence (CytoTox-Glo Cytotoxicity Assay, Promega, cat. NO G9291) was measured. The degree of cell death was calculated using the following mathematical formula 1 with the measured value.

[0321]

[0322] [Mathematical Formula 1]

[0323] % Cytotoxicity = [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)]

[0324]

[0325] Experimental: Luminescence values ​​derived from the medium of target cell and CAR-T cell complex cultures

[0326] Effector Spontaneous: Luminescence derived from CAR-T cell-only medium

[0327] Target Spontaneous: Luminescence value derived from the medium of target cells only

[0328] Target Maximum: Luminescence value derived from 100% lysis of target cells (using lysis reagent)

[0329]

[0330] As a result, as shown in Fig. 6, both IBC301 and IBC302 inteliCAR-T showed apoptotic effects on HepG2 cells derived from human liver cancer, but did not show apoptotic effects on A549 cells derived from human lung adenocarcinoma that express only CD276.

[0331]

[0332] Example 7: In vitro culture profiles and cancer cell killing effects of IBC301, IBC302, and IBC303 inteliCAR-T cells.

[0333] In the present invention, in order to confirm the in vitro culture profile of IBC301, IBC302, and IBC303 inteliCAR-T prepared in the above <Example 5>, a 9-day culture process and a 13-day culture process were performed, and then the total number of viable cells and the survival rate were measured (Fig. 7).

[0334] In addition, IBC301, IBC302, and IBC303 inteliCAR-T cells cultured for 9 and 13 days, respectively, were mixed at ratios of 4:1, 2:1, 1:1, 0.5:1, and 0.25:1 to target cells, respectively, and cultured for 12 hours, and then luminescence (CytoTox-Glo Cytotoxicity Assay, Promega, cat. NO G9291) was measured. The degree of cell death was calculated using the above mathematical formula 1 with the measured value.

[0335]

[0336] As a result, as shown in Fig. 8, it was confirmed that IBC301, IBC302, and IBC303 inteliCAR-T of the 13-day culture process and the 9-day culture process all had excellent cell killing effects on HepG2 cells overexpressing both GPC3 and CD276.

[0337]

[0338] Example 8: Confirmation of the antitumor effects of IBC301 inteliCAR-T and IBC302 inteliCAR-T in animal models.

[0339] In the present invention, the anti-tumor effects of IBC301 inteliCAR-T, IBC302 inteliCAR-T, and IBC303 inteliCAR-T were confirmed using a mouse model in which tumor cells were xenografted (Fig. 9a).

[0340] 1x10 in 9-week-old NOD / SCID mice 6After intravenous injection of HepG23 / Luc cells, IBC301 inteliCAR-T, IBC302 inteliCAR-T, and IBC303 inteliCAR-T were injected intravenously, respectively. The luminescence expressed in HepG2 / Luc cells was photographed in vitro using IVIS SpectrumCT on the day before inteliCAR-T infusion and on days 1, 9, 16, 23, 30, and 37 after inteliCAR-T infusion, and the antitumor effect was observed by inhibition of HepG2 / Luc cell proliferation.

[0341]

[0342] As a result, as shown in Figures 9b and 9c, IBC301 inteliCAR-T and IBC303 inteliCAR-T failed to suppress tumor growth, whereas the animal group injected with IBC302 inteliCAR-T showed excellent anti-tumor effects. In particular, it was observed that most tumor cells were killed 14 days after IBC302 inteliCAR-T injection.

[0343]

[0344] Example 9: Confirmation of memory response to the antitumor effect of IBC302 inteliCAR-T in an animal model.

[0345] In the present invention, a memory response was confirmed when a secondary tumor was formed in a mouse administered with the IBC302 inteliCAR-T of <Example 8>.

[0346] First, on the 51st day after inteliCAR-T injection in the above <Example 8>, 1x10 6HepG23 / Luc cells were intravenously injected. On days 7, 13, 20, 27, and 33 after the second HepG23 / Luc cell injection, luminescence expressed in HepG2 / Luc cells was imaged in vitro using IVIS SpectrumCT to observe the antitumor effect by inhibiting the proliferation of HepG2 / Luc cells. No additional inteliCAR-T administration was performed.

[0347]

[0348] As a result, as shown in FIGS. 10a and 10b, in the IBC302 inteliCAR-T administration group of <Example 8>, HepG2 secondary tumor growth was suppressed without additional administration (the tumor recurred in only one mouse).

[0349] That is, it was confirmed that a memory response was observed due to administration of IBC302 inteliCAR-T of the present invention.

[0350]

[0351] Example 10: Production of IBC302CA inteliCAR-T with Disulphide Bond Blocking

[0352] As shown in Fig. 11, the present invention produced IBC302CA inteliCAR-T with blocked disulfide bonds to reduce the possibility of CD276 signal leakage to the intracellular signaling domain (CD3ζ) of GPC3-CAR.

[0353] A lentiviral vector expressing GPC3-CAR and CD276-CAR simultaneously was prepared using the same method as in <Example 4> above, and then IBC302CA inteliCAR-T, which is an [immune effector cell expressing GPC3-CAR, CD276-CAR, and TGFβDNR], was prepared using the same method as in <Example 5> above.

[0354] To block disulfide bonds, cysteine ​​(C) in the CD28-derived hinge region and CD28-derived transmembrane domain of CD276-CAR was substituted with alanine (A). Specifically, the CD28-derived hinge region (SEQ ID NO: 31) was substituted with cysteine ​​(C) in the CD28 hinge (28th sequence of SEQ ID NO: 31), and the CD28-derived transmembrane domain (SEQ ID NO: 33) was substituted with cysteine ​​(C) in the CD28 transmembrane domain (13th sequence of SEQ ID NO: 33).

[0355]

[0356] Specifically, the IBC302CA lentiviral vector, which blocks disulfide bonds,

[0357] EF1α promoter (SEQ ID NO: 38)

[0358] A polynucleotide encoding an IgGκ signal peptide (SEQ ID NO: 39);

[0359] A polynucleotide encoding a GPC3-binding domain (SEQ ID NO: 40);

[0360] A polynucleotide encoding a CD8α hinge region (SEQ ID NO: 41);

[0361] A polynucleotide encoding a CD8α transmembrane domain (SEQ ID NO: 42);

[0362] A polynucleotide encoding the CD3ζ intracellular signal transduction domain (SEQ ID NO: 43);

[0363] T2A linker (SEQ ID NO: 44);

[0364] A polynucleotide encoding a CD33 signal peptide (SEQ ID NO: 45);

[0365] A polynucleotide encoding a CD276-binding domain (SEQ ID NO: 46);

[0366] A polynucleotide encoding the CD28 hinge region (C->A) (SEQ ID NO: 49);

[0367] A polynucleotide encoding a CD28 transmembrane domain (C->A) (SEQ ID NO: 51);

[0368] A polynucleotide encoding the CD28 intracellular costimulatory domain (SEQ ID NO: 52);

[0369] P2A linker (SEQ ID NO: 53);

[0370] A polynucleotide encoding TGFβDNR (SEQ ID NO: 54); and

[0371] IBC302CA inteli-CAR DNA, consisting of a polynucleotide encoding WPRE (SEQ ID NO: 56), was synthesized in vitro and inserted into a third-generation lentiviral vector.

[0372]

[0373] Example 11: Characterization of IBC302CA inteliCAR-T

[0374] 11-1: Confirmation of signal leakage through CD276 in IBC302CA inteliCAR-T

[0375] The above IBC302CA inteliCAR-T reduces the possibility of disulfide bonds by substituting cysteines in the hinge and TM domains of CD276-CAR with alanines, thereby reducing signal leakage to CD3ζ due to CD276 binding, thereby improving safety.

[0376] To confirm this, IFN-γ production was compared through intracellular cytokine staining after 6 h of co-culture of IBC302 inteliCAR-T and IBC302CA inteliCAR-T with hepatocellular carcinoma cell lines Hep1 (GPC3-CD276+) and HepG2 (GPC3+CD276+), respectively.

[0377]

[0378] As a result, as shown in Fig. 12, signal leakage due to CD276 sole binding (Hep1) was found to be reduced in IBC302CA inteliCAR-T compared to IBC302 inteliCAR-T. Meanwhile, IFN-γ production due to signal transduction by simultaneous binding of GPC3 and CD276 (HepG2) was confirmed to be similar between IBC302 inteliCAR-T and IBC302CA inteliCAR-T.

[0379]

[0380] 11-2: Confirmation of CAR expression level of IBC302CA inteliCAR-T

[0381] The expression levels of GPC3-CAR and CD276-CAR of IBC302CA inteliCAR-T were confirmed through flow cytometry in the same manner as in <Example 5> above.

[0382]

[0383] On days 6, 9, and 19 of T cell activation, both IBC302 and IBC302CA were analyzed by flow cytometry using FITC-conjugated human GPC3 protein (Acrobiosystems), PE-conjugated human CD276 (Acrobiosystems) protein, and BV421-conjugated TGFbR2 mAb (Biolegend), respectively. As shown in Figures 13a to 13c, it was confirmed that GPC3-CAR and CD276-CAR as well as TGFbR2 were similarly expressed on the surface of human T cells.

[0384]

[0385] Example 12: In vitro culture profile and cancer cell killing effect of IBC302CA inteliCAR-T

[0386] In the present invention, in order to confirm the in vitro culture profile of IBC302CA inteliCAR-T manufactured in the above <Example 10>, a 9-day culture process and a 13-day culture process were performed in the same manner as in the above <Example 7>, and then the total number of viable cells and survival rate were measured, and the degree of cancer cell death was calculated.

[0387]

[0388] As a result, as shown in Figs. 14 and 15, it was confirmed that both IBC302 and IBC302CA inteliCAR-T of the 13-day culture process and the 9-day culture process had excellent cell killing effects on HepG2 cells overexpressing both GPC3 and CD276.

[0389]

[0390] Example 13: Confirmation of the antitumor effect of IBC302CA inteliCAR-T in an animal model.

[0391] In the present invention, the anti-tumor effects of IBC302 inteliCAR-T and IBC302CA inteliCAR-T were confirmed using a mouse model in which tumor cells were xenografted (Fig. 16a).

[0392] 1x10 in 9-week-old NOD / SCID mice 6 HepG23 / Luc cells were intravenously injected, and then IBC302 inteliCAR-T, IBC302CA inteliCAR-T, and IBC303 inteliCAR-T were intravenously injected, respectively. The luminescence expressed in HepG2 / Luc cells was imaged in vitro using IVIS SpectrumCT on the day before inteliCAR-T infusion and on days 1, 7, 14, 21, 27, and 34 after inteliCAR-T infusion, and the antitumor effect was observed by inhibition of HepG2 / Luc cell proliferation.

[0393]

[0394] As a result, as shown in FIGS. 16b and 16c, it was confirmed that the animal groups injected with the IBC302 inteliCAR-T and IBC302CA inteliCAR-T of the present invention induced tumor regression and inhibited growth. In particular, it was observed that most tumor cells were killed 14 days after the injection of IBC302CA inteliCAR-T. On the other hand, it was confirmed that the animal group injected with IBC303 inteliCAR-T did not inhibit tumor growth.

[0395]

[0396] Example 14: Confirmation of memory response to the antitumor effect of IBC302CA inteliCAR-T in an animal model.

[0397] In the present invention, when secondary tumors were formed in mice administered with the IBC302 inteliCAR-T and IBC302CA inteliCAR-T of the above <Example 13>, a memory response was confirmed.

[0398] First, on the 51st day after inteliCAR-T injection in the above <Example 8>, 1x10 6 HepG23 / Luc cells were intravenously injected. On days 2, 9, 16, and 23 after the second HepG23 / Luc cell injection, luminescence expressed in HepG2 / Luc cells was imaged in vitro using IVIS SpectrumCT to observe the antitumor effect by inhibiting the proliferation of HepG2 / Luc cells. No additional inteliCAR-T was administered.

[0399]

[0400] As a result, as shown in FIGS. 17a and 17b, in the IBC302 inteliCAR-T and IBC302CA inteliCAR-T administration groups of <Example 13>, both HepG2 secondary tumor growth was inhibited without additional administration.

[0401] That is, it was confirmed that the IBC302 inteliCAR-T and IBC302CA inteliCAR-T administration of the present invention showed a memory response, and in particular, it was confirmed that the IBC302CA inteliCAR-T administration group most effectively suppressed tumor formation without recurrence in mice.

[0402]

[0403] It was confirmed that the immune effector cells manufactured in the present invention not only effectively recognize GPC3 and CD276 to activate CAR-T cells, but also promote apoptosis of liver cancer cells, a type of solid cancer, and effectively suppress tumor growth. In particular, the IBC302CA inteliCAR-T, manufactured to block CD276 signal leakage by blocking disulfide bonds, most effectively suppressed tumor growth and suppressed tumor recurrence through excellent memory response, and thus can be effectively applied to the treatment of cancers or tumors expressing GPC3 and CD276.

Claims

1. A chimeric antigen receptor (GPC3-CAR) targeting GPC3 comprising a signal peptide, a GPC3-binding domain, a hinge region, a transmembrane domain, and an intracellular signal transduction domain; and It consists of a chimeric antigen receptor (CD276-CAR) targeting CD276, which comprises a signal peptide, a CD276-binding domain, a hinge region, a transmembrane domain, and an intracellular costimulatory domain. The above GPC3-binding domain is a GPC3-specific humanized antibody or fragment thereof, which comprises a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region represented by an amino acid sequence of SEQ ID NO:

6. An AND-gate chimeric antigen receptor specific for GPC3 and CD276, characterized in that the CD276-binding domain is a CD276-specific humanized antibody or fragment thereof, comprising a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 11, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 12, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 14, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 15, and a CDR3 region represented by an amino acid sequence of SEQ ID NO:

16.

2. In paragraph 1, The above GPC3-binding domain is represented by the amino acid sequence of SEQ ID NO: 22, An AND-gate chimeric antigen receptor specific for GPC3 and CD276, characterized in that the CD276-binding domain is represented by the amino acid sequence of SEQ ID NO:

28.

3. In paragraph 1, The AND-gated chimeric antigen receptor specific for the above GPC3 and CD276 is TGF-β receptor II (TGFβDNR) consisting of a TGF-β receptor II extracellular domain (TGF-β RII ECD), a TGF-β receptor II transmembrane domain (TGF-β RII TM), and a truncated TGF-β receptor II intracellular domain (Truncated TGF-β RII ICD); or An AND-gated chimeric antigen receptor specific for GPC3 and CD276, characterized in that it further comprises a 4-1BB ligand (4-1BBL) comprising a 4-1BB ligand extracellular domain (4-1BBL ECD), a 4-1BB ligand transmembrane domain (4-1BBL TM), and a 4-1BB ligand intracellular domain (4-1BBL ICD).

4. In paragraph 3, The above TGF-β receptor Ⅱ (TGFβDNR) is represented by the amino acid sequence of sequence number 36. An AND-gate chimeric antigen receptor specific for GPC3 and CD276, characterized in that the above 4-1BB ligand (4-1BBL) is represented by the amino acid sequence of SEQ ID NO:

37.

5. In paragraph 1, The signal peptide is a protein derived from IgGκ or CD33, The above hinge region is a protein derived from CD8α or CD28, The transmembrane domain is a protein derived from any one selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. The intracellular signaling domain is a protein derived from CD3ζ, An AND-gated chimeric antigen receptor specific for GPC3 and CD276, characterized in that the intracellular costimulatory domain is a protein derived from any one selected from the group consisting of a CD28-derived intracellular domain, a 4-1BB-derived intracellular domain, and an OX40-derived intracellular domain.

6. In paragraph 1, To prevent disulfide bonds within the chimeric antigen receptor (CD276-CAR) targeting CD276, the CD28 hinge region in which the 28th sequence of SEQ ID NO: 31 is substituted from cysteine ​​(C) to alanine (A), and An AND-gate chimeric antigen receptor specific for GPC3 and CD276, characterized in that the 13th sequence of SEQ ID NO: 33 comprises a transmembrane domain derived from CD28 in which cysteine ​​is substituted with alanine.

7. A polynucleotide encoding an AND-gate chimeric antigen receptor specific for GPC3 and CD276 of any one of claims 1 to 6.

8. A vector comprising a polynucleotide encoding an AND-gate chimeric antigen receptor specific for GPC3 and CD276 of any one of claims 1 to 6.

9. A chimeric antigen receptor (GPC3-CAR) targeting GPC3 comprising a signal peptide, a GPC3-binding domain, a hinge region, a transmembrane domain, and an intracellular signal transduction domain; and An immune effector cell targeting GPC3 and CD276, expressing a chimeric antigen receptor (CD276-CAR) targeting CD276 comprising a signal peptide, a CD276-binding domain, a hinge region, a transmembrane domain, and an intracellular costimulatory domain. The above GPC3-binding domain is a GPC3-specific humanized antibody or fragment thereof, which comprises a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 1, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 2, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 4, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 5, and a CDR3 region represented by an amino acid sequence of SEQ ID NO:

6. An immune effector cell targeting GPC3 and CD276, characterized in that the CD276-binding domain is a CD276-specific humanized antibody or fragment thereof, comprising a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 11, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 12, and a CDR3 region represented by an amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of SEQ ID NO: 14, a CDR2 region represented by an amino acid sequence of SEQ ID NO: 15, and a CDR3 region represented by an amino acid sequence of SEQ ID NO:

16.

10. In paragraph 9, The above GPC3-binding domain is represented by the amino acid sequence of SEQ ID NO: 22, An immune effector cell targeting GPC3 and CD276, characterized in that the CD276-binding domain is represented by the amino acid sequence of SEQ ID NO:

28.

11. In paragraph 9, The above immune effector cells are TGF-β receptor II (TGFβDNR) consisting of a TGF-β receptor II extracellular domain (TGF-β RII ECD), a TGF-β receptor II transmembrane domain (TGF-β RII TM), and a truncated TGF-β receptor II intracellular domain (Truncated TGF-β RII ICD); or An immune effector cell targeting GPC3 and CD276, characterized by additionally expressing 4-1BB ligand (4-1BBL), which is composed of a 4-1BB ligand extracellular domain (4-1BBL ECD), a 4-1BB ligand transmembrane domain (4-1BBL TM), and a 4-1BB ligand intracellular domain (4-1BBL ICD).

12. In paragraph 11, The above TGF-β receptor Ⅱ (TGFβDNR) is represented by the amino acid sequence of sequence number 36. An immune effector cell targeting GPC3 and CD276, characterized in that the above 4-1BB ligand (4-1BBL) is represented by the amino acid sequence of SEQ ID NO:

37.

13. In paragraph 9, The signal peptide is a protein derived from IgGκ or CD33, The above hinge region is a protein derived from CD8α or CD28, The transmembrane domain is a protein derived from any one selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. The intracellular signaling domain is a protein derived from CD3ζ, An immune effector cell targeting GPC3 and CD276, characterized in that the intracellular costimulatory domain is a protein derived from any one selected from the group consisting of a CD28-derived intracellular domain, a 4-1BB-derived intracellular domain, and an OX40-derived intracellular domain.

14. In paragraph 9, To prevent disulfide bonds within the chimeric antigen receptor (CD276-CAR) targeting CD276, the CD28 hinge region in which the 28th sequence of SEQ ID NO: 31 is substituted from cysteine ​​(C) to alanine (A), and An immune effector cell targeting GPC3 and CD276, characterized in that the 13th sequence of SEQ ID NO: 33 comprises a CD28-derived transmembrane domain in which cysteine ​​is substituted with alanine.

15. A pharmaceutical composition for the prevention or treatment of cancer expressing GPC3 and CD276, comprising an immune effector cell according to any one of claims 9 to 14.

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