Chimeric antigen receptor specifically combined with GPC3 and application of chimeric antigen receptor

Abstract

The present invention relates to chimeric antigen receptors that specifically bind to GPC3, engineered immune cells that express the chimeric antigen receptors, and methods of making the engineered immune cells. These chimeric antigens and immune cells are useful in methods of preventing and / or treating diseases associated with expression of GPC3.

Description

Chimeric antigen receptor specifically binding to GPC3 and its application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to single-chain antibodies and chimeric antigen receptors (CARs) that specifically bind to GPC3. The present invention also relates to immune cells expressing the CARs, nucleic acid molecules encoding such CARs or co-expressed molecules, and methods for preparing the modified immune cells. The present invention also relates to methods for using these CARs and immune cells to prevent and / or treat GPC3-positive tumors such as hepatocellular carcinoma (HCC), melanoma, and ovarian clear cell carcinoma. Background Art

[0002] Glypican-3 (GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS, and SGBS1) is a member of the heparan sulfate proteoglycan family. It is anchored to the cell surface via glycosylated phosphatidylinositol and is currently a representative liver cancer marker in preclinical research. GPC3 is expressed in many human malignant tumor cells and serum, including hepatocellular carcinoma (HCC), melanoma, and ovarian clear cell carcinoma, but is rarely expressed in other cancers and normal tissues. GPC3 is a potential biomarker for HCC. It forms a complex with WNT, activates downstream signaling pathways, promotes the proliferation of liver cancer cells, and participates in the regulation of multiple signaling pathways closely related to tumor occurrence and progression.

[0003] Primary liver cancer is the fourth most common malignant tumor and the third leading cause of cancer-related death in my country, posing a serious threat to the lives and health of the Chinese people. Approximately 466,000 new cases of liver cancer are diagnosed in my country each year, accounting for approximately 55% of the global total. Approximately 422,000 deaths from liver cancer occur in my country each year. A significant number of patients with hepatocellular carcinoma (HCC) still lack precise and effective clinical treatments. Most HCC patients are diagnosed at an advanced or late stage, with only 30% eligible for surgical resection. The metastasis and recurrence rate within 5 years after resection is as high as 60%-70%, and the overall 5-year survival rate is low, at only 7%-10%. Chimeric antigen receptor (CAR)-T cell therapy is considered one of the most promising cancer treatments and offers new hope in the fight against cancer. It involves culturing immune cells collected from a patient in vitro, transducing them with specific exogenous genes, and then amplifying them ex vivo before infusing them back into the patient, achieving tumor therapy in a non-MHC-restricted manner. CAR-T cell therapy has achieved remarkable efficacy in the treatment of hematologic malignancies, with complete remission rates exceeding 90% for relapsed and refractory B-cell leukemia. Solid tumors account for approximately 90% of all malignancies, and the demand for therapeutic agents is high. However, CAR-T cell therapy is currently ineffective in treating solid tumors, primarily due to their complex tumor microenvironment and high tumor heterogeneity.

[0004] The present invention provides a GPC3-targeting CAR or a modified immune cell co-expressing a GPC3-targeting CAR. The modified immune cell can be used to treat GPC3-positive hepatocellular carcinoma (HCC), melanoma, ovarian clear cell carcinoma, and the like.

[0005] Summary of the Invention

[0006] In the present application, the inventors first developed a fully human antibody with low immunogenicity that can specifically recognize / bind to GPC3. On this basis, the present invention further designed and constructed a CAR targeting GPC3. The CAR of the present invention can direct the specificity and reactivity of immune effector cells to cells expressing GPC3 (such as hepatocellular carcinoma (HCC), melanoma and ovarian clear cell carcinoma) in a non-MHC restricted manner, thereby eliminating them. Therefore, the CAR targeting GPC3 of the present invention has the potential to be used for the prevention and / or treatment of GPC3-positive tumors such as hepatocellular carcinoma (HCC), melanoma and ovarian clear cell carcinoma, and has significant clinical value.

[0007] Antigen-binding molecules

[0008] In a first aspect, the present invention provides an antigen-binding molecule comprising a binding domain capable of specifically binding to GPC3; the antigen-binding molecule comprises the following complementarity-determining regions (CDRs):

[0009] (a) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 1; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 2;

[0010] or,

[0011] (b) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 3; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 4;

[0012] or,

[0013] (c) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 5; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 6;

[0014] or,

[0015] (d) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 7; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 8;

[0016] or,

[0017] (e) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 9; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 10;

[0018] or,

[0019] (f) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 11; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 12;

[0020] or

[0021] (g) CDR-H1, CDR-H2 and CDR-H3 contained in the following heavy chain variable region (VH), and / or CDR-L1, CDR-L2 and CDR-L3 contained in the following light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation compared to the heavy chain variable region and / or light chain variable region according to any one of (a) to (f), and the mutation is a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids).

[0022] In certain embodiments, the substitutions are conservative substitutions.

[0023] In certain embodiments, the CDRs are defined according to the Kabat, IMGT, Chothia, or AbM numbering systems.

[0024] In certain embodiments, the antigen binding molecules of the invention comprise a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0025] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof; CDR-H2 of SEQ ID NO: 14 or a variant thereof; CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof; CDR-L2 of SEQ ID NO: 17 or a variant thereof; and CDR-L3 of SEQ ID NO: 18 or a variant thereof;

[0026] or,

[0027] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof; CDR-H2 of SEQ ID NO: 27 or a variant thereof; CDR-H3 of SEQ ID NO: 28 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 29 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; and CDR-L3 of SEQ ID NO: 31 or a variant thereof;

[0028] or,

[0029] (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 39 or a variant thereof; CDR-H2 of SEQ ID NO: 40 or a variant thereof; and CDR-H3 of SEQ ID NO: 41 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 42 or a variant thereof; CDR-L2 of SEQ ID NO: 43 or a variant thereof; and CDR-L3 of SEQ ID NO: 44 or a variant thereof;

[0030] or,

[0031] (1d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof; CDR-H2 of SEQ ID NO: 27 or a variant thereof; CDR-H3 of SEQ ID NO: 52 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 53 or a variant thereof; CDR-L2 of SEQ ID NO: 54 or a variant thereof; and CDR-L3 of SEQ ID NO: 55 or a variant thereof;

[0032] or,

[0033] (1e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 60 or a variant thereof; CDR-H2 of SEQ ID NO: 61 or a variant thereof; CDR-H3 of SEQ ID NO: 62 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 63 or a variant thereof; CDR-L2 of SEQ ID NO: 64 or a variant thereof; and CDR-L3 of SEQ ID NO: 65 or a variant thereof;

[0034] or,

[0035] (1f) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof; CDR-H2 of SEQ ID NO: 27 or a variant thereof; CDR-H3 of SEQ ID NO: 73 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 74 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; and CDR-L3 of SEQ ID NO: 75 or a variant thereof;

[0036] Wherein, the variant described in any one of (1a)-(1f) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0037] In certain embodiments, the antigen binding molecules of the invention comprise a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0038] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 19 or a variant thereof; CDR-H2 of SEQ ID NO: 20 or a variant thereof; CDR-H3 of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 22 or a variant thereof; CDR-L2 of SEQ ID NO: 23 or a variant thereof; and CDR-L3 of SEQ ID NO: 18 or a variant thereof;

[0039] or,

[0040] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 32 or a variant thereof; CDR-H2 of SEQ ID NO: 33 or a variant thereof; CDR-H3 of SEQ ID NO: 34 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 35 or a variant thereof; CDR-L2 of SEQ ID NO: 36 or a variant thereof; and CDR-L3 of SEQ ID NO: 31 or a variant thereof;

[0041] or,

[0042] (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 45 or a variant thereof; CDR-H2 of SEQ ID NO: 46 or a variant thereof; and CDR-H3 of SEQ ID NO: 47 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 48 or a variant thereof; CDR-L2 of SEQ ID NO: 49 or a variant thereof; and CDR-L3 of SEQ ID NO: 44 or a variant thereof;

[0043] or,

[0044] (2d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 32 or a variant thereof; CDR-H2 of SEQ ID NO: 33 or a variant thereof; CDR-H3 of SEQ ID NO: 56 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 57 or a variant thereof; CDR-L2 of SEQ ID NO: 58 or a variant thereof; and CDR-L3 of SEQ ID NO: 59 or a variant thereof;

[0045] or,

[0046] (2e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 66 or a variant thereof; CDR-H2 of SEQ ID NO: 67 or a variant thereof; and CDR-H3 of SEQ ID NO: 68 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 69 or a variant thereof; CDR-L2 of SEQ ID NO: 70 or a variant thereof; and CDR-L3 of SEQ ID NO: 65 or a variant thereof;

[0047] or,

[0048] (2f) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 32 or a variant thereof; CDR-H2 of SEQ ID NO: 33 or a variant thereof; and CDR-H3 of SEQ ID NO: 76 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 77 or a variant thereof; CDR-L2 of SEQ ID NO: 36 or a variant thereof; and CDR-L3 of SEQ ID NO: 75 or a variant thereof;

[0049] Wherein, the variant described in any one of (2a)-(2f) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0050] In certain embodiments, the antigen binding molecules of the invention comprise a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0051] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 24 or a variant thereof; CDR-H2 of SEQ ID NO: 25 or a variant thereof; CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof; CDR-L2 of SEQ ID NO: 17 or a variant thereof; and CDR-L3 of SEQ ID NO: 18 or a variant thereof;

[0052] or,

[0053] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 37 or a variant thereof; CDR-H2 of SEQ ID NO: 38 or a variant thereof; and CDR-H3 of SEQ ID NO: 28 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 29 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; and CDR-L3 of SEQ ID NO: 31 or a variant thereof;

[0054] or,

[0055] (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 50 or a variant thereof; CDR-H2 of SEQ ID NO: 51 or a variant thereof; and CDR-H3 of SEQ ID NO: 41 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 42 or a variant thereof; CDR-L2 of SEQ ID NO: 43 or a variant thereof; and CDR-L3 of SEQ ID NO: 44 or a variant thereof;

[0056] or,

[0057] (3d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 37 or a variant thereof; CDR-H2 of SEQ ID NO: 38 or a variant thereof; and CDR-H3 of SEQ ID NO: 52 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 53 or a variant thereof; CDR-L2 of SEQ ID NO: 54 or a variant thereof; and CDR-L3 of SEQ ID NO: 55 or a variant thereof;

[0058] or,

[0059] (3e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 71 or a variant thereof; CDR-H2 of SEQ ID NO: 72 or a variant thereof; and CDR-H3 of SEQ ID NO: 62 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 63 or a variant thereof; CDR-L2 of SEQ ID NO: 64 or a variant thereof; and CDR-L3 of SEQ ID NO: 65 or a variant thereof;

[0060] or,

[0061] (3f) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 37 or a variant thereof; CDR-H2 of SEQ ID NO: 38 or a variant thereof; and CDR-H3 of SEQ ID NO: 73 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 74 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; and CDR-L3 of SEQ ID NO: 75 or a variant thereof;

[0062] Wherein, the variant described in any one of (3a)-(3f) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0063] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0064] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 80 or a variant thereof; CDR-H2 of SEQ ID NO: 81 or a variant thereof; CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof; CDR-L2 of SEQ ID NO: 17 or a variant thereof; and CDR-L3 of SEQ ID NO: 18 or a variant thereof;

[0065] or,

[0066] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 78 or a variant thereof; CDR-H2 of SEQ ID NO: 79 or a variant thereof; CDR-H3 of SEQ ID NO: 28 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 29 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; and CDR-L3 of SEQ ID NO: 31 or a variant thereof;

[0067] or,

[0068] (4c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 96 or a variant thereof; CDR-H2 of SEQ ID NO: 97 or a variant thereof; and CDR-H3 of SEQ ID NO: 41 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 42 or a variant thereof; CDR-L2 of SEQ ID NO: 43 or a variant thereof; and CDR-L3 of SEQ ID NO: 44 or a variant thereof;

[0069] or,

[0070] (4d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 78 or a variant thereof; CDR-H2 of SEQ ID NO: 79 or a variant thereof; and CDR-H3 of SEQ ID NO: 52 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 53 or a variant thereof; CDR-L2 of SEQ ID NO: 54 or a variant thereof; and CDR-L3 of SEQ ID NO: 55 or a variant thereof;

[0071] or,

[0072] (4e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 98 or a variant thereof; CDR-H2 of SEQ ID NO: 99 or a variant thereof; and CDR-H3 of SEQ ID NO: 62 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 63 or a variant thereof; CDR-L2 of SEQ ID NO: 64 or a variant thereof; and CDR-L3 of SEQ ID NO: 65 or a variant thereof;

[0073] or,

[0074] (4f) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 78 or a variant thereof; CDR-H2 of SEQ ID NO: 79 or a variant thereof; CDR-H3 of SEQ ID NO: 73 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 74 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; and CDR-L3 of SEQ ID NO: 75 or a variant thereof;

[0075] Wherein, the variant described in any one of (4a)-(4f) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0076] In certain embodiments, the antigen binding molecules of the invention comprise:

[0077] (a) a VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof;

[0078] or,

[0079] (b) a VH comprising the sequence shown in SEQ ID NO: 3 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 4 or a variant thereof;

[0080] or,

[0081] (c) a VH comprising the sequence shown in SEQ ID NO: 5 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 6 or a variant thereof;

[0082] or

[0083] (d) a VH comprising the sequence shown in SEQ ID NO: 7 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 8 or a variant thereof;

[0084] or,

[0085] (e) a VH comprising the sequence shown in SEQ ID NO: 9 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 10 or a variant thereof;

[0086] or,

[0087] (f) a VH comprising the sequence shown in SEQ ID NO: 11 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 12 or a variant thereof;

[0088] wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0089] In certain embodiments, the antigen binding molecule described in any of the above embodiments may comprise a constant region from or derived from a human immunoglobulin.

[0090] In certain embodiments, the heavy chain of the antigen binding molecule comprises a heavy chain constant region from or derived from human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4). In certain embodiments, the heavy chain of the antigen binding molecule comprises a wild-type Fc region, or comprises a mutated or chemically modified Fc region having an altered effector function (e.g., enhanced ADCC activity) compared to a wild-type Fc region.

[0091] In certain embodiments, the light chain of the antigen binding molecule comprises a light chain constant region from or derived from a human immunoglobulin (eg, kappa or lambda).

[0092] In certain embodiments, the antigen binding molecule described in any of the above embodiments is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0093] In certain embodiments, the antigen binding molecule is selected from a full-length antibody, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)'3 fragment, a single-chain antibody (e.g., scFv, di-scFv or (scFv)2), a miniantibody, a disulfide-stabilized Fv protein (dsFv) and a single-domain antibody (sdAb, nanobody).

[0094] In certain embodiments, the VH and VL of the antigen binding molecules of the present invention are connected by one or more linkers. Linkers are typically peptide linkers, such as flexible and / or soluble peptide linkers, such as peptide linkers rich in glycine, serine and / or threonine. In some embodiments, linkers also include charged residues (such as lysine and / or glutamic acid), which can improve solubility. In some embodiments, linkers also include one or more proline.

[0095] In certain embodiments, the linker comprises one or more (e.g., 1, 2, or 3) sequences as represented by (GmS)n, wherein m is selected from an integer of 1-6, and n is selected from an integer of 1-6; preferably, m is 3, 4, or 5; preferably, n is 1 or 2. In certain embodiments, the linker has the sequence of SEQ ID NO: 110.

[0096] In certain embodiments, the antigen binding molecules of the invention are single chain antibodies, such as scFv, di-scFv, or (scFv)2.

[0097] In certain embodiments, the single-chain antibody comprises, from its N-terminus to its C-terminus:

[0098] (1) a VH comprising a sequence as shown in SEQ ID NO: 1 or a variant thereof, a linker comprising a VL comprising a sequence as shown in SEQ ID NO: 2 or a variant thereof; or a VL comprising a sequence as shown in SEQ ID NO: 2 or a variant thereof, a linker comprising a VH comprising a sequence as shown in SEQ ID NO: 1 or a variant thereof;

[0099] or

[0100] (2) a VH comprising a sequence as set forth in SEQ ID NO: 3 or a variant thereof, a linker comprising a VL comprising a sequence as set forth in SEQ ID NO: 4 or a variant thereof; or a VL comprising a sequence as set forth in SEQ ID NO: 4 or a variant thereof, a linker comprising a VH comprising a sequence as set forth in SEQ ID NO: 3 or a variant thereof;

[0101] or

[0102] (3) a VH comprising a sequence as set forth in SEQ ID NO: 5 or a variant thereof, a linker comprising a VL comprising a sequence as set forth in SEQ ID NO: 6 or a variant thereof; or a VL comprising a sequence as set forth in SEQ ID NO: 6 or a variant thereof, a linker comprising a VH comprising a sequence as set forth in SEQ ID NO: 5 or a variant thereof;

[0103] or

[0104] (4) a VH comprising a sequence as set forth in SEQ ID NO: 7 or a variant thereof-a linker-a VL comprising a sequence as set forth in SEQ ID NO: 8 or a variant thereof; or a VL comprising a sequence as set forth in SEQ ID NO: 8 or a variant thereof-a linker-a VH comprising a sequence as set forth in SEQ ID NO: 7 or a variant thereof;

[0105] or

[0106] (5) a VH comprising a sequence as set forth in SEQ ID NO: 9 or a variant thereof, a linker comprising a VL comprising a sequence as set forth in SEQ ID NO: 10 or a variant thereof; or a VL comprising a sequence as set forth in SEQ ID NO: 10 or a variant thereof, a linker comprising a VH comprising a sequence as set forth in SEQ ID NO: 9 or a variant thereof;

[0107] or

[0108] (6) a VH comprising a sequence as set forth in SEQ ID NO: 11 or a variant thereof, a linker comprising a VL comprising a sequence as set forth in SEQ ID NO: 12 or a variant thereof; or a VL comprising a sequence as set forth in SEQ ID NO: 12 or a variant thereof, a linker comprising a VH comprising a sequence as set forth in SEQ ID NO: 11 or a variant thereof;

[0109] wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions.

[0110] In certain embodiments, the single-chain antibody comprises a sequence as shown in any one of SEQ ID NOs: 86, 88, 90, 92, 94, 82, or a variant thereof, wherein the variant has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 86, 88, 90, 92, 94, or 82, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions); in certain embodiments, the substitutions are conservative substitutions.

[0111] In certain embodiments, the antigen binding molecules of the present invention further comprise a constant region derived from human immunoglobulin. In certain embodiments, the heavy chain of the antigen binding molecules comprises a heavy chain constant region derived from human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antigen binding molecules comprises a light chain constant region derived from human immunoglobulin (e.g., κ or λ).

[0112] In certain embodiments, the heavy chain of the antigen binding molecule comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived; and / or,

[0113] The light chain of the antigen binding molecule comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions).

[0114] In certain embodiments, the heavy chain constant region is an IgG, IgM, IgE, IgD or IgA heavy chain constant region. In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region.

[0115] In certain embodiments, the light chain constant region is a kappa or lambda light chain constant region. In certain preferred embodiments, the light chain constant region is a human kappa light chain constant region.

[0116] Preparation of antigen-binding molecules

[0117] The antigen binding molecules of the present invention can be prepared by various methods known in the art, for example, by genetic engineering recombinant technology. For example, a DNA molecule encoding the antigen binding molecules is obtained by chemical synthesis or PCR amplification, the resulting DNA molecule is inserted into an expression vector, and then transfected into a host cell. The transfected host cell is then cultured under specific conditions and the antigen binding molecules of the present invention are expressed.

[0118] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be produced directly from recombinant host cells (Reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture fluid. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0119] Therefore, a second aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antigen-binding molecule of the present invention. In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein:

[0120] The nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO: 100, (ii) a sequence substantially identical to SEQ ID NO: 100 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions compared to SEQ ID NO: 100), or (iii) a degenerate sequence of (i) or (ii) above; and / or,

[0121] The nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence set forth in SEQ ID NO: 101, (v) a sequence substantially identical to SEQ ID NO: 101 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO: 101), or (vi) a degenerate sequence of (iv) or (v) above.

[0122] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein:

[0123] The nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence of SEQ ID NO: 102, (ii) a sequence substantially identical to SEQ ID NO: 102 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO: 102), or (iii) a degenerate sequence of (i) or (ii) above; and / or,

[0124] The nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence set forth in SEQ ID NO: 103, (v) a sequence substantially identical to SEQ ID NO: 103 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO: 103), or (vi) a degenerate sequence of (iv) or (v) above.

[0125] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein:

[0126] The nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence of SEQ ID NO: 104, (ii) a sequence substantially identical to SEQ ID NO: 104 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO: 104), or (iii) a degenerate sequence of (i) or (ii) above; and / or,

[0127] The nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence set forth in SEQ ID NO: 105, (v) a sequence substantially identical to SEQ ID NO: 105 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO: 105), or (vi) a degenerate sequence of (iv) or (v) above.

[0128] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein:

[0129] The nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence of SEQ ID NO: 106, (ii) a sequence substantially identical to SEQ ID NO: 106 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO: 106), or (iii) a degenerate sequence of (i) or (ii) above; and / or,

[0130] The nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence set forth in SEQ ID NO: 107, (v) a sequence substantially identical to SEQ ID NO: 107 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO: 107), or (vi) a degenerate sequence of (iv) or (v) above.

[0131] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein:

[0132] The nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence of SEQ ID NO: 108, (ii) a sequence substantially identical to SEQ ID NO: 108 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO: 108), or (iii) a degenerate sequence of (i) or (ii) above; and / or,

[0133] The nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence set forth in SEQ ID NO: 109, (v) a sequence substantially identical to SEQ ID NO: 109 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions compared to SEQ ID NO: 109), or (vi) a degenerate sequence of (iv) or (v) above.

[0134] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein:

[0135] The nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence of SEQ ID NO: 84, (ii) a sequence substantially identical to SEQ ID NO: 84 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions compared to SEQ ID NO: 84), or (iii) a degenerate sequence of (i) or (ii) above; and / or,

[0136] The nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence set forth in SEQ ID NO:85, (v) a sequence substantially identical to SEQ ID NO:85 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions compared to SEQ ID NO:85), or (vi) a degenerate sequence of (iv) or (v) above.

[0137] In certain embodiments, the isolated nucleic acid molecule comprises: (i) the nucleotide sequence set forth in SEQ ID NO:87, (ii) a sequence substantially identical to SEQ ID NO:87 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity, or a sequence having one or more nucleotide substitutions compared to SEQ ID NO:87), or (iii) a degenerate sequence of (i) or (ii) above.

[0138] In certain embodiments, the isolated nucleic acid molecule comprises: (i) the nucleotide sequence set forth in SEQ ID NO:89, (ii) a sequence substantially identical to SEQ ID NO:89 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity, or a sequence having one or more nucleotide substitutions compared to SEQ ID NO:89), or (iii) a degenerate sequence of (i) or (ii) above.

[0139] In certain embodiments, the isolated nucleic acid molecule comprises: (i) the nucleotide sequence set forth in SEQ ID NO:91, (ii) a sequence substantially identical to SEQ ID NO:91 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity, or a sequence having one or more nucleotide substitutions compared to SEQ ID NO:91), or (iii) a degenerate sequence of (i) or (ii) above.

[0140] In certain embodiments, the isolated nucleic acid molecule comprises: (i) the nucleotide sequence set forth in SEQ ID NO:93, (ii) a sequence substantially identical to SEQ ID NO:93 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity, or a sequence having one or more nucleotide substitutions compared to SEQ ID NO:93), or (iii) a degenerate sequence of (i) or (ii) above.

[0141] In certain embodiments, the isolated nucleic acid molecule comprises: (i) the nucleotide sequence set forth in SEQ ID NO:95, (ii) a sequence substantially identical to SEQ ID NO:95 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity, or a sequence having one or more nucleotide substitutions compared to SEQ ID NO:95), or (iii) a degenerate sequence of (i) or (ii) above.

[0142] In certain embodiments, the isolated nucleic acid molecule comprises: (i) the nucleotide sequence set forth in SEQ ID NO:83, (ii) a sequence substantially identical to SEQ ID NO:83 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity, or a sequence having one or more nucleotide substitutions compared to SEQ ID NO:83), or (iii) a degenerate sequence of (i) or (ii) above.

[0143] A third aspect of the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above. In certain embodiments, the vector of the present invention is, for example, a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector, or a viral vector; preferably, the vector is an expression vector; preferably, the vector is an episomal vector; preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector, or a retroviral vector.

[0144] A fourth aspect of the present invention provides a host cell comprising the nucleic acid molecule of the present invention or the vector of the present invention. The host cell can be a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).

[0145] In certain embodiments, the host cell of the present invention can be a chimeric antigen receptor T cell (CAR-T). In such embodiments, the isolated nucleic acid molecule included in the host cell can include a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further includes a nucleotide sequence encoding an antigen binding molecule of the present invention (e.g., ScFv). In certain embodiments, the isolated nucleic acid molecule included in the host cell encodes a chimeric antigen receptor comprising an antigen binding molecule of the present invention (e.g., scFv).

[0146] In another aspect, the present invention also relates to a method for preparing the antigen-binding molecule of the present invention, comprising culturing the host cell as described above under conditions allowing protein expression, and recovering the antigen-binding molecule from the cultured host cell culture.

[0147] Chimeric antigen receptor

[0148] The present invention relates to a CAR targeting GPC3, which has the characteristics of non-MHC-restricted GPC3 recognition ability, which confers the ability of immune cells expressing the CAR (e.g., T cells, NK cells, monocytes, macrophages or dendritic cells) to recognize GPC3-expressing cells (e.g., tumor cells) independently of antigen processing and presentation.

[0149] Therefore, the fifth aspect of the present invention provides a chimeric antigen receptor comprising an antigen binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain.

[0150] I. Extracellular Antigen Binding Domain

[0151] The antigen binding domain contained in the chimeric antigen receptor of the present invention confers the ability of the CAR to recognize GPC3.

[0152] In certain embodiments, the antigen binding domain comprises the antigen binding molecule of the first aspect.

[0153] In certain embodiments, the antigen-binding domain comprises the antigen-binding molecule as a first antigen-binding domain and further comprises a second antigen-binding domain that does not bind to GPC3. In certain embodiments, the second antigen-binding domain binds to an antigen selected from the group consisting of: PD-1, PD-L1, CTLA-, CD3, ASGPR1, CD19, MSLN, PSMA, MUC1, EGFR, HER2, CD276, GD2, BCMA, CD33, or Claudin18.2.

[0154] In certain embodiments, the antigen binding domain is a single-chain antibody.

[0155] In certain embodiments, the first antigen binding domain comprises a sequence as shown in any one of SEQ ID NOs: 86, 88, 90, 92, 94, 82, or a variant thereof, wherein the variant has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 86, 88, 90, 92, 94, 82, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions); preferably, the substitutions are conservative substitutions.

[0156] II. Transmembrane domain

[0157] The transmembrane domain included in the chimeric antigen receptor of the present invention can be any protein structure known in the art, as long as it can be thermodynamically stable in a cell membrane (particularly a eukaryotic cell membrane). The transmembrane domain suitable for the CAR of the present invention can be derived from a natural source. In such embodiments, the transmembrane domain can be derived from any membrane-bound or transmembrane protein. Alternatively, the transmembrane domain can be a synthetic non-naturally occurring protein segment, for example, a protein segment mainly comprising hydrophobic residues such as leucine and valine.

[0158] In certain embodiments, the transmembrane domain is a transmembrane region selected from the following proteins: α, β or ζ chain of the T cell receptor, CD28, CD45, CD3ε, CD3ζ, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD-1 and any combination thereof. In certain preferred embodiments, the transmembrane domain is a transmembrane region selected from the following proteins: CD8, CD28, CD4, PD-1, CD152 and CD154. In certain embodiments, the transmembrane domain comprises a CD8 transmembrane region as shown in SEQ ID NO: 111.

[0159] III. Spacer Domain

[0160] The chimeric antigen receptor of the present invention comprises a spacer domain located between the extracellular antigen-binding domain and the transmembrane domain.

[0161] In certain embodiments, the spacer domain comprises the CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG4). In such embodiments, without being bound by a particular theory, it is believed that CH2 and CH3 extend the antigen binding domain of the CAR from the cell membrane of the cell expressing the CAR and more accurately mimic the size and domain structure of a natural TCR.

[0162] In certain embodiments, the spacer domain comprises a hinge domain. A hinge domain can be an amino acid segment typically found between two domains of a protein that allows the protein to be flexible and allows one or both domains to move relative to each other. Thus, the hinge domain can be any amino acid sequence that provides flexibility to the extracellular antigen-binding domain and mobility relative to the transmembrane domain.

[0163] In certain embodiments, the hinge domain is a hinge region or portion thereof of a naturally occurring protein. In certain embodiments, the spacer domain is selected from a hinge domain and / or an immunoglobulin (e.g., IgG1 or IgG4) CH2 and CH3 region. In certain embodiments, the hinge domain comprises a hinge region of CD8, IgG4, PD-1, CD152, or CD154. In certain embodiments, the hinge domain comprises a CD8 hinge region having a sequence as shown in SEQ ID NO: 112.

[0164] IV. Signal Peptide

[0165] In certain embodiments, the CAR of the present invention may further include a signal peptide at its N-terminus. Typically, a signal peptide is a polypeptide sequence that targets the sequence connected thereto to the desired site. In certain embodiments, the signal peptide can target the CAR connected thereto to the secretory pathway of the cell and allow the CAR to be further integrated and anchored into the lipid bilayer. Signal peptides that can be used for CAR are known to those skilled in the art. In certain embodiments, the signal peptide includes a heavy chain signal peptide (such as a heavy chain signal peptide of IgG1), a granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal peptide, an IL2 signal peptide, or a CD8 alpha signal peptide. In certain preferred embodiments, the signal peptide is selected from a CD8 alpha signal peptide. In certain exemplary embodiments, the signal peptide includes the amino acid sequence shown in SEQ ID NO:116.

[0166] In certain embodiments, the CAR of the present invention can also be co-expressed with another bioactive molecule. The other bioactive molecule may have its own proprietary signal peptide, which is distinguished from the signal peptide in the previous paragraph. This signal peptide is named signal peptide-2. The signal peptide-2 guides the transport of other bioactive molecules to a specific site in the cell or outside the cell membrane. The signal peptide-2 may be the same as or different from the signal peptide described in the previous paragraph. In certain embodiments, the signal peptide-2 may be different from the signal peptide described in the previous paragraph. In certain embodiments, the signal peptide-2 is an IL2 signal peptide (e.g., an amino acid sequence as shown in SEQ ID NO: 129).

[0167] V. Intracellular signaling domain

[0168] The intracellular signaling domain contained in the CAR of the present invention participates in the signal transduction generated by the binding of the CAR of the present invention to GPC3 into the interior of the immune effector cell, activates at least one normal effector function of the immune effector cell expressing CAR, or enhances the secretion of at least one cytokine (e.g., IL-2, IFN-γ) of the immune effector cell expressing CAR.

[0169] In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and / or a costimulatory signaling domain.

[0170] In certain embodiments, the primary signaling domain can be any intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, or CD66d. In certain embodiments, the primary signaling domain comprises the intracellular signaling domain of CD3ζ.

[0171] In certain embodiments, the costimulatory signaling domain can be an intracellular signaling domain from a costimulatory molecule. In certain embodiments, the costimulatory signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS), or DAP10.

[0172] In certain embodiments, the costimulatory signaling domain is selected from the intracellular signaling domain of CD28, or the intracellular signaling domain of CD137 (4-1BB), or a combination of fragments thereof.

[0173] In certain embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In certain embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains. In such embodiments, the two or more costimulatory signaling domains can be the same or different.

[0174] In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and at least one costimulatory signaling domain. The primary signaling domain and at least one costimulatory signaling domain can be connected in series to the carboxyl terminus of the transmembrane domain in any order.

[0175] In certain embodiments, the intracellular signaling domain may comprise the intracellular signaling domain of CD3 zeta and the intracellular signaling domain of CD137 (4-1BB). In certain exemplary embodiments, the intracellular signaling domain of CD3 zeta comprises the amino acid sequence shown in SEQ ID NO: 113. In certain exemplary embodiments, the intracellular signaling domain of CD137 (4-1BB) comprises the amino acid sequence shown in SEQ ID NO: 114.

[0176] In certain exemplary embodiments, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO:115.

[0177] VI. Full-length CAR

[0178] The present invention provides a chimeric antigen receptor capable of specifically binding to GPC3, wherein the chimeric antigen receptor comprises, from its N-terminus to its C-terminus, an antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain. In certain preferred embodiments, the intracellular signaling domain comprises, from the N-terminus to the C-terminus, a costimulatory signaling domain and a primary signaling domain.

[0179] In certain embodiments, the spacer domain comprises the hinge region of CD8 (eg, CD8α) (eg, the hinge region whose sequence is set forth in SEQ ID NO: 112).

[0180] In certain embodiments, the transmembrane domain comprises the transmembrane region of CD8 (eg, CD8α) (eg, the transmembrane region whose sequence is shown in SEQ ID NO: 111).

[0181] In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3ζ (e.g., the sequence shown in SEQ ID NO: 113), and the costimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., the sequence shown in SEQ ID NO: 114); more preferably, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO: 115.

[0182] In certain preferred embodiments, the chimeric antigen receptor comprises the signal peptide, antigen binding domain, spacer domain, transmembrane domain, intracellular signaling domain (from N-terminus to C-terminus, costimulatory signaling domain and primary signaling domain) in sequence from its N-terminus to C-terminus.

[0183] In certain embodiments, the signal peptide comprises an IgG1 heavy chain signal peptide or a CD8α signal peptide (e.g., a signal peptide having a sequence as shown in SEQ ID NO: 116). In certain exemplary embodiments, the CAR of the present invention comprises a sequence as shown in any one of SEQ ID NOs: 117, 119, 121, 123, 125, 127, or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0184] VII. Co-expression of CAR and Additional Biologically Active Molecules

[0185] In some cases, the CAR described in the fifth aspect of the present invention can also be co-expressed with other bioactive molecules. Self-cleaving peptides can prevent amino acids from forming covalent bonds during translation and maintain translation to continue, so that the translation product is "self-cleaved", thereby separating the chimeric antigen receptor of the present invention from other bioactive molecules. Therefore, when the CAR described in the fifth aspect of the present invention can also be co-expressed with other bioactive molecules, the chimeric antigen receptor that can specifically bind to GPC3 becomes an independent CAR having an extracellular antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, while the other bioactive molecules can be secreted outside the cell or expressed as membrane-forming chimeric polypeptides or proteins. As the immune cells expressing CAR proliferate and enrich in the tumor microenvironment, other bioactive molecules are enriched in the tumor microenvironment and synergize with CAR to exert an anti-tumor effect.

[0186] In certain embodiments, the nucleic acid sequence encoding CAR is connected to the nucleic acid sequence of another biologically active molecule through the nucleic acid sequence of a self-cleaving peptide. CAR can be at the N-terminus or C-terminus of another biologically active molecule. In certain exemplary embodiments, CAR is at the 5' end of another biologically active molecule. Any self-cleaving peptide that can cause the fusion protein to be cleaved into two independent proteins can be applied to the present invention. In certain exemplary embodiments, the self-cleaving peptide is P2A, preferably having the sequence shown in SEQ ID NO: 130, and its nucleotide sequence can be optimized according to the needs of gene recombination. In such embodiments, the fusion protein comprising CAR and another biologically active molecule has the following structure:

[0187] N'-signal peptide - extracellular antigen binding domain that specifically binds to GPC3 - spacer domain - transmembrane domain - intracellular signaling domain - self-cleaving peptide - signal peptide-2 - another biologically active molecule - C'. Signal peptide-2 is the same as or different from the N-terminal signal peptide.

[0188] In certain embodiments, the signal peptide-2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (eg, as set forth in SEQ ID NO: 129).

[0189] Preparation of chimeric antigen receptors

[0190] The method for generating a chimeric antigen receptor and an immune effector cell (such as a T cell) comprising the chimeric antigen receptor is known in the art, and may include transfecting cells with at least one polynucleotide encoding CAR and expressing the polynucleotide in the cell. For example, the nucleic acid molecules encoding the CAR of the present invention may be included in an expression vector (for example, a lentiviral vector), which can be expressed in a host cell such as a T cell to manufacture the CAR.

[0191] Therefore, the sixth aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor according to the fifth aspect.

[0192] Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, a nucleotide sequence encoding a chimeric antigen receptor of the present invention may have a variety of different sequences. Therefore, unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0193] In certain exemplary embodiments, the nucleotide sequence encoding the chimeric antigen receptor of the fifth aspect is selected from: (1) a sequence shown in any one of SEQ ID NOs: 118, 120, 122, 124, 126, 128, or a degenerate variant thereof; (2) a sequence substantially identical to the sequence of (1), for example, a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to (1), or a sequence having one or more nucleotide substitutions compared to the sequence of (1); and the sequence substantially retains at least one biological activity of the nucleotide sequence from which it is derived (for example, being able to encode an ability to direct immune effector cell specificity and reactivity to cells expressing GPC3 in a non-MHC restricted manner).

[0194] As described above in the fifth aspect, the CAR of the present invention can also be co-expressed with other biologically active molecules to synergistically exert anti-tumor effects.

[0195] Therefore, the seventh aspect of the present invention further provides a nucleic acid construct comprising a first nucleic acid sequence encoding the chimeric antigen receptor according to the fifth aspect, and further comprising a second nucleic acid sequence encoding another biologically active molecule.

[0196] In certain embodiments, the additional biologically active molecule encoded by the second nucleic acid sequence has anti-tumor activity.

[0197] In certain embodiments, the additional biologically active molecule encoded by the second nucleotide sequence further comprises a signal peptide-2 at its N-terminus.

[0198] In certain embodiments, the first nucleotide sequence is located upstream of the second nucleotide sequence.

[0199] In certain embodiments, the first nucleic acid sequence and the second nucleic acid sequence are connected by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A, or any combination thereof). In certain exemplary embodiments, the sequence encoding the self-cleaving peptide is connected to the 3' end of the first nucleotide sequence and to the 5' end of the second nucleotide sequence.

[0200] In certain embodiments, the self-cleaving peptide is P2A (eg, as set forth in SEQ ID NO: 130).

[0201] In certain exemplary embodiments, the nucleic acid construct of the seventh aspect comprises, from its 5' end to its 3' end, in sequence: a nucleotide sequence encoding the signal peptide, a nucleotide sequence encoding the antigen binding domain, a nucleotide sequence encoding the spacer domain, a nucleotide sequence encoding the transmembrane domain, a nucleotide sequence encoding the intracellular signaling domain, a nucleotide sequence encoding the self-cleaving peptide sequence, a nucleotide sequence encoding the signal peptide-2, and a nucleotide sequence encoding another biologically active molecule.

[0202] The eighth aspect of the present invention provides a vector comprising the isolated nucleic acid molecule described in the second aspect or the sixth aspect, or the nucleic acid construct described in the seventh aspect.

[0203] In certain embodiments, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector, and a viral vector.

[0204] In certain embodiments, the vector is an expression vector.

[0205] In certain embodiments, the vector is an episomal vector.

[0206] In certain embodiments, the vector is a viral vector.

[0207] In certain exemplary embodiments, the viral vector is a lentiviral vector, an adenoviral vector, or a retroviral vector.

[0208] In certain embodiments, the vector is an episomal or non-integrating viral vector, such as an integration-defective retrovirus or lentivirus.

[0209] The ninth aspect of the present invention provides a host cell comprising the isolated nucleic acid molecule as described in the sixth aspect, the nucleic acid construct as described in the seventh aspect, or the vector as described in the eighth aspect. The vector as described above can be introduced into the host cell by various suitable means, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN method, ZFN method, non-viral vector-mediated transfection (e.g., liposome) or viral vector-mediated transfection (e.g., lentiviral infection, retroviral infection, adenoviral infection), and other physical, chemical or biological means for transfer into host cells, such as transposon technology, CRISPR-Cas9 and other technologies.

[0210] In certain embodiments, the host cell comprises the isolated nucleic acid molecule of the sixth aspect or a vector comprising the nucleic acid molecule, and the host cell expresses the chimeric antigen receptor of the present invention.

[0211] In certain embodiments, the host cell comprises the nucleic acid construct of the seventh aspect or a vector comprising the nucleic acid construct, and the host cell expresses the chimeric antigen receptor of the present invention and another biologically active molecule.

[0212] In certain embodiments, the host cell is selected from a mammalian (e.g., human) immune cell. In certain embodiments, the immune cell is derived from a patient or a healthy donor. In certain embodiments, the immune cell is selected from a T lymphocyte, a natural killer (NK) cell, a monocyte, a macrophage, or a dendritic cell, and any combination thereof; preferably, the immune cell is derived from a T lymphocyte or a NK cell.

[0213] The tenth aspect of the present invention provides a method for preparing a cell expressing a chimeric antigen receptor of the present invention, comprising: (1) providing a host cell; (2) introducing the isolated nucleic acid molecule described in the sixth aspect or a vector comprising the nucleic acid molecule into the host cell to obtain a host cell capable of expressing the chimeric antigen receptor. A method for co-expressing a chimeric antigen receptor of the present invention and another biologically active molecule is also provided, comprising: (1) providing a host cell; (2) introducing the nucleic acid construct described in the seventh aspect or a vector comprising the nucleic acid construct into the host cell to obtain a host cell capable of co-expressing the chimeric antigen receptor and another biologically active molecule.

[0214] In certain embodiments, the host cell is selected from immune cells, such as T lymphocytes, NK cells, monocytes, dendritic cells, macrophages and any combination thereof. In certain embodiments, the immune cell is selected from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof.

[0215] In certain embodiments, in step (1), the host cells are provided from a patient or a healthy donor and are pretreated; the pretreatment includes sorting, activation and / or proliferation of immune cells; in certain embodiments, the pretreatment includes contacting the immune cells with anti-CD3 antibodies and anti-CD28 antibodies, thereby stimulating the immune cells and inducing their proliferation, thereby generating pretreated immune cells.

[0216] In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the host cell by viral infection. In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the host cell by non-viral vector transfection, such as a transposon vector system, CRISPR / Cas9 vector, TALEN method, ZFN method, electroporation method, calcium phosphate transfection, DEAE-dextran mediated transfection or microinjection.

[0217] In certain embodiments, after step (2), the method further comprises: amplifying the host cells obtained in step (2).

[0218] Engineered immune cells

[0219] The eleventh aspect of the present invention further provides a modified immune cell comprising and expressing the isolated nucleic acid molecule of the second and sixth aspects of the present invention. The modified immune cell expresses the chimeric antigen receptor of the fifth aspect.

[0220] The twelfth aspect of the present invention further provides a modified immune cell comprising and expressing the nucleic acid construct of the seventh aspect of the present invention. The modified immune cell expresses the chimeric antigen receptor of the fifth aspect and the additional bioactive molecule.

[0221] In certain embodiments, the immune cells are derived from T lymphocytes, NK cells, monocytes, macrophages, or dendritic cells, and any combination thereof. In certain embodiments, the immune cells are obtained from a patient; alternatively, the immune cells are obtained from a healthy donor. In certain embodiments, the immune cells are derived from T lymphocytes or NK cells.

[0222] In certain embodiments, the transcription or expression of one or two target genes in the genes related to immune rejection of the modified immune cells (e.g., TRAC, TRBC, B2M, HLA-A, HLA-B or HLA-C) and the genes of immune co-inhibitory pathways or signaling molecules (e.g., PD-1, CTLA-4 or LAG-3) is inhibited. In certain embodiments, the method used to inhibit the transcription or expression of the target gene is selected from gene knockout (e.g., CRISPR, CRISPR / Cas9), homologous recombination, and interfering RNA.

[0223] The present invention also provides a method for preparing modified immune cells, which comprises: (1) providing immune cells from a patient or a healthy donor; (2) introducing the isolated nucleic acid molecule described in the sixth aspect, or the nucleic acid construct described in the seventh aspect, or a vector containing them into the immune cells described in step (1) to obtain immune cells capable of expressing and optionally expressing additional biologically active molecules.

[0224] In certain embodiments, in step (1), the immune cells are pretreated, and the pretreatment includes sorting, activation and / or proliferation of the immune cells; more preferably, the pretreatment includes contacting the immune cells with anti-CD3 antibodies and anti-CD28 antibodies, thereby stimulating the immune cells and inducing their proliferation, thereby generating pretreated immune cells.

[0225] In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the immune cells by viral infection.

[0226] In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the immune cell by non-viral vector transfection, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, transposon vector system, CRISPR / Cas9 vector, TALEN method, ZFN method or electroporation method.

[0227] In certain embodiments, the method further comprises, after step (2), a step of expanding the immune cells obtained in step (2).

[0228] Immune cell composition

[0229] In the thirteenth aspect, the present invention also provides an immune cell composition, which includes the modified immune cells of any of the aforementioned aspects, and optional unmodified and / or unsuccessfully modified immune cells, which do not express the target CAR. Due to the current level of technology and some unknown reasons, not all immune cells can express the target CAR after modification. Moreover, immune cells that do not express CAR also have certain biological activities. Therefore, the immune cell composition can contain immune cells that express and do not express the target CAR, and the immune cell composition can still meet the needs of clinical applications.

[0230] In certain embodiments, the engineered immune cells expressing the target CAR account for approximately 10%-100%, preferably 40%-80%, of the total cell number of the immune cell composition.

[0231] In certain embodiments, the immune cell composition is cultured into an immune cell line. Therefore, in another aspect, the present invention also provides an immune cell line containing the immune cell composition.

[0232] In a fourteenth aspect, the present invention provides a kit for preparing the modified immune cells described in any of the above aspects. In certain embodiments, the kit includes the isolated nucleic acid molecule described in the sixth aspect, or the nucleic acid construct described in the seventh aspect, or a vector comprising them, and necessary solvents, such as sterile water, physiological saline, or cell culture fluid, such as LB broth, such as the EliteCell Primary T Lymphocyte Culture System (Product No.: PriMed-EliteCell-024), and optionally, instructions for use.

[0233] Pharmaceutical composition

[0234] In the fifteenth aspect, the present invention provides a pharmaceutical composition comprising the antigen-binding molecule described in the first aspect of the invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which a chimeric antigen receptor is co-expressed with another biologically active molecule), the isolated nucleic acid molecule described in the second aspect or the sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third aspect or the eighth aspect, the host cell described in the fourth aspect or the ninth aspect, the modified immune cell described in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0235] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as a drug with anti-tumor activity (e.g., anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-CD3 antibody, anti-ASGPR1 antibody, sorafenib or its derivatives, regorafenib or its derivatives, pemetrexed, cisplatin, paclitaxel, gemcitabine, capecitabine or FOLFIRINOX).

[0236] In certain embodiments, the antigen binding molecule of the first aspect, the chimeric antigen receptor of the fifth aspect (including a CAR construct in which a chimeric antigen receptor is co-expressed with another biologically active molecule), the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third aspect or the eighth aspect, the host cell of the fourth aspect or the ninth aspect, the modified immune cell of the eleventh aspect or the twelfth aspect, or the immune cell composition of the thirteenth aspect and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

[0237] In certain embodiments, the pharmaceutical composition of the present invention comprises: the antigen binding molecule described in the first aspect.

[0238] In certain embodiments, the pharmaceutical composition of the present invention comprises: the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, or a vector comprising the same.

[0239] In certain embodiments, the pharmaceutical composition of the present invention comprises: the modified immune cell of the eleventh or twelfth aspect, or the immune cell composition of the thirteenth aspect.

[0240] The antigen binding molecules described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct co-expressed with a chimeric antigen receptor and another bioactive molecule), the isolated nucleic acid molecules described in the second aspect or the sixth aspect, the nucleic acid construct described in the seventh aspect, the carrier described in the third aspect or the eighth aspect, the host cell described in the fourth aspect or the ninth aspect, the modified immune cell described in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such injections can be sterile injection solutions. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0241] The antigen binding molecules described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including the CAR construct co-expressed by the chimeric antigen receptor and another bioactive molecule), the nucleic acid molecules separated in the second aspect or the sixth aspect, the nucleic acid construct described in the seventh aspect, the carrier described in the third aspect or the eighth aspect, the host cell described in the fourth aspect or the ninth aspect, the immune cell transformed in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect can be applied by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, leaf sheath, endocytoplasmic reticulum groove, groin, bladder, local (such as, powder, ointment or drops), or nasal route. However, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that route of administration and / or mode will change according to the intended purpose. In certain embodiments, the antigen binding molecule of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect (including a CAR construct in which a chimeric antigen receptor is co-expressed with another biologically active molecule), the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third aspect or the eighth aspect, the host cell of the fourth aspect or the ninth aspect, the modified immune cell of the eleventh aspect or the twelfth aspect, or the immune cell composition of the thirteenth aspect is administered by intravenous injection or push injection.

[0242] The pharmaceutical composition of the present invention may include a "therapeutically effective amount" or a "prophylactic effective amount" of the antigen binding molecule of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect (including a CAR construct in which a chimeric antigen receptor is co-expressed with another biologically active molecule), the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third aspect or the eighth aspect, the host cell of the fourth aspect or the ninth aspect, the modified immune cell of the eleventh aspect or the twelfth aspect, or the immune cell composition of the thirteenth aspect. "Prophylactic effective amount" refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease. "Therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the antigen binding molecule described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a CAR construct in which a chimeric antigen receptor is co-expressed with another biologically active molecule), the isolated nucleic acid molecule described in the second aspect or the sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third aspect or the eighth aspect, the host cell described in the fourth aspect or the ninth aspect, the modified immune cell described in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect may vary according to the following factors: the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0243] Treatment methods and uses

[0244] In another aspect, the present invention provides a method for preventing and / or treating a disease associated with GPC3 expression in a subject (e.g., a human), the method comprising administering to a subject in need thereof an effective amount of the antigen-binding molecule of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect (including a CAR construct in which a chimeric antigen receptor is co-expressed with another biologically active molecule), the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third aspect or the eighth aspect, the host cell of the fourth aspect or the ninth aspect, the modified immune cell of the eleventh aspect or the twelfth aspect, or the immune cell composition of the thirteenth aspect, or the pharmaceutical composition of the fifteenth aspect.

[0245] In certain embodiments, the disease associated with GPC3 expression is selected from a proliferative disease, such as a tumor. In certain embodiments, the disease associated with GPC3 expression is a non-tumor-related indication associated with GPC3 expression.

[0246] In certain embodiments, the tumor is a GPC3-positive tumor. In certain embodiments, the tumor is selected from solid tumors (e.g., liver cancer, hepatocellular carcinoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, ovarian clear cell carcinoma, melanoma, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, glioma or a combination thereof. In certain embodiments, the tumor is selected from blood tumors (e.g., leukemia, lymphoma, etc.).

[0247] In certain embodiments, the method comprises administering to the subject an effective amount of the antigen binding molecule of the first aspect.

[0248] In certain embodiments, the method comprises administering to the subject an effective amount of the engineered immune cell of the eleventh or twelfth aspect, or the immune cell composition of the thirteenth aspect.

[0249] In certain embodiments, the method comprises the following steps: (1) providing the subject with the immune cells required (e.g., T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells); (2) introducing the isolated nucleic acid molecule described in the second aspect or the sixth aspect, or the nucleic acid construct described in the seventh aspect, or a vector containing them into the immune cells described in step (1); (3) administering the immune cells obtained in step (2) to the subject for treatment.

[0250] In certain embodiments, the method administers to the subject an immune cell expressing a CAR of interest by fractionating the dose, e.g., administering a partial dose once, twice, three times or more, e.g., administering a first percentage of the total dose on the first day of treatment, administering a second percentage of the total dose on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh day or later) treatment day, e.g., administering a third percentage (e.g., the remaining percentage) of the total dose on a subsequent (e.g., third, fourth, fifth, sixth, seventh, eighth, ninth, tenth day or later) treatment day.

[0251] In certain embodiments, 10% of the total dose of cells is administered on the first day of treatment, 30% of the total dose of cells is administered on the second day, and the remaining 60% of the total dose of cells is administered on the third day.

[0252] In certain embodiments, 50% of the total dose of cells is administered on the first day of treatment, and 50% of the total dose of cells is administered on a subsequent (e.g., second, third, fourth, fifth, sixth, or seventh or later) day of treatment. In certain embodiments, 1 / 3 of the total dose of cells is administered on the first day of treatment, and 1 / 3 of the total dose of cells is administered on a subsequent (e.g., second, third, fourth, fifth, sixth, or seventh or later) day of treatment, and 1 / 3 of the total dose of cells is administered on a subsequent (e.g., third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or later) day of treatment.

[0253] In certain embodiments, the total cell dose comprises 1×10 7 to 10×10 8 CAR-positive immune cells of interest, for example, (1-5)×10 7 to (5-10)×10 8 CAR-positive immune cells.

[0254] In certain embodiments, the physician may adjust the dosage or treatment regimen based on clinical circumstances such as the patient's condition, tumor size and stage, or combination therapy drugs.

[0255] In certain embodiments, the antigen binding molecules described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect (including a chimeric antigen receptor and a CAR construct co-expressed with another bioactive molecule), the isolated nucleic acid molecules described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the carrier described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the transformed immune cell described in the eleventh aspect or the twelfth aspect, or the immune cell composition described in the thirteenth aspect, or the pharmaceutical composition described in the fifteenth aspect are administered in combination with another agent. In certain embodiments, the other agent includes (i) an agent that increases the efficacy of cells comprising CAR nucleic acids or CAR polypeptides (e.g., immune cells expressing the CAR of the present invention, the transformed immune cells or immune cell compositions of the present invention); (ii) an agent that improves one or more side effects associated with the administration of cells comprising CAR nucleic acids or CAR polypeptides (e.g., immune cells expressing the CAR of the present invention, the transformed immune cells or immune cell compositions of the present invention); (iii) another pharmaceutically active agent with anti-tumor activity. These agents may be administered before, simultaneously with, or after administration of the antigen binding molecule of the first aspect of the invention, the chimeric antigen receptor of the fifth aspect (including a CAR construct in which a chimeric antigen receptor is co-expressed with another biologically active molecule), the isolated nucleic acid molecule of the second aspect or the sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third aspect or the eighth aspect, the host cell of the fourth aspect or the ninth aspect, the modified immune cell of the eleventh aspect or the twelfth aspect, or the immune cell composition of the thirteenth aspect, or the pharmaceutical composition of the fifteenth aspect.

[0256] In certain embodiments, the above method further comprises administering to the subject a second therapy, which can be any therapy known for tumors, such as surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

[0257] In certain embodiments, the second therapy can be applied separately from or in combination with the above-described method; or, the second therapy can be applied simultaneously with or sequentially with the above-described method.

[0258] In certain embodiments, the subject can be a mammal, such as a human.

[0259] In another aspect, the present invention provides the use of the antigen-binding molecule of the first aspect, the chimeric antigen receptor of the fifth aspect (including a CAR construct co-expressing a chimeric antigen receptor and another biologically active molecule), the isolated nucleic acid molecule of the second or sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third or eighth aspect, the host cell of the fourth or ninth aspect, the modified immune cell of the eleventh or twelfth aspect, the immune cell composition of the thirteenth aspect, or the pharmaceutical composition of the fifteenth aspect in the preparation of a medicament for preventing and / or treating a disease associated with GPC3 expression. The dosage, dosage form, route of administration, indications, combination therapy, and other aspects of the aforementioned treatment methods can be applied to the use of the medicament.

[0260] In another aspect, the present invention provides the antigen-binding molecule of the first aspect, the chimeric antigen receptor of the fifth aspect (including a CAR construct co-expressing a chimeric antigen receptor and another biologically active molecule), the isolated nucleic acid molecule of the second or sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the third or eighth aspect, the host cell of the fourth or ninth aspect, the modified immune cell of the eleventh or twelfth aspect, or the immune cell composition of the thirteenth aspect, or the pharmaceutical composition of the fifteenth aspect, for use in preventing and / or treating diseases associated with GPC3 expression. The dosage, dosage form, route of administration, indications, combination therapy, and other aspects of the aforementioned treatment methods can be applied to the use of the drug.

[0261] Abbreviations CAR Chimeric Antigen Receptor CDR Complementarity Determining Region in an Immunoglobulin Variable Region CDR-H1 Complementarity Determining Region 1 in an Immunoglobulin Heavy Chain Variable Region CDR-H2 Complementarity Determining Region 2 in an Immunoglobulin Heavy Chain Variable Region CDR-H3 Complementarity Determining Region 3 in an Immunoglobulin Heavy Chain Variable Region CDR-L1 Complementarity Determining Region 1 in an Immunoglobulin Light Chain Variable Region CDR-L2 Complementarity Determining Region 2 in an Immunoglobulin Light Chain Variable Region CDR-L3 Complementarity Determining Region 3 in an Immunoglobulin Light Chain Variable Region FR Antibody Framework Region: amino acid residues in an antibody variable region other than the CDR residues VH Antibody Heavy Chain Variable Region VL Antibody Light Chain Variable Region Kabat Immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). IMGT is based on the international ImMunoGeneTics information system initiated by Lefranc et al. The numbering system for immunoglobulins (IMGT) is described in Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. Chothia The immunoglobulin numbering system proposed by Chothia et al. is a classic rule for identifying CDR region boundaries based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). AbM The AbM CDR definition method is derived from the relevant work of Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272). IL-2 Interleukin-2 IFN Interferon PCR Polymerase chain reaction FACS Flow cytometry sorting

[0262] Definition of terms

[0263] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the procedures in molecular biology, microbiology, cell biology, biochemistry, immunology, and the like used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0264] As used herein, the term "antigen binding molecule" is an antibody molecule or an antigen binding fragment thereof.

[0265] As used herein, the term "antibody" refers to an immunoglobulin molecule that can specifically bind to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) by at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term includes not only complete polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chains (such as scFv, di-scFv, (scFv)2) and domain antibodies (including, for example, shark and camel antibodies), as well as fusion proteins including antibodies, and immunoglobulin molecules including any other modified configurations of antigen recognition sites. The antibodies of the present invention are not limited by any specific method for producing antibodies. Antibodies include antibodies of any type, such as IgG, IgA or IgM (or its subclass), and antibodies do not need to belong to any specific type. Depending on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulins can be assigned to different types. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different types of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. The subunit structures and three-dimensional configurations of the different types of immunoglobulins are well known. The heavy chain constant region is composed of four domains (CH1, hinge region, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant domains are not directly involved in antibody-antigen binding, but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0266] The VH and VL domains of antibodies can be further subdivided into regions of high variability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V LIt consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site. The allocation of amino acids within each region or domain can be based on the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0267] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0268] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof can be identified according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably identified by the Kabat, Chothia or IMGT numbering systems.

[0269] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0270] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide of a fragment of an antibody, such as a polypeptide of a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include camelid Ig, Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv") and single domain antibodies (sdAb, nanobodies) and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0271] As used herein, the term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means the fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0272] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0273] As used herein, the term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0274] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, containing NH2-VH-VH-COOH, NH 2- VL-VL-COOH scFv. The scFv can be formed into any engineeringly feasible structure, including single-chain antibodies (scFv), tandem antibodies (tandem di-scFvs), bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, disulfide-stabilized Fv proteins, camelid Ig, IgNAR, etc. In certain embodiments of the present invention, the scFv can form a di-scFv, which refers to two or more single scFvs connected in series to form an antibody. In certain embodiments of the present invention, the scFv can form a (scFv)2, which refers to two or more single scFvs connected in parallel to form an antibody.

[0275] As used herein, the term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also called nanobodies.

[0276] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0277] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0278] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0279] As used herein, the expression "specifically binds" or "specifically targets" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) indicates. In the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0280] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant K D(See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D , kon and kdis values. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0281] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0282] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0283] The twenty conventional amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0284] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. A vector may include sequences that directly replicate autonomously in a cell, or may include sequences sufficient to allow integration into the host cell DNA. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and viral vectors. Non-limiting examples of viral vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). A vector may contain a variety of elements for controlling expression, including but not limited to a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector may also contain a replication initiation site.

[0285] As used herein, the term "episomal vector" means that the vector is capable of replicating without integrating into the host's chromosomal DNA and being gradually lost by dividing host cells, and also means that the vector replicates extrachromosomally or episomally.

[0286] As used herein, the term "viral vector" is broadly used to refer to a nucleic acid molecule (e.g., a transfer plasmid) that includes virally derived nucleic acid elements that typically facilitate transfer or integration of the nucleic acid molecule into the genome of a cell, or a viral particle that mediates nucleic acid transfer. In addition to the nucleic acid, the viral particle will typically include various viral components and sometimes host cell components.

[0287] The term "viral vector" can refer to a virus or viral particle capable of transferring nucleic acid into a cell, or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses.

[0288] As used herein, the term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements primarily derived from a retrovirus, or portions thereof.

[0289] As used herein, the term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements derived primarily from a lentivirus, or portions thereof (including LTRs). In certain embodiments, the terms "lentiviral vector," "lentiviral expression vector," and "lentiviral transfer plasmid" may be used to refer to lentiviral transfer plasmids and / or infectious lentiviral particles. When referring to elements (e.g., cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc.) herein, it is understood that the sequences of these elements are present in the lentiviral particles of the present invention in the form of RNA and in the DNA plasmids of the present invention in the form of DNA.

[0290] As used herein, an "integration-defective" retrovirus or lentivirus refers to a retrovirus or lentivirus that has an integrase that is unable to integrate the viral genome into the host cell's genome. In certain embodiments, the integrase protein is mutated to specifically reduce its integrase activity. Integration-defective lentiviral vectors can be obtained by modifying the pol gene encoding the integrase protein to produce a mutant pol gene encoding an integration-defective integrase. Such integration-defective viral vectors have been described in patent application WO 2006 / 010834, which is incorporated herein by reference in its entirety.

[0291] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells, immune cells (such as T lymphocytes, NK cells, monocytes, macrophages or dendritic cells, etc.). Host cells can include single cells or cell populations.

[0292] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct comprising at least one extracellular antigen binding domain, a spacer domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain"), which is directed against an antigen of interest (e.g., GPC3) based on antibody-based specificity and immune effector cell activation intracellular domain combination to demonstrate specific immune activity for cells expressing the antigen of interest (e.g., GPC3). In the present invention, the expression "immune effector cells expressing CAR" refers to immune effector cells expressing CAR and having an antigen-specificity determined by the targeting domain of the CAR. Methods for making CARs (e.g., for cancer treatment) are known in the art, see, for example, Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol. Oncol., 6:47, 2013; PCT patent publications WO2012 / 079000, WO2013 / 059593; and U.S. Patent Publication 2012 / 0213783, all of which are incorporated herein by reference in their entirety.

[0293] As used herein, the term "extracellular antigen binding domain" refers to a polypeptide that is capable of specifically binding to an antigen or receptor of interest. This domain will be capable of interacting with cell surface molecules. For example, an extracellular antigen binding domain can be selected to recognize an antigen that is a cell surface marker of a target cell associated with a particular disease state.

[0294] As used herein, the term "intracellular signaling domain" refers to a portion of a protein that conducts effector signaling functions and directs cells to perform specialized functions. Thus, the intracellular signaling domain has the ability to activate at least one normal effector function of an immune effector cell expressing a CAR. For example, the effector function of a T cell can be cytolytic activity or helper activity, including the secretion of cytokines.

[0295] As used herein, the term "primary signaling domain" refers to a portion of a protein that is capable of regulating the primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary signaling domains that act in a stimulatory manner typically contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Non-limiting examples of ITAMs containing primary signaling domains particularly useful in the present invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD22, CD79a, CD79b, and CD66d.

[0296] As used herein, the term "costimulatory signaling domain" refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules that provide the second signal required for efficient activation and function of T lymphocytes after being bound to an antigen, except for antigen receptors or Fc receptors. Non-limiting examples of the costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS), DAP10.

[0297] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, sterile water, physiological saline, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffer. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0298] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or entirely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared to the expected survival (if not receiving treatment), extending the survival period.

[0299] As used herein, the term "subject" refers to a mammal, such as a primate, such as a human. In certain embodiments, the term "subject" refers to a living organism in which an immune response can be elicited. In certain embodiments, the subject (e.g., a human) has a tumor (e.g., a GPC3-associated tumor), or is at risk of having such a disease.

[0300] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0301] As used herein, the term "immune cell" refers to a cell involved in an immune response, such as a cell involved in promoting immune effector functions. Examples of immune cells include T cells (e.g., α / β T cells and γ / δ T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived macrophages.

[0302] The immune cells of the present invention can be self / autologous ("self") or non-self ("non-self," e.g., allogeneic, isogenic, or allogeneic). As used herein, "self" refers to cells from the same subject; "allogeneic" refers to cells of the same species that are genetically different from the comparison cells; "isogenic" refers to cells from a different subject that are genetically identical to the comparison cells; and "allogeneic" refers to cells from a different species than the comparison cells. In preferred embodiments, the cells of the present invention are allogeneic.

[0303] Exemplary immune cells that can be used for CAR as described herein include T lymphocytes and / or NK cells. The term "T cell" or "T lymphocyte" is well known in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4 T cells) CD4 T cells, cytotoxic T cells (CTL; ​​CD8 T cells), CD4CD8 T cells, CD4CD8 T cells or any other T cell subsets. In certain embodiments, T cells can include naive T cells and memory T cells.

[0304] It will be understood by those skilled in the art that other cells can also be used as immune cells with CAR as described herein. Specifically, immune cells also include NK cells, monocytes, macrophages or dendritic cells, NKT cells, neutrophils and macrophages. Immune cells also include the progenitor cells of immune cells, wherein the progenitor cells can be induced in vivo or in vitro to differentiate into immune cells. Therefore, in certain embodiments, immune cells include the progenitor cells of immune cells, such as hematopoietic stem cells (HSC) contained in CD34+ cell colonies derived from umbilical cord blood, bone marrow or flowing peripheral blood, which differentiate into mature immune cells after administration in the subject, or it can be induced in vitro to differentiate into mature immune cells.

[0305] As used herein, the term "modified immune cell" refers to an immune cell expressing any of the CARs described herein, or an immune cell into which any of the isolated nucleic acids or vectors described herein have been introduced. After the polynucleotide encoding the CAR polypeptide is introduced into the cell in a variety of ways, the CAR polypeptide can also be synthesized in situ in the cell. Alternatively, the CAR polypeptide can be produced extracellularly and then introduced into the cell. Methods for introducing polynucleotide constructs into cells are known in the art. In some embodiments, a stable transformation method can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, a transient transformation method can be used for transient expression of the polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell. In other embodiments, a viral-mediated method can be used. The polynucleotide can be introduced into the cell by any suitable method, such as a recombinant viral vector (e.g., retrovirus, adenovirus), liposome, etc. Transient transformation methods include, for example, but not limited to, microinjection, electroporation, or microparticle bombardment. The polynucleotide can be included in a vector, such as a plasmid vector or a viral vector.

[0306] As used herein, the term "immune effector function" refers to the function or response of immune effector cells to enhance or promote an immune attack on a target cell (e.g., killing of a target cell, or inhibiting its growth or proliferation). For example, the effector function of a T cell can be cytolytic activity or helper activity, including the secretion of cytokines.

[0307] Advantageous Effects of the Invention

[0308] CAR-T cell therapy is currently ineffective in treating solid tumors, primarily due to the complex tumor microenvironment and high tumor heterogeneity of solid tumors. The present invention provides a GPC3-targeting CAR or immune cells containing such a CAR. By specifically targeting GPC3, the CAR enhances the killing of cells expressing tumor antigens while reducing off-target toxicity to a certain extent, thereby enhancing the tumor-killing efficacy of CAR-T cells.

[0309] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only intended to illustrate the present invention and are not intended to limit the scope of the invention. Based on the following detailed description of the drawings and preferred embodiments, the various objects and advantages of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0310] Figure 1 shows the results of the cytotoxicity test of CAR-T cells (blank T, CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, and CH3-T) against target cells.

[0311] Figure 2A shows the results of IFN-γ secretion after activation of CAR-T cells (blank T, CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, and CH3-T). Figure 2B shows the results of IL-2 secretion after activation of CAR-T cells (blank T, CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, and CH3-T).

[0312] Figure 3A shows the tumor volume change curves of B-NDG mice treated with PBS, blank T cells, CA3-T, CC8-T, and CH3-T. Figure 3B shows the body weight change curves of B-NDG mice treated with PBS, blank T cells, CA3-T, CC8-T, and CH3-T.

[0313] Sequence information

[0314] The sequence information involved in the present invention is provided as follows:

[0315] Table 1 DETAILED DESCRIPTION

[0316] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0317] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Molecular Biology: A Compendium of Laboratory Manuals, 3rd edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0318] Example 1: Preparation of specific single-chain antibody (scFv) binding to human GPC3

[0319] 1) Phage library screening of GPC3 scFv

[0320] The fully human phage library was screened using biotinylated GPC3 and SV magnetic beads, and the screened products were titrated by plating. The products from the first round of panning were mixed with PBST and panned for the second and third rounds according to the above steps.

[0321] 2) ELISA detection of monoclonal phage

[0322] Single colonies were inoculated from the phage panning product titer plate into 96-deep-well plates, and monoclonal phages were detected by ELISA.

[0323] A total of six fully human anti-GPC3 monoclonal antibodies (CD4, CA3, CB5, CC8, CE9, and CH3) were screened and obtained. Sequencing and analysis of these monoclonal antibodies revealed the VH and VL sequences, as well as the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences according to the Kabat, IMGT, Chothia, and AbM numbering systems (see Tables 1 and 2 for specific sequences).

[0324] Table 2. Sequences of anti-GPC3 monoclonal antibodies

[0325] 3) Construction of scFv-Fc and analysis of antibody aggregates

[0326] The VH and VL of the above fully human antibody were connected via a linker (SEQ ID NO: 110) to obtain scFv. The sequence information of each scFv is shown in the following table.

[0327] Table 3: Structure of scFv

[0328] The candidate scFv sequences were constructed in the TGEX-KAL vector and then transfected into expi293 cells for expression and purification of the scFv-Fc protein. SEC analysis results (Table 4) showed that the main peak area of ​​the six candidate scFv sequences was greater than 80%.

[0329] Table 4: scFv-Fc protein SEC data

[0330] 4) GPC3 cell binding assay

[0331] To characterize the binding affinity of GPC3 scFv-Fc proteins, a GPC3-expressing cell line (293T / GPC3+) was selected for cell-binding assays. Mouse IgG isotype control (Thermo Fisher Scientific) was used as a negative control, and the anti-GPC3 antibody GC33 (Ishiguro et al., 2008; for the GC33 sequence, see US20150259417A1) was used as a positive control. The results in Table 5 show that the six candidate scFvs exhibited significantly better affinity for GPC3-positive 293T / GPC3+ cells than the control.

[0332] Table 5. GPC3 cell binding assay results

[0333] Example 2: Construction of lentiviral plasmid and viral packaging

[0334] 1) Construction of lentiviral plasmid:

[0335] Based on the scFv sequence in the above example, a CAR lentiviral expression vector was further constructed. The intracellular domain of CD137 (4-1BB) and the ITAM region of CD3ζ were used as activation signals and fused with the above scFv. At the same time, the CD8α signal peptide, CD8 hinge region, and CD8 transmembrane region were added to construct a chimeric antigen receptor expression vector. The chimeric antigen receptor structure is shown in Table 6 below.

[0336] Table 6. Structure of chimeric antigen receptors

[0337] 2) Virus packaging:

[0338] The CAR lentiviral plasmid and transfection reagent mixture constructed above was added dropwise to 293T (source: ATCC) cells, and the culture dish was gently shaken to mix thoroughly. The culture dish was placed in a 37°C, 5% CO2 incubator; after 6-8 hours of incubation, the culture medium containing the transfection reagent was removed and replaced with fresh complete culture medium. After continuous incubation for 48 hours, the supernatant containing the virus in the culture dish was collected, filtered with a 0.45μm filter membrane, transferred to a centrifuge tube, balanced, and centrifuged at 20,000×g at 4°C for 2 hours. After centrifugation, in a biosafety cabinet, the liquid in the centrifuge tube was carefully aspirated, the pellet was resuspended in 500μL PBS buffer, and the virus was stored at -80°C.

[0339] Experimental Example 3: CAR-T cell preparation

[0340] 1) Isolation of primary T cells:

[0341] (1) Human PBMC cells were isolated using lymphocyte separation medium (GE) and cultured in an incubator at 37°C and 5% CO2. 100 μL / mL of CD3 antibody and CD28 antibody were added, mixed thoroughly, and incubated at room temperature for 15 minutes.

[0342] (2) Remove the magnetic beads and mix thoroughly by pipetting up and down at least 5 times.

[0343] (3) Pipette 50 μL of magnetic beads / mL into the above sample, mix thoroughly, and incubate at room temperature for 10 minutes.

[0344] (4) Add complete medium to a total volume of 2.5 mL in the tube, insert the tube (with the lid open) into the magnet, and let it stand at room temperature for 5 minutes.

[0345] (5) After incubation, the tube remains in the magnet and is gently inverted to pour out the cells.

[0346] (6) Resuspend the cells in X-vivo 15 medium and add 10% FBS, 300 U / mL IL-2, 5 ng / mL IL-15, and 10 ng / mL IL-7.

[0347] 2) T cell activation:

[0348] Adjust the cell density to 1×10 6 cells / mL, and cytokine and antibody complexes (configured at a final concentration of 300 U / mL of IL-2, 10 ng / mL IL-7, 5 ng / mL IL-15, 500 ng / mL Anti-CD3 (OKT3), and 2 μg / mL Anti-CD28) were added and cultured for 48 hours.

[0349] 3) Viral infection:

[0350] (1) Calculate the required amount of virus according to MOI = 5. The calculation formula is as follows: Required amount of virus (mL) = (MOI * number of cells) / virus titer

[0351] (2) Rapidly rewarm the virus to 37°C. Add the calculated amount of virus to a six-well plate, add DEAE at a final concentration of 5 μg / mL, mix thoroughly, and centrifuge.

[0352] (3) After centrifugation, place the six-well plate in a 37°C 5% CO2 incubator and continue culturing until ready for use.

[0353] (4) Centrifuge at 250 × g for 10 minutes, remove the virus-containing culture medium supernatant, resuspend the cell pellet with fresh culture medium, transfer the cells to a new six-well plate, and continue culturing for 3-6 days.

[0354] CAR-T cells expressing the CARs described in Example 2 (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, and CH3-T) were obtained by the above method.

[0355] Experimental Example 4: Detection of the Positive Rate of CAR-T Cells

[0356] The nucleic acid sequence encoding CAR is expressed under the drive of the promoter, and the T cells transfected with the lentivirus are labeled with the GPC3 antigen and measured by flow cytometry to reflect the expression level of CAR on the surface of the T cells. The CAR positive rate of the CAR-T cells obtained in Example 3 was detected by the above method, and the FACS test results are shown in Table 7 below. The results showed that the CAR positive rate of all CAR-T cells was greater than 10%, indicating that after the lentivirus was transfected into the effector cells, CAR was successfully expressed, and 6 types of GPC3-CAR chimeric antigen receptor T cells (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, CH3-T) were successfully constructed.

[0357] Table 7: CAR positive rate test results

[0358] Experimental Example 5: Evaluation of CAR-T's killing activity against HepG2 target cells

[0359] HEPG2-luc cells were digested with 0.25% trypsin and digested with 1640 medium containing 10% FBS. After centrifugation, the cells were resuspended and the cell density was adjusted to 1×10 5 Target HEPG2-luc cells were inoculated at 100 μL / well in a 96-well plate and incubated in a 5% CO2, 37°C incubator for 30 minutes. CAR-T cells were harvested by centrifugation and resuspended in 1640 medium containing 10% FBS. CAR-T cells, GPC3-CAR, and untransfected CAR-positive T cells (UTD) were then added to a 96-well plate containing HEPG2-luc at varying E / T (effector cell / target cell) ratios at 100 μL / well, bringing the final volume to 200 μL / well. The plates were then incubated in a 5% CO2, 37°C incubator for 18-24 hours. After incubation, the plates were removed from the incubator, 20 μL of fluorescence detection reagent was added, and fluorescence readings were measured using a microplate reader.

[0360] The results of CAR-T cell killing activity testing are shown in Figure 1. The six types of CAR-T cells constructed in this application (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, and CH3-T) can effectively lyse tumor cells at different E / T ratios. Among them, CA3-T, CC8-T, CH3-T, and CE9-T are particularly effective, with a tumor cell lysis rate of up to 98% at an effector cell / target cell ratio of 10.

[0361] Experimental Example 6: Cytokine Release During Co-incubation of CAR-T Cells with HepG2 Target Cells

[0362] Collect HepG2-luc cells and adjust the cell density to 1×105 / mL, inoculate target cells in a 96-well plate at an amount of 100 μL / well, and resuspend CAR-T cells, GPC3-CAR and blank T cells that are not transfected with CAR in the culture medium as effector cells, and then add them to the 96-well plate containing target cells at an E / T (effector cell / target cell) ratio of 1:1, 100 μL / well, and the final volume is supplemented to 200 μL / well, and cultured overnight in a 5% CO2 37°C incubator. After the culture is completed, the well plate is removed from the incubator, centrifuged, the supernatant is taken, and cytokine release is detected using an ELISA kit (IL2, IFN-γ). The six CAR-T cells (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, CH3-T) constructed in this application can kill tumor cells to varying degrees and release IFN-γ (test results are shown in Figure 2A) or IL-2 (test results are shown in Figure 2B).

[0363] Experimental Example 7: In vivo model to evaluate the killing ability of CAR-T cells on target cells

[0364] Thirty-six B-NDG mice were subcutaneously inoculated with 5 × 10 6 HepG2 tumor cells, and wait until the average tumor volume reaches 100-150 mm 3 The mice were randomly divided into 6 groups, and each mouse was given cyclophosphamide 100 mg / kg intraperitoneally and 5×10 6 CAR-T cells and blank T cells that were not transfected with CAR. The tumor diameter and mouse weight were measured with a vernier caliper twice a week. The results of the killing ability of CAR-T cells on target cells are shown in Figure 3A, and the changes in mouse weight are shown in Figure 3B. Compared with the negative control group, the CAR-T cells (CA3-T, CC8-T, CH3-T) constructed in this application have a good inhibitory effect on tumor cells. There was no animal death or significant animal weight loss in all treatment groups during the observation period, no obvious drug toxicity was observed, and the mice tolerated the treatment well.

[0365] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A chimeric antigen receptor comprising an antigen binding domain comprising the following complementarity determining regions (CDRs): (a) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 1; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 2; or, (b) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 3; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 4; or, (c) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 5; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 6; or, (d) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 7; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 8; or, (e) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 9; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 10; or, (f) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 11; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 12; or (g) CDR-H1, CDR-H2, and CDR-H3 contained in the following heavy chain variable region (VH), and / or CDR-L1, CDR-L2, and CDR-L3 contained in the following light chain variable region (VL), wherein: The heavy chain variable region (VH) and / or light chain variable region (VL) comprises at least one CDR comprising a mutation compared to the heavy chain variable region and / or light chain variable region described in any one of (a) to (f), wherein the mutation is a substitution, deletion or addition of one or more amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitution is a conservative substitution; Preferably, the CDRs are defined according to the Kabat, IMGT, Chothia or AbM numbering systems.

2. The chimeric antigen receptor according to claim 1, wherein The antigen binding domain comprises: (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof; CDR-H2 of SEQ ID NO: 14 or a variant thereof; CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof; CDR-L2 of SEQ ID NO: 17 or a variant thereof; and CDR-L3 of SEQ ID NO: 18 or a variant thereof; or, (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof; CDR-H2 of SEQ ID NO: 27 or a variant thereof; CDR-H3 of SEQ ID NO: 28 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 29 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; and CDR-L3 of SEQ ID NO: 31 or a variant thereof; or, (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 39 or a variant thereof; CDR-H2 of SEQ ID NO: 40 or a variant thereof; and CDR-H3 of SEQ ID NO: 41 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 42 or a variant thereof; CDR-L2 of SEQ ID NO: 43 or a variant thereof; and CDR-L3 of SEQ ID NO: 44 or a variant thereof; or, (1d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof; CDR-H2 of SEQ ID NO: 27 or a variant thereof; CDR-H3 of SEQ ID NO: 52 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 53 or a variant thereof; CDR-L2 of SEQ ID NO: 54 or a variant thereof; CDR-H4 of SEQ ID NO: 55 or a variant thereof. CDR-L3 of 55 or a variant thereof; or, (1e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 60 or a variant thereof; CDR-H2 of SEQ ID NO: 61 or a variant thereof; CDR-H3 of SEQ ID NO: 62 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 63 or a variant thereof; CDR-L2 of SEQ ID NO: 64 or a variant thereof; and CDR-L3 of SEQ ID NO: 65 or a variant thereof; or, (1f) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof; CDR-H2 of SEQ ID NO: 27 or a variant thereof; CDR-H3 of SEQ ID NO: 73 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 74 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; and CDR-L3 of SEQ ID NO: 75 or a variant thereof; or, (2) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 19 or a variant thereof; CDR-H2 of SEQ ID NO: 20 or a variant thereof; CDR-H3 of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 22 or a variant thereof; CDR-L2 of SEQ ID NO: 23 or a variant thereof; and CDR-L3 of SEQ ID NO: 18 or a variant thereof; or, (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 32 or a variant thereof; CDR-H2 of SEQ ID NO: 33 or a variant thereof; CDR-H3 of SEQ ID NO: 34 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 35 or a variant thereof; CDR-L2 of SEQ ID NO: 36 or a variant thereof; and CDR-L3 of SEQ ID NO: 31 or a variant thereof; or, (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 45 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 46 or a variant thereof; and CDR-H3 having a sequence of SEQ ID NO: 47 or a variant thereof. and / or a light chain variable region (VL) comprising the following three CDRs: a CDR-L1 having a sequence of SEQ ID NO: 48 or a variant thereof; a CDR-L2 having a sequence of SEQ ID NO: 49 or a variant thereof; and a CDR-L3 having a sequence of SEQ ID NO: 44 or a variant thereof; or, (2d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 32 or a variant thereof; CDR-H2 of SEQ ID NO: 33 or a variant thereof; CDR-H3 of SEQ ID NO: 56 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 57 or a variant thereof; CDR-L2 of SEQ ID NO: 58 or a variant thereof; and CDR-L3 of SEQ ID NO: 59 or a variant thereof; or, (2e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 66 or a variant thereof; CDR-H2 of SEQ ID NO: 67 or a variant thereof; and CDR-H3 of SEQ ID NO: 68 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 69 or a variant thereof; CDR-L2 of SEQ ID NO: 70 or a variant thereof; and CDR-L3 of SEQ ID NO: 65 or a variant thereof; or, (2f) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 32 or a variant thereof; CDR-H2 of SEQ ID NO: 33 or a variant thereof; and CDR-H3 of SEQ ID NO: 76 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 77 or a variant thereof; CDR-L2 of SEQ ID NO: 36 or a variant thereof; and CDR-L3 of SEQ ID NO: 75 or a variant thereof; or, (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 24 or a variant thereof; CDR-H2 of SEQ ID NO: 25 or a variant thereof; CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof; CDR-L2 of SEQ ID NO: 17 or a variant thereof; and CDR-L3 of SEQ ID NO: 18 or a variant thereof; or, (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 37 or a variant thereof; CDR-H2 of SEQ ID NO: 38 or a variant thereof; and CDR-H3 of SEQ ID NO: 28 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 29 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; and CDR-L3 of SEQ ID NO: 31 or a variant thereof; or, (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 50 or a variant thereof; CDR-H2 of SEQ ID NO: 51 or a variant thereof; and CDR-H3 of SEQ ID NO: 41 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 42 or a variant thereof; CDR-L2 of SEQ ID NO: 43 or a variant thereof; and CDR-L3 of SEQ ID NO: 44 or a variant thereof; or, (3d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 37 or a variant thereof; CDR-H2 of SEQ ID NO: 38 or a variant thereof; and CDR-H3 of SEQ ID NO: 52 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 53 or a variant thereof; CDR-L2 of SEQ ID NO: 54 or a variant thereof; and CDR-L3 of SEQ ID NO: 55 or a variant thereof; or, (3e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 71 or a variant thereof; CDR-H2 of SEQ ID NO: 72 or a variant thereof; and CDR-H3 of SEQ ID NO: 62 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 63 or a variant thereof; CDR-L2 of SEQ ID NO: 64 or a variant thereof; and CDR-L3 of SEQ ID NO: 65 or a variant thereof; or, (3f) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 37 or a variant thereof; CDR-H2 of SEQ ID NO: 38 or a variant thereof; and CDR-H3 of SEQ ID NO: 73 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 74 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; and CDR-L3 of SEQ ID NO: 75 or a variant thereof; or, (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system: (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 80 or a variant thereof; CDR-H2 of SEQ ID NO: 81 or a variant thereof; CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof; CDR-L2 of SEQ ID NO: 17 or a variant thereof; and CDR-L3 of SEQ ID NO: 18 or a variant thereof; or, (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 78 or a variant thereof; CDR-H2 of SEQ ID NO: 79 or a variant thereof; and CDR-H3 of SEQ ID NO: 28 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 29 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; and CDR-L3 of SEQ ID NO: 31 or a variant thereof; or, (4c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 96 or a variant thereof; CDR-H2 of SEQ ID NO: 97 or a variant thereof; and CDR-H3 of SEQ ID NO: 41 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 42 or a variant thereof; CDR-L2 of SEQ ID NO: 43 or a variant thereof; and CDR-L3 of SEQ ID NO: 44 or a variant thereof; or, (4d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 78 or a variant thereof; CDR-H2 of SEQ ID NO: 79 or a variant thereof; and CDR-H3 of SEQ ID NO: 52 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 53 or a variant thereof; CDR-L2 of SEQ ID NO: 54 or a variant thereof; and CDR-L3 of SEQ ID NO: 55 or a variant thereof; or, (4e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 98 or a variant thereof; CDR-H2 of SEQ ID NO: 99 or a variant thereof; CDR-H3 of SEQ ID NO: 62 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 63 or a variant thereof; CDR-L2 of SEQ ID NO: 64 or a variant thereof; CDR-H4 of SEQ ID NO: 65 or a variant thereof. CDR-L3 of 65 or a variant thereof; or, (4f) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 78 or a variant thereof; CDR-H2 of SEQ ID NO: 79 or a variant thereof; CDR-H3 of SEQ ID NO: 73 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 74 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; and CDR-L3 of SEQ ID NO: 75 or a variant thereof; Wherein, the variant described in any one of (1a)-(1f), (2a)-(2f), (3a)-(3f), (4a)-(4f) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

3. The chimeric antigen receptor according to claim 1 or 2, wherein The antigen binding domain comprises: (a) a VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof; or, (b) a VH comprising the sequence shown in SEQ ID NO: 3 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 4 or a variant thereof; or, (c) a VH comprising the sequence shown in SEQ ID NO: 5 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 6 or a variant thereof; or (d) a VH comprising the sequence shown in SEQ ID NO: 7 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 8 or a variant thereof; or, (e) a VH comprising the sequence shown in SEQ ID NO: 9 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 10 or a variant thereof; or, (f) a VH comprising the sequence shown in SEQ ID NO: 11 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 12 or a variant thereof; wherein the variant has at least 80%, at least 85%, at least 90%, at least At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

4. The chimeric antigen receptor according to any one of claims 1 to 3, wherein The antigen binding domain is selected from a full-length antibody, a Fab fragment, a Fab' fragment, a Fab'-SH, a F(ab)'2 fragment, a F(ab)'3 fragment, an Fv fragment, a single-chain antibody (e.g., scFv, di-scFv or (scFv)2), a miniantibody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb, nanobody), a diabody, a bispecific antibody and a multispecific antibody.

5. The chimeric antigen receptor according to any one of claims 1 to 4, wherein The antigen binding domain is a single-chain antibody, such as scFv, di-scFv or (scFv)2; Preferably, the single-chain antibody comprises, from its N-terminus to its C-terminus: (1) a VH comprising a sequence as shown in SEQ ID NO: 1 or a variant thereof, a linker comprising a VL comprising a sequence as shown in SEQ ID NO: 2 or a variant thereof; or a VL comprising a sequence as shown in SEQ ID NO: 2 or a variant thereof, a linker comprising a VH comprising a sequence as shown in SEQ ID NO: 1 or a variant thereof; or (2) a VH comprising a sequence as set forth in SEQ ID NO: 3 or a variant thereof, a linker comprising a VL comprising a sequence as set forth in SEQ ID NO: 4 or a variant thereof; or a VL comprising a sequence as set forth in SEQ ID NO: 4 or a variant thereof, a linker comprising a VH comprising a sequence as set forth in SEQ ID NO: 3 or a variant thereof; or (3) a VH comprising a sequence as set forth in SEQ ID NO: 5 or a variant thereof, a linker comprising a VL comprising a sequence as set forth in SEQ ID NO: 6 or a variant thereof; or a VL comprising a sequence as set forth in SEQ ID NO: 6 or a variant thereof, a linker comprising a VH comprising a sequence as set forth in SEQ ID NO: 5 or a variant thereof; or (4) a VH comprising a sequence as set forth in SEQ ID NO: 7 or a variant thereof, a linker comprising a VL comprising a sequence as set forth in SEQ ID NO: 8 or a variant thereof; or a VL comprising a sequence as set forth in SEQ ID NO: 8 or a variant thereof, a linker comprising a VH comprising a sequence as set forth in SEQ ID NO: 7 or a variant thereof; or (5) a VH-linker comprising a sequence as shown in SEQ ID NO: 9 or a variant thereof; a VH-linker comprising a sequence as shown in SEQ ID NO: 10 or a VL comprising a sequence as shown in SEQ ID NO: 10 or a variant thereof; or a VL-linker comprising a sequence as shown in SEQ ID NO: 10 or a variant thereof; or a VH comprising a sequence as shown in SEQ ID NO: 9 or a variant thereof; or (6) a VH comprising a sequence as set forth in SEQ ID NO: 11 or a variant thereof, a linker comprising a VL comprising a sequence as set forth in SEQ ID NO: 12 or a variant thereof; or a VL comprising a sequence as set forth in SEQ ID NO: 12 or a variant thereof, a linker comprising a VH comprising a sequence as set forth in SEQ ID NO: 11 or a variant thereof; wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; Preferably, the linker is a polypeptide; preferably, the linker comprises one or several (e.g., 1, 2 or 3) sequences as shown in (GmS)n, wherein m is selected from an integer of 1-6, and n is selected from an integer of 1-6; preferably, m is 3, 4, or 5; preferably, n is 1 or 2; more preferably, the linker has a sequence of SEQ ID NO:

110.

6. The chimeric antigen receptor according to any one of claims 1 to 5, wherein The antigen-binding domain is a single-chain antibody, comprising a sequence as shown in any one of SEQ ID NOs: 86, 88, 90, 92, 94, 82, or a variant thereof, wherein the variant has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NOs: 86, 88, 90, 92, 94 or 82, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

7. The chimeric antigen receptor of any one of claims 1 to 6, further comprising a transmembrane domain selected from the group consisting of the transmembrane regions of the α, β, or ζ chains of the T cell receptor, CD28, CD45, CD3ε, CD3ζ, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD-1; Preferably, the transmembrane domain is selected from the transmembrane regions of the following proteins: CD8, CD28, CD4, PD-1, CD152 and CD154; Preferably, the transmembrane domain comprises the CD8 transmembrane region shown in SEQ ID NO:

111.

8. The chimeric antigen receptor of any one of claims 1 to 7, further comprising a spacer domain, wherein the spacer domain is located between the antigen binding domain and the transmembrane domain, and the spacer domain is selected from the hinge domain and / or the CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG4); Preferably, the hinge domain comprises the hinge region of CD8, IgG4, PD-1, CD152 or CD154; more preferably, the hinge domain comprises the CD8 hinge region shown in SEQ ID NO:

112.

9. The chimeric antigen receptor of any one of claims 1 to 8, further comprising an intracellular signaling domain comprising a primary signaling domain and / or a costimulatory signaling domain; Preferably, the intracellular signaling domain comprises a costimulatory signaling domain and a primary signaling domain in sequence from N-terminus to C-terminus; Preferably, the intracellular signaling domain comprises a primary signaling domain and at least one costimulatory signaling domain; Preferably, the primary signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM); Preferably, the primary signaling domain comprises an intracellular signaling domain selected from the group consisting of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, or CD66d; more preferably, the primary signaling domain comprises the CD3ζ intracellular signaling domain shown in SEQ ID NO: 113; Preferably, the costimulatory signaling domain comprises an intracellular signaling domain selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS) or DAP10; Preferably, the costimulatory signaling domain is selected from the intracellular signaling domain of CD28 or the intracellular signaling domain of CD137 (4-1BB), or a combination of fragments thereof; more preferably, the costimulatory signaling domain comprises the CD137 (4-1BB) intracellular signaling domain shown in SEQ ID NO: 114; More preferably, the intracellular signaling domain sequence comprises the sequence shown in SEQ ID NO:

115.

10. The chimeric antigen receptor according to any one of claims 1 to 9, wherein The chimeric antigen receptor further comprises a signal peptide at its N-terminus; Preferably, the signal peptide comprises a heavy chain signal peptide (e.g., a heavy chain signal peptide of IgG1), a granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal peptide, an IL2 signal peptide, or a CD8α signal peptide; more preferably .... The signal peptide comprises the sequence shown in SEQ ID NO:

116.

11. The chimeric antigen receptor according to any one of claims 1 to 10, wherein The chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain from its N-terminus to its C-terminus; Preferably, the signal peptide comprises an IgG1 heavy chain signal peptide or a CD8α signal peptide (eg, a signal peptide having a sequence as shown in SEQ ID NO: 116); Preferably, the antigen binding domain is as defined in any one of claims 1 to 6 (e.g., comprising a sequence as shown in any one of SEQ ID NOs: 86, 88, 90, 92, 94, 82); Preferably, the spacer domain comprises the hinge region of CD8 (eg, CD8α) (eg, the hinge region whose sequence is shown in SEQ ID NO: 112); Preferably, the transmembrane domain comprises the transmembrane region of CD8 (eg, CD8α) (eg, the transmembrane region whose sequence is shown in SEQ ID NO: 111); Preferably, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3ζ (e.g., the sequence shown in SEQ ID NO: 113), and the costimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., the sequence shown in SEQ ID NO: 114); more preferably, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO: 115; Preferably, the chimeric antigen receptor comprises the sequence shown in any one of SEQ ID NOs: 117, 119, 121, 123, 125, 127, or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

12. The chimeric antigen receptor according to any one of claims 1 to 11, wherein The antigen-binding domain comprises the antigen-binding domain defined in any one of claims 1 to 6 as a first antigen-binding domain, and further comprises a second antigen-binding domain that does not bind to GPC3; more preferably, the antigen bound by the second antigen-binding domain is selected from the group consisting of: PD-1, PD-L1, CTLA-4, CD3, ASGPR1, CD19, MSLN, PSMA, MUC1, EGFR, HER2, CD276, GD2, BCMA, CD33, or Claudin18.2; Preferably, the antigen binding domain is a single-chain antibody, such as scFv, di-scFv or (scFv)2; Preferably, the VH and VL in the antigen binding domain are connected by a linker; preferably, the linker comprises one or several (eg, 1, 2 or 3) such as (G m S) n The sequence shown, wherein m is selected from an integer of 1-6, and n is selected from an integer of 1-6; preferably, m is 3, 4, or 5; preferably, n is 1 or 2; more preferably, the linker has the sequence of SEQ ID NO: 110; Preferably, the first antigen-binding domain and the second antigen-binding domain are linked by a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A or any combination thereof); Preferably, the self-cleaving peptide is P2A; for example, the amino acid sequence of the self-cleaving peptide is shown in SEQ ID NO: 130; Preferably, the second antigen-binding domain further comprises a signal peptide at its N-terminus; preferably, the signal peptide is different from the signal peptide comprised by the GPC3-specific chimeric antigen receptor; preferably, the signal peptide at the N-terminus of the second antigen-binding domain is an IL2 signal peptide (e.g., the amino acid sequence is shown in SEQ ID NO: 129).

13. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of any one of claims 1 to 12.

14. The isolated nucleic acid molecule of claim 13, comprising a nucleotide sequence selected from the group consisting of: (1) a sequence set forth in any one of SEQ ID NOs: 118, 120, 122, 124, 126, 128, or a degenerate variant thereof; (2) a sequence substantially identical to the sequence set forth in any one of (1) (e.g., a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of (1), or a sequence having one or more nucleotide substitutions to the sequence set forth in any one of (1)).

15. A nucleic acid construct comprising a nucleic acid sequence encoding a GPC3-specific chimeric antigen receptor; wherein: The GPC3-specific chimeric antigen receptor comprises a GPC3-targeting antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain. The GPC3-targeting antigen binding domain comprises the antigen binding domain defined in any one of claims 1 to 6.

16. The nucleic acid construct of claim 15, which has one or more of the following characteristics: (1) The transmembrane domain is as defined in claim 7; (2) the spacer domain as defined in claim 8; (3) the intracellular signaling domain as defined in claim 9; (4) The GPC3-specific chimeric antigen receptor encoded by the nucleic acid sequence further comprises a signal peptide at its N-terminus, and the signal peptide is as defined in claim 10.

17. The nucleic acid construct according to any one of claims 15 to 16, wherein The nucleic acid construct comprises, from its 5' end to its 3' end, a nucleotide sequence encoding the signal peptide, a nucleotide sequence encoding the antigen-binding domain targeting GPC3, a nucleotide sequence encoding the spacer domain, a nucleotide sequence encoding the transmembrane domain, and a nucleotide sequence encoding the intracellular signaling domain; Preferably, the signal peptide comprises an IgG1 heavy chain signal peptide or a CD8α signal peptide (eg, a signal peptide having a sequence as shown in SEQ ID NO: 116); Preferably, the antigen-binding domain targeting GPC3 is selected from the antigen-binding domain defined in any one of claims 1 to 6 (e.g., comprising the sequence shown in any one of SEQ ID NOs: 86, 88, 90, 92, 94, 82); Preferably, the spacer domain comprises the hinge region of CD8 (eg, CD8α) (eg, the hinge region whose sequence is shown in SEQ ID NO: 112); Preferably, the transmembrane domain comprises the transmembrane region of CD8 (eg, CD8α) (eg, the transmembrane region whose sequence is shown in SEQ ID NO: 111); Preferably, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3ζ (e.g., the sequence shown in SEQ ID NO: 113), and the costimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., the sequence shown in SEQ ID NO: 114); more preferably, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO:

115.

18. The nucleic acid construct according to any one of claims 15 to 17, wherein The nucleic acid construct comprises a nucleotide sequence selected from the following: (1) a sequence shown in any one of SEQ ID NOs: 118, 120, 122, 124, 126, 128, or a degenerate variant thereof; (2) a sequence substantially identical to the sequence shown in any one of (1) (e.g., a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence shown in any one of (1), or a sequence having one or more nucleotide substitutions compared to the sequence shown in any one of (1)).

19. The nucleic acid construct of any one of claims 15 to 18, comprising: (1) a first nucleic acid sequence encoding a GPC3-specific chimeric antigen receptor; and (2) a second nucleic acid sequence encoding another biologically active molecule; Preferably, the GPC3-specific chimeric antigen receptor comprises a GPC3-targeting antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, and the GPC3-targeting antigen binding domain comprises the first antigen binding domain defined in any one of claims 1 to 7; Preferably, the additional biologically active molecule encoded by the second nucleic acid sequence is selected from one or more of the following components: immune checkpoint inhibitors (e.g., anti-PD-1, PD-L1, CTLA-4 or LAG3 antibodies or antigen-binding fragments thereof, cytokines (e.g., IL-15, IL-7, IL-12, IL-18 or IL-21), T cell surface antigens (e.g., CD3, etc.) or membrane chimeric polypeptides (e.g., mIL-15, mIL-7, mIL-12, mIL-18 or mIL-21); Preferably, the first nucleic acid sequence and the second nucleic acid sequence are linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A or any combination thereof); Preferably, the self-cleaving peptide is P2A; for example, the amino acid sequence of the self-cleaving peptide is shown in SEQ ID NO: 130; Preferably, the additional biologically active molecule encoded by the second nucleotide sequence further comprises a signal peptide at its N-terminus; preferably, the signal peptide is different from the signal peptide contained in the GPC3-specific chimeric antigen receptor encoded by the first nucleic acid sequence; preferably, the signal peptide at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (for example, the amino acid sequence of the IL2 signal peptide is shown in SEQ ID NO: 129).

20. A vector comprising the isolated nucleic acid molecule of claim 13 or 14, or the nucleic acid construct of any one of claims 15 to 19; Preferably, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector, or a viral vector; Preferably, the vector is an expression vector; Preferably, the vector is an episomal vector; Preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector.

21. A host cell comprising the isolated nucleic acid molecule of claim 13 or 14, or the nucleic acid construct of any one of claims 15 to 19, or the vector of claim 20.

22. A method for preparing a cell expressing a chimeric antigen receptor, comprising: (1) providing host cells; (2) introducing the isolated nucleic acid molecule of claim 13 or 14, or the nucleic acid construct of any one of claims 15 to 19, or a vector comprising the same into the host cell of step (1), to obtain a host cell capable of co-expressing the chimeric antigen receptor and optionally another biologically active molecule; Preferably, the host cell is selected from immune cells, such as T lymphocytes, NK cells, monocytes, dendritic cells, macrophages, and any combination thereof; Preferably, the immune cells are selected from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination of these cells; Preferably, in step (1), the host cells are provided from a patient or a healthy donor and are pretreated; the pretreatment includes sorting, activation and / or proliferation of immune cells; preferably, the pretreatment includes contacting the immune cells with anti-CD3 antibodies and anti-CD28 antibodies, thereby stimulating the immune cells and inducing their proliferation, thereby generating pretreated immune cells; Preferably, in step (2), the nucleic acid molecule or vector is introduced into the host cell by viral infection; Preferably, in step (2), the nucleic acid molecule or vector is introduced into the host cell by non-viral vector transfection, such as a transposon vector system, CRISPR / Cas9 vector, TALEN method, ZFN method, electroporation method, calcium phosphate transfection, DEAE-dextran-mediated transfection or microinjection; Preferably, after step (2), the method further comprises: amplifying the host cells obtained in step (2).

23. An engineered immune cell comprising the isolated nucleic acid molecule of claim 13 or 14, or the nucleic acid construct of any one of claims 15 to 19, or a vector comprising the same; Preferably, the engineered immune cells express the chimeric antigen receptor according to any one of claims 1 to 12.

24. The modified immune cell according to claim 23, wherein The immune cells are derived from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof; preferably, the immune cells are obtained from patients; alternatively, the immune cells are obtained from healthy donors; preferably, the immune cells are derived from T lymphocytes or NK cells.

25. The modified immune cell according to claim 23 or 24, wherein The immune cells also express a CAR that is not specific for GPC3; preferably, the CAR that is not specific for GPC3 has specificity for targets selected from the following targets: PD-1, PD-L1, CTLA-4, CD3, ASGPR1, CD19, MSLN, PSMA, MUC1, EGFR, HER2, CD276, GD2, BCMA, CD33 or Claudin18.

2.

26. The modified immune cell according to any one of claims 23 to 25, wherein The transcription or expression of one or two target genes among the immune rejection-related genes (e.g., TRAC, TRBC, B2M, HLA-A, HLA-B or HLA-C) and the genes of the immune co-inhibitory pathway or signaling molecules (e.g., PD-1, CTLA-4 or LAG-3) of the modified immune cells is inhibited; preferably, the method used to inhibit the transcription or expression of the target genes is selected from gene knockout (e.g., CRISPR, CRISPR / Cas9), homologous recombination, and interfering RNA.

27. A method for preparing an engineered immune cell, comprising: (1) Providing immune cells from patients or healthy donors; (2) introducing the isolated nucleic acid molecule of claim 13 or 14 or a vector comprising the same into the immune cell of step (1) to obtain an immune cell capable of expressing a chimeric antigen receptor; or introducing the nucleic acid construct of any one of claims 15 to 19 or a vector comprising the same into the immune cell of step (1) to obtain an immune cell capable of co-expressing a chimeric antigen receptor and another biologically active molecule; Preferably, in step (1), the immune cells are pretreated, and the pretreatment includes sorting, activation and / or proliferation of the immune cells; more preferably, the pretreatment includes contacting the immune cells with anti-CD3 antibodies and anti-CD28 antibodies, thereby stimulating the immune cells and inducing their proliferation, thereby generating pretreated immune cells; Preferably, in step (2), the nucleic acid molecule or vector is introduced into the immune cell via viral infection; Preferably, in step (2), the nucleic acid molecule or vector is introduced into the immune cell by non-viral vector transfection, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, transposon vector system, CRISPR / Cas9 vector, TALEN method, ZFN method or electroporation method; Preferably, after step (2), the method further includes a step of amplifying the immune cells obtained in step (2).

28. An immune cell composition comprising the modified immune cells of any one of claims 23-26; optionally, the composition further comprises unmodified and / or unsuccessfully modified immune cells; preferably, the number of the modified immune cells accounts for 10%-100%, more preferably 40%-80% of the total number of cells in the immune cell composition.

29. A kit comprising the chimeric antigen receptor according to any one of claims 1 to 12, or the nucleic acid construct according to any one of claims 15 to 19; Preferably, the kit comprises the isolated nucleic acid molecule according to claim 13 or 14 or a vector comprising the isolated nucleic acid molecule; the kit is used to prepare the chimeric antigen receptor according to any one of claims 1 to 12; Preferably, the kit comprises the isolated nucleic acid molecule of claim 13 or 14 or a vector comprising the isolated nucleic acid molecule; the kit is used to prepare the modified immune cell of claims 23-26 or an immune cell composition comprising the modified immune cell; Preferably, the kit comprises the nucleic acid construct according to any one of claims 15 to 19 or a vector comprising the nucleic acid construct; the kit is used to prepare the modified immune cells according to claims 23 to 26 or an immune cell composition comprising the modified immune cells.

30. A pharmaceutical composition comprising the chimeric antigen receptor of any one of claims 1 to 12, or the isolated nucleic acid molecule of claim 13 or 14, or the nucleic acid construct of any one of claims 15 to 19, or the vector of claim 20, or the host cell of claim 21, or the engineered immune cell of any one of claims 23 to 26, or the immune cell composition of claim 28, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises another pharmaceutically active agent, such as a drug having anti-tumor activity; preferably, the other pharmaceutically active agent comprises anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-CD3 antibody, anti-ASGPR1 antibody, sorafenib or its derivatives, regorafenib or its derivatives, pemetrexed, cisplatin, paclitaxel, gemcitabine, capecitabine or FOLFIRINOX; Alternatively, the chimeric antigen receptor, or isolated nucleic acid molecule, or vector, or host cell, or nucleic acid construct, or engineered immune cell, or immune cell composition comprised in the pharmaceutical composition can be administered simultaneously, separately or sequentially with the additional pharmaceutically active agent.

31. Use of the chimeric antigen receptor of any one of claims 1-12, or the isolated nucleic acid molecule of claim 13 or 14, or the nucleic acid construct of any one of claims 15-19, or the vector of claim 20, or the host cell of claim 21, or the engineered immune cell of any one of claims 23-26, or the immune cell composition of claim 28, or the pharmaceutical composition of claim 30 in the preparation of a medicament for preventing and / or treating a disease associated with GPC3 expression; Preferably, the disease associated with GPC3 expression is selected from a proliferative disease, such as a tumor, or a non-tumor-related indication associated with GPC3 expression; Preferably, the tumor is a GPC3-positive tumor; Preferably, the tumor is selected from solid tumors; preferably, the solid tumor is selected from one or a combination of liver cancer, hepatocellular carcinoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, ovarian clear cell carcinoma, melanoma, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, and glioma; Preferably, the tumor is selected from blood tumors; preferably, the blood tumor is selected from leukemia and lymphoma.

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