Antibodies targeting GPC3 and uses thereof

Abstract

The invention relates to an antibody specifically binding to GPC3 or an antigen binding fragment thereof and a chimeric antigen receptor (CAR) comprising the antibody or the antigen binding fragment thereof, in particular to a modified immune cell expressing the CAR and a method for preparing the modified immune cell. The invention further relates to application of the antibodies, the CAR and the immune cells in preventing and / or treating diseases related to expression of the GPC3, such as cancers such as liver cancer, melanoma and ovarian cancer, and a method for preventing and / or treating GPC3 positive tumors such as liver cancer, melanoma and ovarian cancer.

Description

Antibodies targeting GPC3 and their uses Technical Field

[0001] The present application relates to the field of biomedicine. Specifically, the present application relates to antibodies or antigen-binding fragments thereof that specifically bind to GPC3. The present invention also relates to the use of these antibodies or antigen-binding fragments thereof for preventing and / or treating diseases associated with GPC3 expression, such as liver cancer, melanoma, ovarian cancer, and other cancers, as well as methods for preventing and / or treating GPC3-positive tumors, such as liver cancer, melanoma, and ovarian cancer. 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 in the advanced or late stages, with only 30% eligible for surgical resection. The metastasis and recurrence rate within five years after resection is as high as 60% to 70%, resulting in a low overall five-year survival rate of only 7% to 10%.

[0004] Based on the specific expression of GPC3, targeted therapy against GPC3 is expected to become one of the ways to conquer GPC3-positive tumors. Therefore, providing an antibody or antigen-binding fragment targeting GPC3 is urgent and necessary for the treatment of GPC3-positive hepatocellular carcinoma (HCC), melanoma, and ovarian clear cell carcinoma.

[0005] Summary of the Invention

[0006] In the present application, the inventors have developed a fully human antibody that has low immunogenicity and is highly specific for GPC3 and can specifically recognize / bind to GPC3. This antibody has the potential to prevent and / or treat diseases associated with GPC3 expression, such as liver cancer, melanoma, ovarian cancer, and other cancers, and has significant clinical value.

[0007] Antibodies of the present invention

[0008] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to GPC3, wherein the antibody or antigen-binding fragment thereof 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] (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;

[0011] (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;

[0012] (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;

[0013] (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;

[0014] (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

[0015] (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).

[0016] In certain embodiments, the substitutions are conservative substitutions.

[0017] In certain embodiments, the CDRs are defined according to the IMGT, Kabat, Chothia, or AbM numbering systems.

[0018] 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 Kabat numbering system:

[0019] (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;

[0020] (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;

[0021] (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;

[0022] (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;

[0023] (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; CDR-L3 of SEQ ID NO: 65 or a variant thereof; or,

[0024] (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;

[0025] Wherein, the variant described in any one of (1a) to (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. In certain embodiments, the substitutions are conservative substitutions.

[0026] 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 IMGT numbering system:

[0027] (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;

[0028] (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;

[0029] (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;

[0030] (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;

[0031] (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; 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; CDR-L3 of SEQ ID NO: 65 or a variant thereof; or

[0032] (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;

[0033] 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; in certain embodiments, the substitutions are conservative substitutions.

[0034] 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 Chothia numbering system:

[0035] (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;

[0036] (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;

[0037] (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;

[0038] (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;

[0039] (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; 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-L3 of SEQ ID NO: 65 or a variant thereof; or

[0040] (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;

[0041] 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. In certain embodiments, the substitutions are conservative substitutions.

[0042] 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:

[0043] (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;

[0044] (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;

[0045] (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;

[0046] (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;

[0047] (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-L3 of SEQ ID NO: 65 or a variant thereof; or

[0048] (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;

[0049] 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.

[0050] In certain embodiments, the antibody or antigen-binding fragment thereof comprises framework regions (FRs) from human immunoglobulins.

[0051] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0052] (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;

[0053] (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;

[0054] (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;

[0055] (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;

[0056] (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;

[0057] (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; or,

[0058] 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.

[0059] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention are murine antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0060] In certain embodiments, the antibodies of the present invention or their antigen-binding fragments are selected from full-length antibodies, single-chain antibodies (e.g., scFv, di-scFv or (scFv)2), Fab, Fab', Fab'-SH, (Fab')2, F(ab)'3 fragments, Fv fragments, miniantibodies, disulfide-linked Fv (dsFv), single-domain antibodies (sdAb, nanoantibodies), diabodies, bispecific antibodies and multispecific antibodies.

[0061] In certain embodiments, the VH and VL of the antibodies or antigen-binding fragments thereof of the present invention are connected by one or more linkers. The linker is typically a peptide linker, such as a flexible and / or soluble peptide linker, such as a peptide linker rich in glycine, serine and / or threonine. In some embodiments, the linker also includes charged residues (such as lysine and / or glutamic acid), which can improve solubility. In some embodiments, the linker also includes one or more proline.

[0062] 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.

[0063] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention is a single-chain antibody, such as scFv, di-scFv, or (scFv)2.

[0064] In certain embodiments, the single-chain antibody comprises, from its N-terminus to its C-terminus:

[0065] (1) VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof - linker - VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof;

[0066] (2) VH comprising the sequence shown in SEQ ID NO: 3 or a variant thereof - linker - VL comprising the sequence shown in SEQ ID NO: 4 or a variant thereof;

[0067] (3) VH comprising the sequence shown in SEQ ID NO: 5 or a variant thereof - linker - VL comprising the sequence shown in SEQ ID NO: 6 or a variant thereof;

[0068] (4) VH comprising the sequence shown in SEQ ID NO: 7 or a variant thereof - linker - VL comprising the sequence shown in SEQ ID NO: 8 or a variant thereof;

[0069] (5) VH comprising the sequence shown in SEQ ID NO: 9 or a variant thereof - linker - VL comprising the sequence shown in SEQ ID NO: 10 or a variant thereof;

[0070] (6) VH comprising the sequence shown in SEQ ID NO: 11 or a variant thereof - linker - VL comprising the sequence shown in SEQ ID NO: 12 or a variant thereof;

[0071] (7) VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof - linker - VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof;

[0072] (8) VL comprising the sequence shown in SEQ ID NO: 4 or a variant thereof - linker - VH comprising the sequence shown in SEQ ID NO: 3 or a variant thereof;

[0073] (9) VL comprising the sequence shown in SEQ ID NO: 6 or a variant thereof - linker - VH comprising the sequence shown in SEQ ID NO: 5 or a variant thereof;

[0074] (10) a VL-linker comprising the sequence shown in SEQ ID NO: 8 or a variant thereof-a VH comprising the sequence shown in SEQ ID NO: 7 or a variant thereof;

[0075] (11) a VL comprising the sequence shown in SEQ ID NO: 10 or a variant thereof-a linker-a VH comprising the sequence shown in SEQ ID NO: 9 or a variant thereof; or

[0076] (12) a VL-linker comprising the sequence shown in SEQ ID NO: 12 or a variant thereof-a VH comprising the sequence shown in SEQ ID NO: 11 or a variant thereof;

[0077] 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.

[0078] In certain embodiments, the single-chain antibody comprises an amino acid sequence selected from the group consisting of: (1) an amino acid sequence as set forth in any one of SEQ ID NOs: 86, 88, 90, 92, 94, 82; (2) 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 the amino acid sequence as set forth in any one of SEQ ID NOs: 86, 88, 90, 92, 94, 82; or (3) a sequence that 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) compared to the amino acid sequence as set forth in any one of SEQ ID NOs: 86, 88, 90, 92, 94, 82. In certain embodiments, the substitutions are conservative substitutions.

[0079] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention further comprise a constant region derived from a human immunoglobulin. In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (e.g., κ or λ).

[0080] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, said variant having 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,

[0081] The light chain of the antibody or antigen-binding fragment thereof 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; such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions).

[0082] 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.

[0083] 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.

[0084] Derivatized antibodies

[0085] The antibodies or antigen-binding fragments thereof of the present invention may be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to GPC3 (particularly human GPC3). Therefore, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms. For example, an antibody or antigen-binding fragment thereof of the present invention may be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., to form a bispecific antibody), a detection reagent, a pharmaceutical agent, and / or a protein or polypeptide capable of mediating binding of the antibody or antigen-binding fragment to another molecule (e.g., an avidin or polyhistidine tag).

[0086] As one of the derivatives of the antibody, the present invention provides a conjugate comprising the antibody or antigen-binding fragment thereof of the present invention and a conjugated moiety.

[0087] In certain embodiments, the coupling moiety is selected from a detectable label. The detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In certain embodiments, the detectable label is selected from a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme. In certain embodiments, the detectable label as described above can be linked to the antibody or antigen-binding fragment thereof of the present invention by linkers of varying lengths to reduce potential steric hindrance.

[0088] In certain embodiments, the conjugated moiety is selected from a therapeutic agent. In certain embodiments, the therapeutic agent is preferably an anti-tumor agent, such as a cytotoxic agent, a cytokine, a toxin or a radionuclide.

[0089] In certain embodiments, the conjugated moiety is selected from substances that can improve the biological properties of the antibody (eg, increase serum half-life), for example, a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a sugar group.

[0090] As one of the antibody derivatives, the present invention provides a multispecific antibody comprising the antibody of the present invention or an antigen-binding fragment thereof.

[0091] In certain embodiments, the multispecific antibody comprises an antibody of the present invention or an antigen-binding fragment thereof as a first antigen-binding domain and further comprises at least one second antigen-binding domain directed against another target.

[0092] In certain embodiments, each antigen-binding domain of the multispecific antibody retains its respective original binding specificity.

[0093] In certain embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0094] Antibody preparation

[0095] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0096] 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.

[0097] Therefore, the second aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof, or the heavy chain variable region and / or light chain variable region thereof of the present invention.

[0098] In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:

[0099] (a) the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence of 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 more 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 the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of 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 more 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;

[0100] or

[0101] (b) 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 more 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 the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of 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 more 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;

[0102] or

[0103] (c) 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 more 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 the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of 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 more 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;

[0104] or

[0105] (d) 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 more 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 the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of 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 more 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;

[0106] or

[0107] (e) 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 more 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 the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of 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 more 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;

[0108] or

[0109] (f) the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence set forth in 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 more 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, 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 more 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.

[0110] In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (1) a nucleotide sequence set forth in any one of SEQ ID NOs: 87, 89, 91, 93, 95, and 83; or (2) 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 nucleotide sequence set forth in any one of SEQ ID NOs: 87, 89, 91, 93, 95, and 83.

[0111] 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. In certain embodiments, the vector is an expression vector. In certain embodiments, the vector is an episomal vector. In certain embodiments, the vector is a viral vector. In certain embodiments, the viral vector is a lentiviral vector, an adenoviral vector, or a retroviral vector.

[0112] A fourth aspect of the present invention provides a host cell comprising an isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0113] In another aspect, the present invention also relates to a method for preparing the antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell as described above under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0114] Chimeric Antigen Receptor (CAR)

[0115] 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.

[0116] Therefore, the fifth aspect of the present invention provides a chimeric antigen receptor (CAR) capable of specifically binding to GPC3, which comprises an extracellular antigen binding domain (anti-GPC3 binding domain), a spacer domain, a transmembrane domain and an intracellular signaling domain.

[0117] I. Extracellular Antigen Binding Domain

[0118] The antigen binding domain contained in the chimeric antigen receptor of the present invention confers the ability of the CAR to recognize GPC3.

[0119] In certain embodiments, the antigen-binding domain comprises an anti-GPC3 binding domain, which comprises an antibody or antigen-binding fragment thereof that specifically binds to GPC3 (e.g., human GPC3). In certain embodiments, the antibody or antigen-binding fragment thereof is selected from the antibody or antigen-binding fragment thereof described in the first aspect.

[0120] In certain embodiments, the antibody or antigen-binding fragment thereof is a single-chain antibody, such as scFv, di-scFv, or (scFv)2.

[0121] In certain embodiments, the VH and VL of the antibody or antigen-binding fragment thereof are connected by a linker. In certain embodiments, the linker comprises one or several (e.g., 1, 2, or 3) such as (G m S) n In some embodiments, m is selected from an integer of 1 to 6, and n is selected from an integer of 1 to 6. In some embodiments, m is 3, 4, or 5. In some embodiments, n is 1 or 2. In some embodiments, the linker has the sequence of SEQ ID NO: 110.

[0122] In certain embodiments, the antigen binding domain comprises an amino acid sequence selected from the group consisting of: (1) an amino acid sequence as set forth in any one of SEQ ID NOs: 86, 88, 90, 92, 94, 82; (2) a sequence having 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% identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 86, 88, 90, 92, 94, 82; or (3) a sequence having 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) as compared to the amino acid sequence as set forth in any one of SEQ ID NOs: 86, 88, 90, 92, 94, 82. In certain embodiments, the substitutions are conservative substitutions.

[0123] II. Transmembrane domain

[0124] 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.

[0125] In certain embodiments, the transmembrane domain is selected from the transmembrane region of 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 selected from the transmembrane region of the following proteins: CD8, CD4, PD-1, CD152 and CD154. In certain preferred embodiments, the transmembrane domain comprises the transmembrane region of CD8. In certain preferred embodiments, the transmembrane domain comprises the CD8 transmembrane region whose sequence is shown in SEQ ID NO: 111.

[0126] III. Spacer Domain

[0127] The chimeric antigen receptor of the present invention comprises a spacer domain located between the extracellular antigen-binding domain and the transmembrane domain.

[0128] 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.

[0129] 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.

[0130] In certain embodiments, the hinge domain is a hinge region or portion thereof of a naturally occurring protein. In certain embodiments, the hinge domain comprises a hinge region or portion thereof of CD8, for example, a fragment of at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of a hinge region containing CD8. In certain embodiments, the hinge domain comprises a hinge region of CD8, IgG4, PD-1, CD152, or CD154. In certain exemplary embodiments, the spacer domain comprises the amino acid sequence shown in SEQ ID NO: 112.

[0131] IV. Signal Peptide

[0132] 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.

[0133] In certain embodiments, the CAR of the present invention is 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 by being named signal peptide-2. 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. Preferably, 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).

[0134] V. Intracellular signaling domain

[0135] 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.

[0136] In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and / or a costimulatory signaling domain.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] In certain embodiments, the intracellular signaling domain may comprise the intracellular signaling domain of CD3 zeta and the intracellular signaling domain of CD 137. In certain exemplary embodiments, the intracellular signaling domain of CD3 zeta comprises the amino acid sequence set forth in SEQ ID NO: 113. In certain exemplary embodiments, the intracellular signaling domain of CD 137 comprises the amino acid sequence set forth in SEQ ID NO: 114.

[0143] In certain exemplary embodiments, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO:115.

[0144] VI. Full-length CAR

[0145] 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.

[0146] In certain embodiments, 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).

[0147] In certain embodiments, the spacer domain comprises the hinge region of CD8 (eg, CD8α) (eg, the hinge region of the sequence set forth in SEQ ID NO: 112).

[0148] In certain embodiments, the transmembrane domain comprises the transmembrane region of CD8 (eg, CD8α) (eg, the transmembrane region of the sequence shown in SEQ ID NO: 111).

[0149] 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., as shown in SEQ ID NO: 113), and the costimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., as shown in SEQ ID NO: 114). In certain embodiments, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO: 115.

[0150] 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.

[0151] In certain exemplary embodiments, the signal peptide comprises an IgG1 heavy chain signal peptide or a CD8α signal peptide. In certain exemplary embodiments, the signal peptide comprises a CD8α signal peptide having a sequence as shown in SEQ ID NO: 116.

[0152] In certain exemplary embodiments, the CAR of the present invention comprises an amino acid sequence selected from the group consisting of:

[0153] (1) the amino acid sequence shown in any one of SEQ ID NOs: 117, 119, 121, 123, 125, and 127,

[0154] (2) a sequence that 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 to the amino acid sequence set forth in any one of SEQ ID NOs: 117, 119, 121, 123, 125, and 127, and that substantially retains at least one biological activity of the amino acid sequence from which it is derived (e.g., the ability to direct the specificity and reactivity of immune effector cells to cells expressing MSLN in a non-MHC restricted manner); or

[0155] (3) having 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) compared to the amino acid sequence shown in any one of SEQ ID NOs: 117, 119, 121, 123, 125, 127; in certain embodiments, the substitutions are conservative substitutions.

[0156] Preparation of chimeric antigen receptors

[0157] 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.

[0158] 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.

[0159] 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.

[0160] The seventh aspect of the present invention further provides a nucleic acid construct comprising a nucleic acid sequence encoding the chimeric antigen receptor described in the fifth aspect.

[0161] The eighth aspect of the present invention provides a vector comprising the isolated nucleic acid molecule described in the sixth aspect, or the nucleic acid construct described in the seventh aspect.

[0162] 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.

[0163] In certain embodiments, the vector is an expression vector.

[0164] In certain embodiments, the vector is an episomal vector.

[0165] In certain embodiments, the vector is a viral vector.

[0166] In certain exemplary embodiments, the viral vector is a lentiviral vector, an adenoviral vector, or a retroviral vector.

[0167] In certain embodiments, the vector is an episomal or non-integrating viral vector, such as an integration-defective retrovirus or lentivirus.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] In certain embodiments, in step (1), the immune cells are provided from a patient or a healthy donor and are pretreated; the pretreatment includes sorting, activation and / or proliferation of the 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.

[0175] In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the immune cell by viral infection. 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 a transposon vector system, CRISPR / Cas9 vector, TALEN method, ZFN method, electroporation method, calcium phosphate transfection, DEAE-dextran-mediated transfection or microinjection.

[0176] In certain embodiments, after step (2), the method further comprises: expanding the immune cells obtained in step (2).

[0177] Engineered immune cells

[0178] The preparation method provided by the present invention can transform immune cells derived from patients or healthy donors into immune cells that express CAR that specifically binds to GPC3 and optionally other biologically active molecules.

[0179] Therefore, the eleventh aspect of the present invention also provides a modified immune cell that expresses the CAR of the present invention that specifically binds to GPC3.

[0180] In certain embodiments, the engineered immune cell comprises the isolated nucleic acid molecule of the sixth aspect or a vector comprising the nucleic acid molecule.

[0181] In certain embodiments, the engineered immune cell comprises the nucleic acid construct of the seventh aspect or a vector comprising the nucleic acid construct.

[0182] In certain embodiments, the immune cells are derived from T lymphocytes, NK cells, monocytes, macrophages, or dendritic cells, or any combination thereof, from a patient or a healthy donor. These immune cells are introduced into the isolated nucleic acid molecule of the sixth aspect, the nucleic acid construct of the seventh aspect, or the vector of the eighth aspect by the method provided in the tenth aspect to prepare modified immune cells.

[0183] In certain embodiments, the transcription or expression of one or two target genes among the genes related to immune rejection (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) of the modified immune cells is inhibited, so that the signal transduction mediated by the target genes is blocked in the modified immune cells; 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.

[0184] Immune cell composition

[0185] In the twelfth aspect, the present invention also provides an immune cell composition, which includes the aforementioned modified immune cells, and optional unmodified and / or unsuccessfully modified immune cells, which do not express CARs specific for GPC3. Due to the limitations of the current level of technology and some unknown reasons, not all immune cells can express CARs specific for GPC3 after modification. Moreover, immune cells that do not express CARs also have certain biological activities. Therefore, the immune cell composition can contain immune cells that express and do not express CARs specific for GPC3, and the immune cell composition can still meet the needs of clinical applications.

[0186] In certain embodiments, the engineered immune cells expressing a CAR specific for GPC3 account for approximately 10%-100%, preferably 40%-80%, of the total cell number of the immune cell composition.

[0187] 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.

[0188] In another aspect, the present invention provides a kit for preparing a chimeric antigen receptor that specifically binds to GPC3, or for preparing cells expressing the chimeric antigen receptor, or immune cells co-expressing the chimeric antigen receptor and another bioactive molecule. In certain embodiments, the kit includes the isolated nucleic acid molecule described in the sixth aspect, the nucleic acid construct described in the seventh aspect, or the vector described in the eighth aspect, or the host cell described in the ninth aspect, 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.

[0189] In another aspect, the present invention provides use of the aforementioned kit for preparing a chimeric antigen receptor capable of specifically binding to GPC3, or a cell expressing the chimeric antigen receptor, or an immune cell co-expressing the chimeric antigen receptor and another bioactive molecule.

[0190] Pharmaceutical composition

[0191] In the thirteenth aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in the first aspect of the invention, the chimeric antigen receptor described in the fifth aspect (including a bispecific chimeric antigen receptor or a CAR construct 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 immune cell composition described in the twelfth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0192] 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).

[0193] In certain embodiments, the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the fifth aspect, 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 immune cell composition of the twelfth aspect and the additional pharmaceutically active agent can be administered simultaneously, separately, or sequentially.

[0194] In certain embodiments, the pharmaceutical composition of the present invention comprises: the isolated nucleic acid molecule of the sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the eighth aspect, or the host cell of the ninth aspect.

[0195] In certain embodiments, the pharmaceutical composition of the present invention comprises: the engineered immune cell or immune cell composition of the present invention.

[0196] The antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect, the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the modified immune cell described in the eleventh aspect, or the immune cell composition described in the twelfth 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 route of administration and therapeutic use. The pharmaceutical composition of the present invention should be sterile and stable under the conditions of production and storage. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for ease of 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.

[0197] The antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect, the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the modified immune cell described in the eleventh aspect, or the immune cell composition described in the twelfth aspect can be administered by any suitable method known in the art, including but not limited to, oral, oral, sublingual, ophthalmic, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powder, ointment or drops), or nasal route. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). It will be understood by the skilled person that the route and / or mode of administration will vary depending on the intended purpose. In certain embodiments, the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the fifth aspect, 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 immune cell composition of the twelfth aspect of the present invention is administered by intravenous injection or push injection.

[0198] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the chimeric antigen receptor of the fifth aspect, 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 engineered immune cell of the eleventh aspect, or the immune cell composition of the twelfth aspect. A "prophylactically effective amount" means an amount sufficient to prevent, inhibit, or delay the onset of a disease. A "therapeutically effective amount" means an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the fifth aspect, 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 engineered immune cell of the eleventh aspect, or the immune cell composition of the twelfth aspect of the present invention may vary depending on factors such as 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 method of drug administration, and other concurrently administered therapies.

[0199] Treatment methods and uses

[0200] 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 antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the fifth aspect, 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 engineered immune cell of the eleventh aspect, the immune cell composition of the twelfth aspect, or the pharmaceutical composition of the present invention.

[0201] 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.

[0202] In certain embodiments, the tumor is a GPC3-positive tumor. In certain embodiments, the tumor is selected from solid tumors. In certain embodiments, the tumor is selected from liver cancer (e.g., hepatocellular carcinoma), melanoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, glioma, and ovarian cancer (e.g., ovarian clear cell carcinoma). In certain embodiments, the tumor is selected from hematological tumors; preferably, the hematological tumor is selected from leukemia and lymphoma.

[0203] In certain embodiments, the method comprises administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of the first aspect.

[0204] In certain embodiments, the method comprises administering to the subject an effective amount of the chimeric antigen receptor of the fifth aspect, the isolated nucleic acid molecule of the sixth aspect, the nucleic acid construct of the seventh aspect, the vector of the eighth aspect, the host cell of the ninth aspect, the modified immune cell of the eleventh aspect, or the immune cell composition of the twelfth aspect. In certain embodiments, the host cell is an immune cell (e.g., a human immune cell).

[0205] 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 sixth aspect of the present invention, the nucleic acid construct described in the seventh aspect, or the vector described in the eighth aspect into the immune cells described in step (1) to obtain cells expressing the chimeric antigen receptor and optionally additional biologically active molecules; (3) administering the immune cells obtained in step (2) to the subject for treatment.

[0206] In certain embodiments, the method administers to the subject an immune cell expressing a CAR of the present invention 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.

[0207] 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.

[0208] 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.

[0209] In certain embodiments, the total cell dose comprises 1×10 7 to 10×10 8 CAR-positive immune cells, for example, containing (1-5)×10 7 to (5-10)×10 8 CAR-positive immune cells.

[0210] 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.

[0211] In certain embodiments, the antibody or its antigen binding fragment described in the first aspect of the present invention, the chimeric antigen receptor described in the fifth aspect, the isolated nucleic acid molecule described in the second or 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 described in the eleventh aspect or the immune cell composition described in the twelfth aspect, or the pharmaceutical composition are co-administered with another agent. In certain embodiments, the other agent includes (i) an agent for increasing the efficacy of cells comprising CAR nucleic acids or CAR polypeptides (e.g., immune cells expressing CAR of the present invention, immune cells or immune cell compositions of the present invention); (ii) an agent for improving one or more side effects associated with administering cells comprising CAR nucleic acids or CAR polypeptides (e.g., immune cells expressing CAR of the present invention, immune cells or immune cell compositions of the present invention); (iii) another pharmaceutically active agent with anti-tumor activity. These agents can be administered before, simultaneously with, or after administration of the antibody or antigen-binding fragment thereof described in the first aspect, the chimeric antigen receptor described in the fifth aspect, the isolated nucleic acid molecule described in the second or sixth aspect, the nucleic acid construct described in the seventh aspect, the vector described in the third or eighth aspect, the host cell described in the fourth or ninth aspect, the modified immune cell described in the eleventh aspect, or the immune cell composition described in the twelfth aspect, or the pharmaceutical composition of the present invention.

[0212] 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.

[0213] 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.

[0214] In certain embodiments, the subject can be a mammal, such as a human.

[0215] In another aspect, provided is the use of the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the fifth aspect, 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, the immune cell composition of the twelfth aspect, or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating tumors.

[0216] In another aspect, provided is the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the fifth aspect, 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 immune cell composition of the twelfth aspect, or a pharmaceutical composition for preventing and / or treating tumors.

[0217] The dosage, dosage form, administration route, indications, combination therapy and other aspects of the aforementioned treatment methods can be applied to the use of the drug.

[0218] Abbreviations

[0219] CAR Chimeric Antigen Receptor

[0220] CDR Complementarity determining region in immunoglobulin variable region

[0221] CDR-H1 Complementarity-determining region 1 in the immunoglobulin heavy chain variable region

[0222] CDR-H2 Complementarity-determining region 2 in the immunoglobulin heavy chain variable region

[0223] CDR-H3 Complementarity determining region 3 in the immunoglobulin heavy chain variable region

[0224] CDR-L1 Complementarity-determining region 1 in the immunoglobulin light chain variable region

[0225] CDR-L2 Complementarity determining region 2 in the immunoglobulin light chain variable region

[0226] CDR-L3 Complementarity determining region 3 in the immunoglobulin light chain variable region

[0227] FR Antibody framework region: amino acid residues in the variable region of an antibody other than CDR residues

[0228] VH antibody heavy chain variable region

[0229] VL Antibody light chain variable region

[0230] Kabat The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, eg, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0231] IMGT is based on the international ImMunoGeneTics information system initiated by Lefranc et al. For the Immunofluorescence Immunofluorescence (IMGT) numbering system, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0232] Chothia The immunoglobulin numbering system proposed by Chothia et al. is a classical rule identifying CDR region boundaries based on the position of structural loop regions (see, eg, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0233] AbM The AbM CDR definition method is derived from Martin's related research (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268–9272).

[0234] IL-2 Interleukin-2

[0235] IFN interferon

[0236] PCR polymerase chain reaction

[0237] FACS flow cytometry

[0238] Definition of terms

[0239] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA 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.

[0240] 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.

[0241] The VH and VL regions of an antibody can be further subdivided into regions of high variability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL region 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 / light chain pair form the antigen-binding site, respectively. 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)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0242] 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).

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] 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, scFv comprising NH2-VH-VH-COOH, NH2-VL-VL-COOH. The scFv can form any engineering possible structure, single-chain antibody (scFv), tandem antibody (tandem di-scFvs), bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide bond stabilized Fv protein, camel Ig, IgNAR, etc. In certain embodiments of the present invention, scFv can form di-scFv, which refers to two or more single scFvs connected in series to form an antibody. In certain embodiments of the present invention, scFv can form (scFv)2, which refers to two or more single scFvs connected in parallel to form an antibody.

[0250] As used herein, the term "bifunctional antibody" refers to an antibody fragment with two antigen-binding sites, wherein the fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domain is forced to pair with the complementary domain of another chain, and two antigen-binding sites are generated. Bifunctional antibodies can be bivalent or bispecific. Bifunctional antibodies are more fully described in, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., PNAS USA 90: 6444-6448 (1993). Trifunctional antibodies and tetrafunctional antibodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

[0251] 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.

[0252] 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.

[0253] 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.

[0254] As used herein, the expression "specific binding" or "specific for" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the equilibrium constant for the dissociation 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.

[0255] The specific binding properties between two molecules can be measured using methods well known in the art. One method involves measuring the speed of formation and dissociation of antigen binding sites / antigen complexes. Both "association rate constant" (ka or kon) and "dissociation rate constant" (kdis or koff) can be calculated by concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon and kdis values ​​can be measured by any effective method. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can also be measured using bioluminescence interferometry or Kinexa.

[0256] 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.

[0257] 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).

[0258] 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] As used herein, the term "intracellular signaling domain" refers to a protein portion that conducts effector signaling function signals and guides 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 auxiliary activity, including the secretion of cytokines.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] 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.

[0274] 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.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] Advantageous Effects of the Invention

[0282] The present invention provides antibodies or antigen-binding fragments thereof targeting GPC3, which can be used to prepare chimeric antigen receptors. Immune effector cells expressing the chimeric antigen receptors of the present invention have improved effector functions (e.g., tumor killing activity and cytokine release activity). In addition, in vivo experiments have also demonstrated that immune effector cells expressing chimeric antigen receptors prepared based on the antibodies or antigen-binding fragments of the present invention have a stronger tumor killing effect.

[0283] 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 intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0284] 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, CH3-T) against target cells.

[0285] 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, 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, CH3-T).

[0286] 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.

[0287] Sequence information

[0288] The sequence information involved in the present invention is provided in Table 1 below.

[0289] Table 1: Description of sequences DETAILED DESCRIPTION

[0290] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit it.

[0291] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention can be found in Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)); and ANIMAL CELL CULTURE. CULTURE) (RI Freshney, ed. (1987)).

[0292] In addition, if specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially. It is understood that the examples describe the present invention by way of example and are not intended to limit the scope of the present invention. All publications and other references mentioned herein are incorporated herein by reference in their entirety.

[0293] Example 1. Preparation of human GPC3-targeted scFv antibody

[0294] 1) Phage library screening of GPC3 scFv

[0295] 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.

[0296] 2) ELISA detection of monoclonal phage

[0297] Single colonies were inoculated from the phage panning product titer plate into 96-deep-well plates, and monoclonal phages were detected by ELISA.

[0298] In summary, 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).

[0299] Table 2. Sequences of anti-GPC3 monoclonal antibodies

[0300] 3) Construction of scFv-Fc and analysis of antibody aggregates

[0301] 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.

[0302] Table 3: Structure of scFv

[0303] 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%.

[0304] Table 4. SEC data of 7 scFv-Fc proteins

[0305] 4) GPC3 cell binding assay

[0306] 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 demonstrate that the six candidate scFvs exhibited significantly superior affinity for GPC3-positive 293T / GPC3+ cells compared to the control.

[0307] Table 5. GPC3 cell binding assay results

[0308] Example 2. Construction and preparation of chimeric antigen receptor (CAR) lentiviral expression vector

[0309] 1) Construction of lentiviral plasmid:

[0310] 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.

[0311] Table 6. Structure of chimeric antigen receptors

[0312] 2) Virus packaging:

[0313] The CAR lentiviral plasmid and transfection reagent mixture constructed above was added dropwise to 293T (ATCC) cells. Gently shake the culture dish to mix thoroughly. Place the culture dish in a 37°C, 5% CO2 incubator. After 6-8 hours of incubation, remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium. After 48 hours of continuous incubation, collect the supernatant containing the virus from the culture dish, filter it through a 0.45μm filter, transfer it to a centrifuge tube, balance it, and centrifuge it at 20,000×g at 4°C for 2 hours. After centrifugation, carefully aspirate the liquid from the centrifuge tube in a biosafety cabinet, resuspend the pellet in 500μL of PBS buffer, and store the virus at -80°C.

[0314] Example 3. Preparation of CAR-T cells

[0315] 1) Primary T Cell Isolation: Human PBMCs were isolated using lymphocyte separation medium (GE) and cultured in a 37°C, 5% CO2 incubator. 100 μl / mL of CD3 and CD28 antibodies were added, mixed thoroughly, and incubated at room temperature for 15 minutes. The magnetic beads were removed and pipetted up and down at least 5 times to mix thoroughly. 50 μl of magnetic beads / mL were added to the sample, mixed thoroughly, and incubated at room temperature for 10 minutes. Complete medium was added to the tube to a total volume of 2.5 mL. The tube (opened) was placed in a magnet and allowed to stand at room temperature for 5 minutes. After incubation, the tube, while still in the magnet, was gently inverted to remove the cells. The cells were resuspended in X-vivo 15 medium supplemented with 10% FBS, 300 U / mL IL-2, 5 ng / mL IL-15, and 10 ng / mL IL-7.

[0316] 2) T cell activation:

[0317] Adjust the cell density to 1×10 6 cells / mL, cytokine and antibody complexes (configured at a final concentration of 300 U / mL of IL-2, 10 ng / mL of IL-7, 5 ng / mL of IL-15, 500 ng / mL of Anti-CD3 (OKT3), and 2 μg / mL of Anti-CD28) were added to six-well plates and cultured for 48 hours.

[0318] 3) Viral infection:

[0319] (1) Calculate the required amount of virus based on MOI = 5. The calculation formula is as follows: Required amount of virus (mL) = (MOI * number of cells) / virus titer.

[0320] (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.

[0321] (3) After centrifugation, place the six-well plate in a 37°C 5% CO2 incubator and continue culturing until ready for use.

[0322] (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 before use.

[0323] CAR-T cells expressing the CARs described in Example 2 (CD4-T, CA3-T, CB5-T, CC8-T, CE9-T, and CH3-T) and positive control GC33-T cells were obtained by the above method.

[0324] Example 4. Detection of the positive rate of CAR-T cells

[0325] The nucleic acid sequence encoding CAR is expressed under the drive of the promoter, and the lentiviral transfected T cells are labeled with GPC3 antigen and measured by flow cytometry to reflect the expression level of CAR on the T cell surface. 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 or equal to 10%, indicating that after the lentiviral transfection 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.

[0326] Table 7: CAR positive rate test results

[0327] Example 5. Evaluation of CAR-T cell killing activity against HepG2 target cells

[0328] 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 5Target 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.

[0329] 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.

[0330] Example 6. Cytokine release when CAR-T cells are co-incubated with HepG2 target cells

[0331] Collect HepG2-luc cells and adjust the cell density to 1×10 5 / mL, inoculate target cells in a 96-well plate at a volume of 100 μL / well, and resuspend CAR-T cells, GPC3-CAR and blank T cells 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 test results are shown in Figure 2. 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).

[0332] Example 7. In vivo model to evaluate the killing ability of CAR-T cells on target cells

[0333] Thirty-six B-NDG mice were subcutaneously inoculated with 5 × 10 6HepG2 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.

[0334] 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. An antibody or antigen-binding fragment thereof that specifically binds to GPC3, wherein: The antibody or antigen-binding fragment thereof comprises 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; (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; (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; (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; (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; (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, compared with the heavy chain variable region and / or light chain variable region according to any one of (a) to (f), at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation, and the mutation is a substitution, deletion or addition of one or several 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 IMGT, Kabat, Chothia or AbM numbering systems.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof 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; (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; (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; (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; (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; 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 having a sequence of SEQ ID NO: 26 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 27 or a variant thereof; and CDR-H3 having a sequence of SEQ ID NO: 73 or a variant thereof. variant CDR-H3; 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; 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; (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; (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; (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; (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; 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; 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; (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; (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; (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; (3e) a heavy chain variable region (VH) comprising the following three CDRs: a sequence of SEQ ID NO: 71 or a variant thereof CDR-H1; CDR-H2 of SEQ ID NO: 72 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-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; (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; (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; (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; 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-L3 of SEQ ID NO: 55 or a variant thereof; (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-L3 of SEQ ID NO: 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 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The antibody or antigen-binding fragment thereof 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; (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; (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; (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; (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 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.

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

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The antibody or antigen-binding fragment thereof 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) VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof - linker - VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof; (2) VH comprising the sequence shown in SEQ ID NO: 3 or a variant thereof - linker - VL comprising the sequence shown in SEQ ID NO: 4 or a variant thereof; (3) VH comprising the sequence shown in SEQ ID NO: 5 or a variant thereof - linker - VL comprising the sequence shown in SEQ ID NO: 6 or a variant thereof; (4) VH comprising the sequence shown in SEQ ID NO: 7 or a variant thereof - linker - VL comprising the sequence shown in SEQ ID NO: 8 or a variant thereof; (5) VH comprising the sequence shown in SEQ ID NO: 9 or a variant thereof - linker - VL comprising the sequence shown in SEQ ID NO: 10 or a variant thereof; (6) VH comprising the sequence shown in SEQ ID NO: 11 or a variant thereof - linker - VL comprising the sequence shown in SEQ ID NO: 12 or a variant thereof; (7) VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof - linker - VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof; (8) VL comprising the sequence shown in SEQ ID NO: 4 or a variant thereof - linker - VH comprising the sequence shown in SEQ ID NO: 3 or a variant thereof; (9) VL comprising the sequence shown in SEQ ID NO: 6 or a variant thereof - linker - VH comprising the sequence shown in SEQ ID NO: 5 or a variant thereof; (10) a VL-linker comprising the sequence shown in SEQ ID NO: 8 or a variant thereof-a VH comprising the sequence shown in SEQ ID NO: 7 or a variant thereof; (11) a VL comprising the sequence shown in SEQ ID NO: 10 or a variant thereof-a linker-a VH comprising the sequence shown in SEQ ID NO: 9 or a variant thereof; or (12) a VL-linker comprising the sequence shown in SEQ ID NO: 12 or a variant thereof-a VH comprising the sequence shown 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 antibody or antigen-binding fragment thereof according to claim 5, wherein The single-chain antibody comprises an amino acid sequence selected from the group consisting of: (1) an amino acid sequence as set forth in any one of SEQ ID NOs: 86, 88, 90, 92, 94, and 82; (2) a sequence having 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% identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 86, 88, 90, 92, 94, and 82; or (3) an amino acid sequence as set forth in SEQ ID NOs: The amino acid sequence shown in any one of NOs:86, 88, 90, 92, 94, and 82 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 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region (CH) and a light chain constant region (CL); Preferably, the heavy chain constant region is selected from IgG, IgM, IgE, IgD and IgA; Preferably, the light chain constant region is selected from kappa or lambda.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein The antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen-binding fragment thereof is detectably labeled, such as an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance) or biotin.

9. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof, the heavy chain and / or light chain thereof, or the heavy chain variable region and / or light chain variable region thereof according to any one of claims 1 to 8.

10. The isolated nucleic acid molecule of claim 9, comprising 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: (a) the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence of 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 more 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 the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of 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 more 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; or (b) 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 more 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 the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of 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 more 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; or (c) 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 more 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 the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of 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 more 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; or (d) 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 more 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 the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of 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 more 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; or (e) 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 more 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 the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of 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 more 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; or (f) 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., having at least (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more 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.

11. The isolated nucleic acid molecule of claim 9 or 10, comprising a nucleotide sequence selected from the group consisting of: (1) a nucleotide sequence as set forth in any one of SEQ ID NOs: 87, 89, 91, 93, 95, and 83; or (2) 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 nucleotide sequence as set forth in any one of SEQ ID NOs: 87, 89, 91, 93, 95, and 83.

12. A vector comprising the isolated nucleic acid molecule according to any one of claims 9 to 11; 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.

13. A host cell comprising the isolated nucleic acid molecule of any one of claims 9 to 11, or the vector of claim 12.

14. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising culturing the host cell according to claim 13 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell.

15. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and a conjugated moiety connected thereto; Preferably, the conjugated moiety is selected from a detectable label (such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme) or a therapeutic agent (such as a cytotoxic agent, a cytokine, a toxin or a radionuclide).

16. A multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; Preferably, the multispecific antibody comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 as a first antigen-binding domain, and further comprises at least one second antigen-binding domain directed against another target; Preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

17. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the isolated nucleic acid molecule according to any one of claims 9 to 11, or the vector according to claim 12, or the host cell according to claim 13, or the conjugate according to claim 15, or the multispecific antibody according to claim 16, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity; Preferably, the additional pharmaceutically active agent is selected from the group consisting of: 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, FOLFIRINOX or any combination thereof; Preferably, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as mixed components.

18. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the isolated nucleic acid molecule according to any one of claims 9 to 11, or the vector according to claim 12, or the host cell according to claim 13, or the conjugate according to claim 15, or the multispecific antibody according to claim 16, or the pharmaceutical composition according to claim 17, and optionally instructions for use and / or an administration device.

19. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the isolated nucleic acid molecule according to any one of claims 9 to 11, or the vector according to claim 12, or the host cell according to claim 13, or the conjugate according to claim 15, or the multispecific antibody according to claim 16, or the pharmaceutical composition according to claim 17, 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 liver cancer (e.g., hepatocellular carcinoma), melanoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, glioma and ovarian cancer (e.g., ovarian clear cell carcinoma); Preferably, the tumor is selected from hematological tumors; preferably, the hematological tumor is selected from leukemia and lymphoma; Preferably, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, conjugate, multispecific antibody, or pharmaceutical composition is administered in combination with another pharmaceutically active agent, e.g., simultaneously, separately or sequentially; Preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity; Preferably, the additional pharmaceutically active agent is selected from: 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, FOLFIRINOX or any combination thereof.

20. 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 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the isolated nucleic acid molecule according to any one of claims 9 to 11, or the vector according to claim 12, or the host cell according to claim 13, or the conjugate according to claim 15, or the multispecific antibody according to claim 16, or the pharmaceutical composition according to claim 17; 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 liver cancer (e.g., hepatocellular carcinoma), melanoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, glioma and ovarian cancer (e.g., ovarian clear cell carcinoma); Preferably, the tumor is selected from hematological tumors; preferably, the hematological tumor is selected from leukemia and lymphoma; Preferably, the method further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy, and any combination thereof.

21. A method for detecting the presence or level of GPC3 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 under conditions that allow formation of a complex between the antibody or antigen-binding fragment thereof and GPC3, and detecting formation of the complex; Preferably, the method is used to diagnose a tumor, such as a GPC3-positive tumor, such as liver cancer (e.g., hepatocellular carcinoma), melanoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, glioma, and ovarian cancer (e.g., ovarian clear cell carcinoma), leukemia, lymphoma, or any combination thereof; Preferably, the method comprises detecting the expression level of GPC3 in a test sample from a subject, and comparing the expression level with a reference value, wherein an increase in the expression level compared to the reference value is indicative of a tumor.

22. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the isolated nucleic acid molecule according to any one of claims 9 to 11, or the vector according to claim 12, or the host cell according to claim 13, or the conjugate according to claim 15, or the multispecific antibody according to claim 16 in the preparation of a detection kit for detecting the presence or level of GPC3 in a sample and / or diagnosing a tumor; Preferably, the tumor is a GPC3-positive tumor; Preferably, the tumor is selected from liver cancer (e.g., hepatocellular carcinoma), melanoma, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, renal cell carcinoma, colorectal cancer, gastric cancer, glioma and ovarian cancer (e.g., ovarian clear cell carcinoma), leukemia, lymphoma or any combination thereof.

23. The antibody or antigen-binding fragment thereof of any one of claims 1-8, or the isolated nucleic acid molecule of any one of claims 9-11, or the vector of claim 12, or the host cell of claim 13, or the conjugate of claim 15, or the multispecific antibody of claim 16, or the pharmaceutical composition of claim 17, for use in 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 disease associated with Non-tumor related indications related to 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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