Anti-GPC3 monoclonal / bispecific antibody or antigen-binding fragment thereof and use thereof

Abstract

The present invention provides a monoclonal antibody (in particular a mouse, chimeric or humanized monoclonal antibody) / bispecific antibody or antigen-binding fragment thereof, which specifically binds to human GPC3 with high affinity and functional activity. The invention also provides a nucleotide molecule for coding the antibody, an expression vector and a host cell for expressing the antibody, and a preparation method of the antibody. In addition, the invention also provides a pharmaceutical composition containing the antibody, and application of the antibody in preparation of drugs for treating cancers.

Description

Anti-GPC3 monoclonal antibody / bispecific antibody or antigen-binding fragment thereof and use thereof Technical Field

[0001] The present invention relates to a monoclonal antibody (particularly a mouse, chimeric, or humanized monoclonal antibody) / bispecific antibody, or an antigen-binding fragment thereof, that specifically binds to human GPC3 with high affinity and functional activity. The present invention also provides nucleic acid molecules encoding the antibody or antigen-binding fragment, as well as expression vectors, host cells, and methods for expressing the antibody or antigen-binding fragment. The present invention further provides drug conjugates, monoclonal antibodies, bispecific molecules, and pharmaceutical compositions comprising the antibody or antigen-binding fragment thereof, as well as diagnostic and therapeutic methods using the anti-GPC3 monoclonal antibody / bispecific antibody, or antigen-binding fragment thereof, of the present invention. Background Art

[0002] GPC3 is a heparan sulfate proteoglycan expressed on the surface of various malignant cells, such as hepatocellular carcinoma (HCC) cells. Glypican-3 is linked to the cell surface via a glycosylphosphatidylinositol (GPI) anchor. GPC3 has been shown to be highly expressed in over 70% of HCC biopsies but absent in adjacent non-tumor tissue. Patients with GPC3-positive HCC have significantly lower disease-free survival than those with GPC3-negative HCC.

[0003] It has been discovered that certain types of antibodies that bind to GPC3 have cytostatic activity through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities (International Patent Application WO 2003 / 000883). Furthermore, it has been shown that GPC3 is cleaved in vivo and secreted into the blood as a secreted form of GPC3, and that tumor diagnosis can be performed using antibodies capable of detecting the secreted form of GPC3 (International Patent Applications WO 2004 / 022739, WO 03 / 100429, and WO 2004 / 018667). For example:

[0004] Patent CN1842540B discloses an anti-GPC3 antibody that has higher ADCC and CDC activities than traditional antibodies, but its binding ability to the GPC3 epitope is still weak;

[0005] Patent CN104520331B discloses a high-affinity antibody specific for glypican 3 (GPC3). Although its binding ability to the GPC3 epitope has been improved, its cytotoxicity still needs to be improved.

[0006] Despite this, due to primary or secondary tumor resistance and tumor heterogeneity, it has become increasingly difficult to inhibit tumor growth by relying on single-epitope antibodies and the physiological effects of the antibodies themselves. Bispecific antibodies that simultaneously recognize two epitopes of GPC3 can have stronger binding ability to tumor cells, thereby enabling the development of more lethal drug compositions.

[0007] Antibody-drug conjugates (ADCs) are a novel targeted drug therapy, combining antibodies with highly cytotoxic small-molecule drugs. They combine the potent lethality of small-molecule drugs with the high targeting properties of monoclonal antibodies, making them a hot topic in the research and development of targeted cancer therapy. ADCs generally consist of three components, linked in a specific manner: an antibody or antibody-like ligand, a linker, and a small-molecule drug. The targeting properties of ADCs come from the antibody component, while the toxicity primarily comes from the small-molecule drug, although the antibody component can also be toxic. After binding to tumor cell surface antigens, the antibody component is internalized and then degraded in the lysosome, releasing active chemical toxins that damage DNA or inhibit tumor cell division, ultimately killing the cells. Compared to other therapeutic modalities, ADCs offer the following advantages: strong therapeutic efficacy; high tumor cell specificity, low false negative rates, and a wider therapeutic safety window; low immunogenicity, making them less susceptible to drug resistance; long circulation time in serum (shorter than naked antibodies); and low toxicity to non-target cells.

[0008] Therefore, at present, there is an urgent need to develop an anti-GPC3 monoclonal / bispecific antibody or its antigen-binding fragment, as well as an ADC drug containing the antibody or antigen-binding fragment, which has both strong binding ability to the GPC3 epitope and good cytotoxicity.

[0009] Summary of the Invention

[0010] The present invention provides an anti-GPC3 monoclonal / bispecific antibody or an antigen-binding fragment thereof, such as a mouse, human, chimeric or humanized monoclonal antibody or an antigen-binding fragment thereof, and a bispecific antibody containing two antigen-binding domains. The antibody or antigen-binding fragment thereof binds to the GPC3 protein and has good binding affinity to the GPC3 protein.

[0011] In one aspect, the present invention provides an anti-GPC3 monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof binds to the GPC3 protein.

[0012] In one aspect, the present invention provides an anti-GPC3 monoclonal antibody or an antigen-binding fragment thereof, characterized in that: the anti-GPC3 monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a chain variable region (VL);

[0013] The heavy chain variable region (VH) comprises a CDR region, wherein the CDR region comprises a CDR region identical to any one of the amino acid sequences of SEQ ID Nos: 14 or SEQ ID Nos: 16, wherein the CDR region comprises a CDR1 region, a CDR2 region, and a CDR3 region, wherein the CDR1, CDR2, and CDR3 regions are defined according to IMGT, Kabat, Chothia, AbM, or Contact;

[0014] The light chain variable region (VL) comprises a CDR region, which comprises a CDR region identical to any one of the amino acid sequences of SEQ ID Nos: 15, SEQ ID Nos: 17, SEQ ID Nos: 44, SEQ ID Nos: 45, SEQ ID Nos: 46, SEQ ID Nos: 56, SEQ ID Nos: 57, SEQ ID Nos: 58, SEQ ID Nos: 59 or SEQ ID Nos: 60, and the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, and the CDR1, CDR2 and CDR3 regions are defined according to IMGT, Kabat, Chothia, AbM or Contact.

[0015] In one aspect, as a preferred technical solution of the present invention, the CDR region of the heavy chain variable region (VH) is selected from:

[0016] 1) any group of amino acid residues 26-33, 51-58, and 97-104; or amino acid residues 31-35, 50-66, and 99-104; or amino acid residues 26-32, 52-57, and 99-104; or amino acid residues 26-35, 50-59, and 99-104; or amino acid residues 30-35, 47-59, and 97-103 of SEQ ID Nos: 14;

[0017] 2) any group of amino acid residues 26-33, 51-58, and 97-104; or amino acid residues 31-35, 50-66, and 99-104; or amino acid residues 26-32, 52-57, and 99-104; or amino acid residues 26-35, 50-59, and 99-104; or amino acid residues 30-35, 47-59, and 97-103 of SEQ ID Nos: 16;

[0018] The CDR region of the light chain variable region (VL) is selected from:

[0019] 1) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 15;

[0020] 2) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 17;

[0021] 3) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 44;

[0022] 4) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 45;

[0023] 5) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 46;

[0024] 6) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 56;

[0025] 7) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 57;

[0026] 8) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 58;

[0027] 9) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 59;

[0028] 10) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 60.

[0029] In one aspect, as a preferred technical solution of the present invention, the CDR region of the heavy chain variable region (VH) is selected from:

[0030] 1) any group of amino acid residues 26-33, 51-58, and 97-104; or amino acid residues 31-35, 50-66, and 99-104; or amino acid residues 26-32, 52-57, and 99-104; or amino acid residues 26-35, 50-59, and 99-104; or amino acid residues 30-35, 47-59, and 97-103 of SEQ ID Nos: 14;

[0031] The CDR region of the light chain variable region (VL) is selected from:

[0032] 5) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 15;

[0033] 6) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 44;

[0034] 7) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 45;

[0035] 8) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 46.

[0036] In one aspect, as a preferred technical solution of the present invention, the heavy chain variable region (VH) comprises a sequence identical to the amino acid sequence of SEQ ID Nos: 14 or SEQ ID Nos: 16;

[0037] The light chain variable region (AVL) comprises a sequence identical to the amino acid sequence of SEQ ID Nos: 15, SEQ ID Nos: 17, SEQ ID Nos: 44, SEQ ID Nos: 45, SEQ ID Nos: 46, SEQ ID Nos: 56, SEQ ID Nos: 57, SEQ ID Nos: 58, SEQ ID Nos: 59 or SEQ ID Nos: 60.

[0038] In one aspect, as a preferred technical solution of the present invention, the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof further comprises a heavy chain (H) and a light chain (L), wherein the heavy chain (H) comprises a sequence identical to the amino acid sequence of SEQ ID Nos: 61 or SEQ ID Nos: 71;

[0039] The light chain (L) comprises the same sequence as SEQ ID Nos:62, SEQ ID Nos:63, SEQ ID Nos:64, SEQ ID Nos:65, SEQ ID Nos:66, SEQ ID Nos:67, SEQ ID Nos:68, SEQ ID Nos:69 or SEQ ID Nos:70.

[0040] In one aspect, as a preferred technical solution of the present invention, the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof further comprises a heavy chain (H) and a light chain (L), wherein the heavy chain (H) comprises a sequence identical to the amino acid sequence of SEQ ID Nos: 61;

[0041] The light chain (L) comprises a sequence identical to SEQ ID Nos: 62, SEQ ID Nos: 63, SEQ ID Nos: 64 or SEQ ID Nos: 65.

[0042] In another aspect, the present invention also provides an anti-GPC3 bispecific antibody (e.g., a mouse, chimeric, or humanized antibody) or an antigen-binding fragment thereof, which binds to a GPC3 protein and contains a first GPC3 antigen-binding domain and a second GPC3 antigen-binding domain.

[0043] Furthermore, the first GPC3 antigen-binding domain and the second GPC3 antigen-binding domain may each comprise or consist of the following: (a) a heavy chain variable region (VH) and a light chain variable region (VL); (b) a single-chain antibody (scFv); (c) a diabody; (d) a miniantibody; (e) F(ab')2; (f) F(ab), or other antibody derivatives known in the art.

[0044] Furthermore, the first GPC3 antigen-binding domain and the second GPC3 antigen-binding domain may be or consist of the following: an animal-derived antibody, a humanized antibody, a fully human antibody, a chimeric antibody, or an affinity-optimized antibody. More preferably, it is a humanized antibody, a human-animal chimeric antibody (such as a human-mouse chimeric antibody), and more preferably a fully human antibody. The antibody may be any one or more of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD antibodies, or antibodies of other subtypes. Preferably, it is of the IgG1, IgG2, or IgG4 subtype. More preferably, the IgG1 antibody is an hIgG1 antibody or a variant thereof. In some embodiments, the amino acid sequence of the hIgG1 is as shown in SEQ ID NO: 30, or has 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%, or at least 99% sequence identity with SEQ ID NO: 30.

[0045] Furthermore, the first GPC3 antigen binding domain and the second GPC3 antigen binding domain specifically bind to different epitopes of GPC3;

[0046] Furthermore, in the bispecific anti-GPC3 antibody, the connection relationship between the first GPC3 antigen-binding domain and the second GPC3 antigen-binding domain is as follows (a1) or (a2):

[0047] (a1) the heavy chain variable region and the light chain variable region of the first GPC3 antigen-binding domain are connected via a linker L1 to form an scFv, and the scFv is further connected to the heavy chain or light chain of the second GPC3 antigen-binding domain via a linker L2;

[0048] (a2) the heavy chain variable region and the light chain variable region of the second GPC3 antigen-binding domain are connected via a linker L1 to form an scFv, and the scFv is further connected to the heavy chain or light chain of the first GPC3 antigen-binding domain via a linker L2;

[0049] The linkers L1 and L2 are independently selected from (GGGGS)n, wherein n is an integer of 1, 2, 3, 4, 5 or 6;

[0050] Preferably, the scFv is further connected to the heavy chain or light chain end of the first or second GPC3 antigen binding domain via a linker L2;

[0051] More preferably, the heavy chain or light chain terminus is selected from the heavy chain or light chain C-terminus or N-terminus.

[0052] Still further, the anti-GPC3 bispecific antibody comprises a first polypeptide chain and a second polypeptide chain;

[0053] The structure of the first polypeptide chain can be selected from any one of the following (b1) to (b8):

[0054] (b1)[BVH]-[L1]-[BVL]-[L2]-[AVH]-[CH1]-[Fcx];

[0055] (b2)[BVL]-[L1]-[BVH]-[L2]-[AVH]-[CH1]-[Fcx];

[0056] (b3)[AVH]-[L1]-[AVL]-[L2]-[BVH]-[CH1]-[Fcx];

[0057] (b4)[AVL]-[L1]-[AVH]-[L2]-[BVH]-[CH1]-[Fcx];

[0058] (b5)[BVH]-[CH1]-[Fcx]-[L2]-[AVH]-[L1]-[AVL];

[0059] (b6)[BVH]-[CH1]-[Fcx]-[L2]-[AVL]-[L1]-[AVH];

[0060] (b7)[AVH]-[CH1]-[Fcx]-[L2]-[BVH]-[L1]-[BVL];

[0061] (b8)[AVH]-[CH1]-[Fcx]-[L2]-[BVL]-[L1]-[BVH];

[0062] Wherein, [L1] and [L2] independently represent a linker, and the linker is selected from (GGGGS)n, wherein n is an integer of 1, 2, 3, 4, 5 or 6; the CH1 is the antibody heavy chain constant region 1; the [Fcx] represents the Fc domain;

[0063] [AVH] and [AVL] represent the heavy chain variable region (AVH) and light chain variable region (AVL) of the first GPC3 antigen-binding domain, respectively;

[0064] [BVH] and [BVL] represent the heavy chain variable region (BVH) and light chain variable region (BVL) of the second GPC3 antigen-binding domain, respectively;

[0065] [Fcx] indicates the Fc domain, which includes CH2 and CH3;

[0066] The structure of the second polypeptide chain may be selected from the following (c1) or (c2):

[0067] (c1)[AVL]-[CL];

[0068] (c2)[BVL]-[CL];

[0069] [CL] is the antibody light chain constant region;

[0070] [AVL]-[CL] represents the light chain variable region (AVL) and light chain constant region (CL) of the first GPC3 antigen-binding domain;

[0071] [BVL]-[CL] represents the light chain variable region (BVL) and light chain constant region (CL) of the second GPC3 antigen-binding domain.

[0072] The first polypeptide chain (heavy chain) and the second polypeptide chain (light chain) are connected by a disulfide bond.

[0073] Further, the CDR region of the first heavy chain variable region (AVH) is selected from the CDR region of any one of SEQ ID Nos: 14, 16, 34, 49 or 51, wherein the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, wherein each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact;

[0074] Further, the CDR region of the first light chain variable region (AVL) is selected from the CDR region of any one of SEQ ID Nos: 15, 17, 35, 44, 45, 46, 50, 52, 56, 57, 58, 59 or 60, wherein the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, wherein each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact;

[0075] Further, the CDR region of the second heavy chain variable region (BVH) is selected from the CDR region of any one of SEQ ID Nos: 1, 3, 5, 6, 8, 12, 18, 20, 22, 24, 26, 28, 30, 32 or 47, wherein the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, wherein each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact;

[0076] Further, the CDR region of the second light chain variable region (BVL) is selected from the CDR region of any one of SEQ ID Nos: 2, 4, 7, 9, 10, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33 or 48, and the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, wherein each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact.

[0077] Furthermore, the CDR region of the first heavy chain variable region (AVH) is selected from:

[0078] 1) any group of amino acid residues 26-33, 51-58, and 97-104; or amino acid residues 31-35, 50-66, and 99-104; or amino acid residues 26-32, 52-57, and 99-104; or amino acid residues 26-35, 50-59, and 99-104; or amino acid residues 30-35, 47-59, and 97-103 of SEQ ID Nos: 14 (h1B12);

[0079] 2) any group of amino acid residues 26-33, 51-58, and 97-104; or amino acid residues 31-35, 50-66, and 99-104; or amino acid residues 26-32, 52-57, and 99-104; or amino acid residues 26-35, 50-59, and 99-104; or amino acid residues 30-35, 47-59, and 97-103 of SEQ ID Nos: 16(m1B12);

[0080] Furthermore, the CDR region of the first light chain variable region (AVL) is selected from:

[0081] 1) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 15 (h1B12);

[0082] 2) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 17(m1B12);

[0083] 3) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 44 (h1B12-G34R);

[0084] 4) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 45 (h1B12-G34K);

[0085] 5) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 46 (h1B12-G34A);

[0086] 6) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 56;

[0087] 7) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 57;

[0088] 8) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 58;

[0089] 9) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 59;

[0090] 10) amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or any group of amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 60;

[0091] Furthermore, the CDR region of the second heavy chain variable region (BVH) is selected from:

[0092] 1) any one of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 1(YP7);

[0093] 2) any group of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 3 (hYP7);

[0094] 3) any group of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 5 (hYP7HM);

[0095] 4) any one of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 6(YP8);

[0096] 5) any one of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 8(YP9);

[0097] 6) amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or any group of amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 12 (YP9.1);

[0098] 7) any group of amino acid residues 26-33, 51-58, and 97-103; or amino acid residues 31-35, 50-66, and 99-103; or amino acid residues 26-32, 52-57, and 99-103; or amino acid residues 26-35, 50-59, and 99-103; or amino acid residues 30-35, 47-59, and 97-102 of SEQ ID Nos: 18 (h2D8);

[0099] 8) any group of amino acid residues 26-33, 51-58, and 97-103; or amino acid residues 31-35, 50-66, and 99-103; or amino acid residues 26-32, 52-57, and 99-103; or amino acid residues 26-35, 50-59, and 99-103; or amino acid residues 30-35, 47-59, and 97-102 of SEQ ID Nos: 20(m2D8);

[0100] Furthermore, the CDR region of the second light chain variable region (BVL) is selected from:

[0101] 1) any one of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 2 (YP7);

[0102] 2) any group of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 4 (hYP7);

[0103] 3) any group of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 7(YP8);

[0104] 4) any group of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 9 (YP9 clone 9);

[0105] 5) any one of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 10 (YP9 clone 10);

[0106] 6) any one of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 11 (YP9 clone 1);

[0107] 7) any one of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 13 (YP9.1);

[0108] 8) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 19 (h2D8);

[0109] 9) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 21 (m2D8).

[0110] Preferably, the CDR region of the first heavy chain variable region (AVH) is selected from: amino acid residues 26-33, 51-58 and 97-104; or amino acid residues 31-35, 50-66 and 99-104; or amino acid residues 26-32, 52-57 and 99-104; or amino acid residues 26-35, 50-59 and 99-104; or any one group of amino acid residues 30-35, 47-59 and 97-103 of SEQ ID Nos: 14 (h1B12).

[0111] The CDR region of the first light chain variable region (AVL) is selected from:

[0112] 1) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 15 (h1B12);

[0113] 2) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 44 (h1B12-G34R);

[0114] 3) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 45 (h1B12-G34K);

[0115] 4) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 46 (h1B12-G34A).

[0116] Preferably, the CDR region of the second heavy chain variable region (BVH) is selected from:

[0117] 1) any one of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 1 (YP7);

[0118] 2) any group of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 3 (hYP7);

[0119] 3) any group of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 5 (hYP7HM);

[0120] 4) any group of amino acid residues 26-33, 51-58, and 97-103; or amino acid residues 31-35, 50-66, and 99-103; or amino acid residues 26-32, 52-57, and 99-103; or amino acid residues 26-35, 50-59, and 99-103; or amino acid residues 30-35, 47-59, and 97-102 of SEQ ID Nos: 18 (h2D8);

[0121] 5) any group of amino acid residues 26-33, 51-58, and 97-103; or amino acid residues 31-35, 50-66, and 99-103; or amino acid residues 26-32, 52-57, and 99-103; or amino acid residues 26-35, 50-59, and 99-103; or amino acid residues 30-35, 47-59, and 97-102 of SEQ ID Nos: 20(m2D8);

[0122] Preferably, the CDR region of the second light chain variable region (BVL) is selected from:

[0123] 1) any one of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 2 (YP7);

[0124] 2) any one of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 4 (hYP7)

[0125] 3) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 19 (h2D8);

[0126] 4) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 21 (m2D8).

[0127] Preferably, the CDR region of the first heavy chain variable region (AVH) is selected from the group consisting of amino acid residues 26-33, 51-58, and 97-104; or amino acid residues 31-35, 50-66, and 99-104; or amino acid residues 26-32, 52-57, and 99-104; or amino acid residues 26-35, 50-59, and 99-104; or any one of amino acid residues 30-35, 47-59, and 97-103 of SEQ ID Nos: 14 (h1B12);

[0128] The CDR region of the first light chain variable region (AVL) is selected from the group consisting of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or any one of amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 44 (h1B12-G34R);

[0129] Preferably, the CDR region of the second heavy chain variable region (BVH) is selected from:

[0130] 1) any group of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 5 (hYP7HM);

[0131] 2) any group of amino acid residues 26-33, 51-58, and 97-103; or amino acid residues 31-35, 50-66, and 99-103; or amino acid residues 26-32, 52-57, and 99-103; or amino acid residues 26-35, 50-59, and 99-103; or amino acid residues 30-35, 47-59, and 97-102 of SEQ ID Nos: 18 (h2D8);

[0132] 3) any group of amino acid residues 26-33, 51-58, and 97-103; or amino acid residues 31-35, 50-66, and 99-103; or amino acid residues 26-32, 52-57, and 99-103; or amino acid residues 26-35, 50-59, and 99-103; or amino acid residues 30-35, 47-59, and 97-102 of SEQ ID Nos: 20(m2D8);

[0133] The CDR region of the second light chain variable region (BVL) is selected from:

[0134] 1) any group of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 4 (hYP7);

[0135] 2) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 19 (h2D8);

[0136] 3) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 21 (m2D8).

[0137] Preferably, the CDR region of the first heavy chain variable region (AVH) is selected from: the CDR region of the heavy chain variable region of any one of h1B12 or m1B12, wherein the CDR region includes a CDR1 region, a CDR2 region, and a CDR3 region, and each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact;

[0138] Preferably, the CDR region of the first light chain variable region (AVL) is selected from the CDR region of the light chain variable region of any one of h1B12, h1B12-G34R, h1B12-G34K, h1B12-G34A or m1B12, wherein the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, and each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact;

[0139] Preferably, the CDR region of the second heavy chain variable region (BVH) is selected from the CDR region of the heavy chain variable region of any one of YP7, hYP7, hYP7HM, YP8, YP9, YP9.1, h2D8 or m2D8, wherein the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, and each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact;

[0140] Preferably, the CDR region of the second light chain variable region (BVL) is selected from: the CDR region of the light chain variable region of any one of YP7, hYP7, YP8, YP9, YP9.1, h2D8 or m2D8, and the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, and each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact.

[0141] Preferably, the first heavy chain variable region (AVH) is selected from any one of SEQ ID Nos: 14, 16, 34, 49 or 51;

[0142] Preferably, the first light chain variable region (AVL) region is selected from any one of SEQ ID Nos: 15, 17, 35, 44, 45, 46, 50, 52, 56, 57, 58, 59 or 60;

[0143] Preferably, the second heavy chain variable region (BVH) is selected from any one of SEQ ID Nos: 1, 3, 5, 6, 8, 12, 18, 20, 22, 24, 26, 28, 30, 32 or 47;

[0144] Preferably, the second light chain variable region (BVL) is selected from any one of SEQ ID Nos: 2, 4, 7, 9, 10, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33 or 48.

[0145] Preferably, the first polypeptide chain of the anti-GPC3 bispecific antibody comprises any one of SEQ ID Nos: 36, SEQ ID Nos: 37, SEQ ID Nos: 38, SEQ ID Nos: 39, SEQ ID Nos: 40, SEQ ID Nos: 41, SEQ ID Nos: 42 or SEQ ID Nos: 43.

[0146] Preferably, the second polypeptide chain of the bispecific anti-GPC3 antibody consists of a light chain variable region (VL) and a light chain constant region (CL), wherein VL comprises any one of SEQ ID Nos: 15, SEQ ID Nos: 17, SEQ ID Nos: 44, SEQ ID Nos: 45, SEQ ID Nos: 46, SEQ ID Nos: 56, SEQ ID Nos: 57, SEQ ID Nos: 58, SEQ ID Nos: 59 or SEQ ID Nos: 60, and CL comprises SEQ ID Nos: 55;

[0147] More preferably, the second polypeptide chain of the bispecific anti-GPC3 antibody consists of a light chain variable region (VL) and a light chain constant region (CL), wherein VL comprises: SEQ ID Nos: 15; SEQ ID Nos: 17; SEQ ID Nos: 44; any one of SEQ ID Nos: 45 or SEQ ID Nos: 46, and CL comprises: SEQ ID Nos: 55;

[0148] In another aspect, the present invention further provides a chimeric antigen receptor, characterized in that the chimeric antigen receptor comprises the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof or the anti-GPC3 bispecific antibody or antigen-binding fragment thereof described in the present invention.

[0149] In another aspect, the present invention further provides a polynucleotide encoding the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof, or the anti-GPC3 bispecific antibody or antigen-binding fragment thereof, described herein. The polynucleotide of the present invention may be, for example, DNA or RNA, and may or may not contain intronic sequences. In a preferred embodiment, the polynucleotide is a cDNA molecule.

[0150] In another aspect, the present invention further provides an expression vector comprising the polynucleotide of the present invention, wherein the expression vector comprises a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.

[0151] In another aspect, the present invention further provides a host cell comprising the polynucleotide or expression vector of the present invention. The host cell comprises a prokaryotic cell, yeast, or mammalian cell, such as a CHO cell, NSO cell, or other mammalian cell, preferably a CHO cell.

[0152] In another aspect, the present invention further provides an antibody-drug conjugate, characterized in that it comprises the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof described in the present invention, or the anti-GPC3 bispecific antibody or antigen-binding fragment thereof described in the present invention, and a drug or toxin.

[0153] On the other hand, as a preferred technical solution of the present invention, the drug or toxin is selected from one or more of SN-38, MMAE, PBD dimer, DX-8951 (DXd) or DUBA.

[0154] The anti-GPC3 bispecific antibody can be prepared according to a method comprising the following steps:

[0155] 1) constructing an expression vector, wherein the expression vector is obtained by ligating a nucleotide sequence encoding any of the anti-GPC3 bispecific antibodies described above to a basic vector;

[0156] 2) transfecting or transforming the expression vector constructed in step 1) into host cells, and culturing the host cells;

[0157] 3) Isolation and purification of anti-GPC3 bispecific antibodies.

[0158] The DNA sequence of the anti-GPC3 bispecific antibody or antigen-binding fragment thereof of the present invention can be obtained using conventional techniques, such as hybridoma PCR amplification or phage display library screening. Furthermore, the coding sequences for the light and heavy chains can be fused together to form a single-chain antibody (e.g., scFv).

[0159] Once the relevant sequence is obtained, it can be cloned into a vector, then transferred into host bacteria, and then the relevant vector can be extracted from the host bacteria by conventional methods.

[0160] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is relatively short. Currently, DNA sequences encoding the antibodies of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely through chemical synthesis. In addition, mutations can also be introduced into the protein sequences of the present invention through chemical synthesis.

[0161] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0162] The host cell can be a prokaryotic cell (such as bacteria), a lower eukaryotic cell (such as yeast), or a higher eukaryotic cell (such as mammalian cell). Preferred animal cells include (but are not limited to): CHO and HEK-293 cells.

[0163] The anti-GPC3 bispecific antibodies or antigen-binding fragments thereof of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified using various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose affinity chromatography, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and other conventional separation and purification methods, or a combination of these methods, to obtain the anti-GPC3 bispecific antibodies or antigen-binding fragments thereof of the present invention.

[0164] As a preferred embodiment of the method for preparing the anti-GPC3 bispecific antibody or antigen-binding fragment thereof of the present invention, the method for separating and purifying the anti-GPC3 bispecific antibody or antigen-binding fragment thereof is protein A affinity chromatography, cation exchange method or anion exchange method.

[0165] The resulting monoclonal antibodies or bispecific antibodies can be characterized by conventional means. For example, the binding specificity of the antibody can be determined by immunoprecipitation or in vitro binding assays such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). The binding affinity of the antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0166] The present invention provides an immunoconjugate, comprising:

[0167] 1) the anti-GPC3 bispecific antibody, monoclonal antibody, or antigen-binding fragment thereof according to the present invention; and

[0168] 2) a conjugated moiety selected from the group consisting of a drug, a toxin, a detectable label, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

[0169] Preferably, the radioactive nuclides include:

[0170] (a) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0171] (b) therapeutic isotopes selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0172] Preferably, the conjugated moiety is a drug or a toxin.

[0173] Preferably, the drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In one embodiment, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In the case where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0174] Preferably, the cytotoxic drug is selected from the group consisting of anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, DNA alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0175] Preferred examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids, or combinations thereof.

[0176] Preferably, the toxin is selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansinoids, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxybenzoate, daptomycin, acetaminophen ... anthracnose dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, a Sapaonaria officinalis inhibitor, a glucocorticoid, or a combination thereof.

[0177] Preferably, the drug or toxin is selected from one or more of SN-38 (NK012, CAS No.: 86639-52-3), MMAE (Monomethyl auristatin E, CAS No.: 474645-27-7), PBD dimer (SG3199, CAS No.: 1595275-71-0), DX-8951 (Exatecan, CAS No.: 171335-80-1) or DUBA (duocarmycin-hydroxybenzamide-azaindole).

[0178] The antibody can be conjugated to the drug to form an antibody-drug conjugate (ADC). Typically, the ADC comprises an anti-GPC3 bispecific antibody, monoclonal antibody, or antigen-binding fragment of the present invention, connected to a drug or toxin via a linker. The linker can be a degradable or non-degradable linker. Degradable linkers typically readily degrade in the intracellular environment, thereby releasing the therapeutic agent from the antibody. Suitable degradable linkers include enzymatically degradable linkers, such as linkers containing peptidyl groups that can be degraded by intracellular lysosomal proteases, or sugar linkers, such as linkers containing glucuronides that can be degraded by glucuronidases. Peptide linkers can include dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include pH-sensitive linkers (e.g., hydrazone linkers that hydrolyze at a pH below 5.5) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug when the antibody is hydrolyzed by proteases.

[0179] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Preferred are sulfhydryl-specific active reactive groups, such as maleimides, halogenated amides, halogenated esters, halogenated methyl ketones, benzyl halides, vinyl sulfones, pyridyl disulfides, mercury derivatives, and polymethylene dimethyl sulfide thiosulfonates. The linker can include, for example, a maleimide attached to the antibody via thiosuccinimide.

[0180] Preferably, the linker-connected drug or toxin is selected from the group consisting of: CL2A-SN-38 (CAS No.: 1279680-68-0), mc-vc-PAB-MMAE (CAS No.: 646502-53-6), Tesirine (SG3249, CAS No.: 1595275-62-9), Deruxtecan (CAS No.: 1599440-13-7), and Vc-seco-DUBA (SYD985, CAS No.: 1345681-58-4). The molecular structure is shown in the figure below:

[0181] In the present invention, the anti-GPC3 bispecific antibody or monoclonal antibody is coupled to SN-38 via a CL2A linker.

[0182] In the present invention, the anti-GPC3 bispecific antibody or monoclonal antibody is coupled to MMAE via a mc-VC-PAB linker.

[0183] In the present invention, the anti-GPC3 bispecific antibody or monoclonal antibody is coupled to the PBD dimer via a maleimide-dPEG8-VA-PABA linker.

[0184] In the present invention, the anti-GPC3 bispecific antibody or monoclonal antibody is coupled to DX-8951 (DXd) via a maleimide-GGFG linker.

[0185] In the present invention, the anti-GPC3 bispecific antibody or monoclonal antibody is coupled to DUBA via a Vc-seco linker.

[0186] In the present invention, the antibody-drug conjugate is prepared according to a method comprising the following steps:

[0187] The interchain disulfide bonds of any of the aforementioned anti-GPC3 bispecific antibodies are reduced to generate 2n (e.g., 2, 4, 6, 8) thiol groups;

[0188] The drug-linker compound cross-links with the reduced antibody sulfhydryl group to generate the corresponding antibody drug conjugate;

[0189] The product was further purified by ultrafiltration and desalting.

[0190] In one aspect, the present invention also provides a pharmaceutical composition comprising the anti-GPC3 monoclonal antibody or its antigen-binding fragment or the anti-GPC3 bispecific antibody or its antigen-binding fragment, or an antibody-drug conjugate, or an oncolytic virus according to the present invention, and a pharmaceutically acceptable carrier. When the composition comprises more than one antibody (or its antigen-binding fragment, or antibody-drug conjugate, or oncolytic virus), the antibody (or its antigen-binding fragment, or antibody-drug conjugate, or oncolytic virus) can be administered in batches. The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug, such as an anti-tumor drug.

[0191] The pharmaceutical composition may comprise any number of excipients. Useful excipients include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavorings, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, or combinations thereof. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0192] In another aspect, the present invention provides a nucleic acid molecule encoding an anti-GPC3 bispecific antibody or antigen-binding fragment thereof of the present invention. The nucleic acid molecule can be present in intact cells or cell lysates, or in partially purified or substantially pure form. A nucleic acid is "isolated" or "deemed substantially pure" when it is separated from other cellular components or other contaminants (e.g., other nucleic acids or proteins within the cell) using standard techniques, including alkaline / SDS lysis, cesium chloride density gradient centrifugation (CsCl banding), column chromatography, agarose gel electrophoresis, and other methods known in the art. See F. Ausubel et al. (eds.) (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acid of the present invention can be, for example, DNA or RNA and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0193] In one aspect, the present invention provides a vector comprising the nucleotide sequence of the present invention, including bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0194] Another aspect of the present invention provides a host cell comprising the above-mentioned vector or antibody; the host cell comprises a prokaryotic cell, yeast or mammalian cell, such as a CHO cell, NSO cell or other mammalian cell, preferably a CHO cell.

[0195] The anti-GPC3 bispecific antibodies or antigen-binding fragments thereof disclosed in the present invention can also be used to prepare chimeric antigen receptors (CARs; also known as chimeric T cell receptors, artificial T cell receptors, or chimeric immunoreceptors).

[0196] Another aspect of the present invention provides use of the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof, or anti-GPC3 bispecific antibody or antigen-binding fragment thereof, antibody-drug conjugate, nucleotide, vector, or host cell of the present invention in the preparation of a therapeutic drug or diagnostic reagent for autoimmune diseases, viral infections, or cancer, wherein the cancer is liver cancer, gastric cancer, colorectal cancer, lung cancer, ovarian cancer, or any other type of cancer expressing GPC3.

[0197] The present invention also provides a method for treating a subject suffering from cancer, such as liver cancer, by selecting a subject suffering from a cancer expressing GPC3, and administering to the subject a therapeutically effective amount of an anti-GPC3 bispecific antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate comprising the antibody.

[0198] In another aspect, the present invention provides a method for inhibiting tumor growth in a subject, comprising administering to the subject an effective dose of a medicament comprising the anti-GPC3 bispecific antibody or antigen-binding fragment thereof of the present invention, or a composition thereof. The tumor can be a solid tumor or a non-solid tumor, including but not limited to liver cancer, B-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, melanoma, colon adenocarcinoma, pancreatic cancer, colon cancer, gastrointestinal cancer, prostate cancer, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, and nasopharyngeal carcinoma. In some embodiments, the method comprises administering a composition of the present invention, an anti-GPC3 bispecific antibody or antigen-binding fragment thereof, an antibody-drug conjugate, or an oncolytic virus encoding or carrying the antibody, or a nucleic acid molecule capable of expressing the same in the subject. In some embodiments, at least one additional anti-tumor antibody can be administered in combination with the anti-GPC3 bispecific antibody of the present invention or its antigen-binding fragment, wherein the at least one additional anti-tumor antibody can be selected from anti-VISTA antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, anti-CD40 antibodies, anti-TIM-3 antibodies, anti-STAT3 antibodies and / or anti-ROR1 antibodies. In another embodiment, the antibody of the present invention or its antigen-binding fragment is administered in combination with a chemotherapeutic agent, which can be a cytotoxic agent such as epirubicin, oxaliplatin and / or 5-fluorouracil (5-FU). The antibody of the present invention can be a mouse, human, chimeric or humanized antibody.

[0199] To ensure that the present invention may be more readily understood, certain terms are first defined, with additional definitions set forth throughout the detailed description:

[0200] The term "GPC3" (also known as glypican 3) refers to a member of the heparan sulfate proteoglycan family, anchored to the cell membrane by glycosylphosphatidylinositol (GPI). The human GPC3 gene is located on chromosome X (Xp26) and encodes a 70 kDa protein containing 580 amino acids. This protein is cleaved endonucleolytically by a furin-like convertase between Arg358 and Ser359 to produce a 40 kDa N-terminal subunit and a 30 kDa C-terminal subunit, which also contains two heparan sulfate (HS) chains.

[0201] As used herein, an antibody that "specifically binds to human GPC3" refers to an antibody that binds to human GPC3 protein (and potentially GPC3 proteins from one or more non-human species) but does not substantially bind to non-GPC3 proteins.

[0202] The term "antibody" as used herein includes intact antibodies and any antigen-binding fragments thereof (i.e., "antigen-binding portions") or single chains thereof. An intact antibody is a glycoprotein comprising two heavy (H) chains and two light (L) chains connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), separated by more conserved regions, called framework regions (FRs). Each VH and VL 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 of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.

[0203] The term "bispecific antibody" refers to an antibody molecule that can bind to two separate antigens or has binding specificities for different epitopes within the same antigen. For example, in some embodiments, one antigen-binding fragment of a bispecific antibody molecule binds to the first antigen-binding domain of GPC3, while the other antigen-binding fragment binds to the second antigen-binding domain of GPC3, thereby further enhancing affinity for the cell surface GPC3 antigen.

[0204] As used herein, the term "antigen-binding fragment" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that specifically binds to an antigen (e.g., a GPC3 protein). It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity-determining region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains VL and VH of the FV fragment are encoded by separate genes, they can be connected by a linker using recombinant methods to form a single protein chain, wherein the VL region and the VH region are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also included in the term "antigen-binding fragment" of an antibody. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragment screening for use is the same as that for intact antibodies.

[0205] The term "scFv" refers to a genetically engineered molecule comprising the variable regions of the light and heavy chains of an immunoglobulin, connected by a suitable polypeptide linker as a genetically fused single-chain antibody.

[0206] The term "Fc": The Fc region refers to the Fc fragment region of an antibody molecule, which includes the hinge region, part of the CH2 and CH3 domains, and has a molecular weight of approximately 50,000 Da. When the antibody is digested with papain, the human IgG heavy chain Fc region is from Threonine 225 to the C-terminus.

[0207] The term "IgG1" refers to the constant region of a human or murine heavy chain, wherein "hIgG1" refers to the constant region of a human heavy chain, said "IgG1" or "hIgG1" including the CH1, CH2 and CH3 domains;

[0208] The term "Fab" refers to a fragment comprising a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digesting an intact antibody with the enzyme papain to produce an intact light chain and a portion of one heavy chain.

[0209] The term "Fab'" refers to a fragment of an antibody molecule that can be obtained by treating an intact antibody with pepsin followed by reduction to produce an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule.

[0210] The term "(Fab')2" refers to an antibody fragment that can be obtained by treating an intact antibody with pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments linked together by two disulfide bonds.

[0211] The term "Fv" refers to a gene fragment containing the light chain variable region and the heavy chain variable region expressed as two chains.

[0212] The term "h1B12-G34R, h1B12-G34K, h1B12-G34A, h1B12-G34H, h1B12-G34M, h1B12-G34L, h1B12-G34S or h1B12-N33R" refers to an antibody or antigen-binding fragment thereof in which the 34th position of the light chain of h1B12 is mutated from glycine (G) to R, K, A, H, M, L or S, or the 33rd position of the light chain of h1B12 is mutated from asparagine (N) to R.

[0213] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities. For example, an isolated antibody that specifically binds to a GPC3 protein is substantially free of antibodies that specifically bind to antigens other than GPC3. However, for example, in some embodiments, an isolated antibody that specifically binds to a human GPC3 protein may have cross-reactivity with other antigens (e.g., GPC3 proteins from other species). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemical substances.

[0214] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0215] As used herein, the term "mouse antibody" is intended to include antibodies with variable regions, wherein the framework region and CDR regions are all derived from the immunoglobulin sequences of mouse germline. In addition, if the antibody comprises a constant region, the constant region is also derived from the immunoglobulin sequences of mouse germline. Mouse antibodies of the present invention can include amino acid residues that are not encoded by mouse germline immunoglobulin sequences, such as mutations introduced in vitro by random or site-directed mutagenesis or in vivo by somatic mutation. However, as used herein, the term "mouse antibody" is not intended to include antibodies whose CDR sequences from another mammalian germline have been transplanted onto the mouse framework sequences.

[0216] The term "chimeric antibody" refers to an antibody made by combining genetic material from a non-human source with genetic material from a human. Or more generally, a chimeric antibody is an antibody that has genetic material from one species and genetic material from another species.

[0217] As used herein, the term "humanized antibody" refers to an antibody from a non-human species whose protein sequence has been modified to increase its similarity to naturally occurring antibody variants in humans.

[0218] The term "antibody-drug conjugate" refers to the use of antibodies to specifically recognize specific antigens on the surface of tumor cells, thereby achieving accurate delivery of anti-tumor therapeutic agents (such as cytotoxins or cytostatics, radioactive isotopes, small molecule chemotherapy drugs, etc.) to tumor target cells, resulting in intracellular accumulation and release, to achieve the purpose of accurately killing tumors. ADC is also considered to be the most promising anti-tumor drug because of its suitable molecular weight, high stability, strong targeting, and low toxic side effects. In addition to monoclonal antibodies, bispecific antibodies can also be coupled to therapeutic agents. In some embodiments, the part coupled to the antibody or bispecific antibody of the present invention to form an antibody conjugate is a cytotoxin, which refers to a substance that inhibits or prevents cell function and / or causes cell destruction, and includes small molecule cytotoxins. In some embodiments, the cytotoxin is selected from SN-38, MMAE, PBD dimer, DX-8951 (DXd) or DUBA.

[0219] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, for example, non-human primates, rodents, rabbits, pigs, dogs, cats, chickens, amphibians and reptiles, although mammals such as non-human primates and rodents are preferred.

[0220] The term "therapeutically effective amount" refers to an amount of the anti-GPC3 bispecific antibody or antigen-binding fragment thereof of the present invention sufficient to prevent or ameliorate symptoms associated with a disease or condition (e.g., cancer) and / or lessen the severity of the disease or condition. A therapeutically effective amount should be understood in the context of the condition being treated, wherein those skilled in the art can readily identify the actual effective amount.

[0221] The antibodies of the present invention are monoclonal antibodies that have been structurally and chemically characterized as described below and in the Examples. The amino acid sequence ID numbers of the heavy and light chain variable regions of the antibodies are summarized in Table 1. Some antibodies have the same VH or VL. For example, hYP7 and its mutant hYP7HM have the same light chain (hYP7-L), and h1B12 and its mutants h1B12-G34R, h1B12-G34K, and h1B12-G34A have the same heavy chain (h1B12-H). The heavy chain constant region of the antibody can be, for example, a human IgG1 heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 54, and the light chain constant region can be a human immunoglobulin kappa light chain constant region having the amino acid sequence set forth in SEQ ID NO: 55.

[0222] The heavy chain variable region CDRs and light chain variable region CDRs in Table 1 are defined by the Kabat, Chothia, IMGT, AbM or Contact numbering systems / methods. However, as is known in the art, CDR regions may also be defined by other, such as different, numbering systems based on heavy chain / light chain variable region sequences. The CDR regions of the antibodies of the present invention are detailed in Table 2.

[0223] Table 1 Amino acid sequences of the heavy chain / light chain variable regions of anti-GPC3 bispecific antibodies

[0224] Table 2 Numbering system / method definition of CDRs of the heavy chain variable region and light chain variable region of anti-GPC3 bispecific antibodies

[0225] By the following detailed description and examples, other features and advantages disclosed by the present invention will become apparent, which should not be construed as limiting. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0226] 1) Figure 1. Schematic diagram of bispecific antibody (heavy chain) construction. The heavy chain of the h1B12 antibody (1B12-VH) is linked to different scFV formats of a second antibody (e.g., hYP7, h2D8) via two GGGGS linkers. The linker between the VH and VL variable regions of the second antibody can be three or four GGGGS. YP7-HM refers to YP7-VH with the A68G mutation, and IgG1-CH refers to the heavy chain constant region of human IgG1.

[0227] 2) Figure 2 shows the ELISA binding curves of the bispecific antibody and the humanized monoclonal antibody to human GPC3 protein. Each antibody has high affinity for human GPC3 (EC50 is at the sub-nM level).

[0228] 3) Figures 3A and 3B show the binding of bispecific antibodies and humanized mAbs to human hepatoma cells HepG2 or Hep3B. The bs-FH series of bispecific antibodies (based on h1B12 + h2D8) exhibited higher cell affinity (EC50 values ​​around 2 nM for both HepG2 and Hep3B cells) compared to the bsDH series of bispecific antibodies (based on h1B12 + h2D8) or individual mAbs.

[0229] 4) Figures 4A and 4B show the internalization of bispecific antibodies and humanized mAbs in human hepatoma cells HepG2 or Hep3B. Compared with the bsDH series of bispecific antibodies (based on h1B12 + h2D8) or individual mAbs, the bs-FH series of bispecific antibodies (based on h1B12 + hYP7) exhibited superior antibody internalization.

[0230] 5) Figures 5A and 5B show the binding of the antibody-drug conjugates to human hepatoma cells Hep3B or Huh-7. Compared to the monoclonal antibody-drug conjugate, the bispecific antibody-drug conjugates exhibited higher cellular affinity in both Hep3B and Huh-7 cell lines with high GPC3 antigen expression.

[0231] 6) Figures 6A-6D show the cytotoxicity of antibody-drug conjugates against human hepatocellular carcinoma cells, HepG2, Hep3B, or Huh-7. At both high concentrations (Figures 6A-6C, 66.7 nM) and low concentrations (Figure 6D, 1 nM), the bispecific antibody-drug conjugates demonstrated superior cytotoxicity compared to the monoclonal antibody-drug conjugates.

[0232] 7) Figures 7A and 7B show that unconjugated bispecific antibodies and monoclonal antibodies have no direct killing effect on human liver cancer cells HepG2 (Figure 7A) or Hep3B (Figure 7B).

[0233] 8) Figure 8A shows the ELISA binding curves of humanized mAbs 1B12 and its variants to human GPC3 protein.

[0234] 9) Figures 8B, 8C and 8D show the binding of humanized mAbs 1B12 and its variants to human hepatoma cells HepG2, Hep3B or Huh-7.

[0235] 10) Figure 9 shows the internalization of humanized mAb 1B12 in human hepatoma cell lines Hep3B. Specific embodiments

[0236] The present invention is explained below by means of examples and drawings, but the present invention is not limited thereto.

[0237] Example 1

[0238] Generation of mouse anti-GPC3 monoclonal antibodies using hybridoma technology

[0239] 1.1 Immunization of mice

[0240] Mice were immunized according to the method described in E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988. BALB / c mice were immunized with the human GPC3 protein (Antigen 1), which contains amino acid residues Gln at position 25 to His at position 559 of the extracellular full-length domain. Serum titers were measured after the second and third immunizations, and the two mice with the highest antibody titers were electrofused. Antigen 1 or a peptide antigen containing amino acid residues Ser at position 359 to His at position 559 of the extracellular C-terminal domain (Antigen 7) was used to determine antiserum titers and screen for hybridoma cells secreting antigen-specific antibodies.

[0241] 1.2 Hybridoma cell fusion and screening

[0242] Prior to cell fusion, cells of a murine myeloma cell line (SP2 / 0-Ag14, ATCC #CRL-1581) were cultured to the logarithmic growth phase. Immunized mice were sacrificed, and spleens were removed under sterile conditions according to the method described in Kohler G and Milstein C, "Continuous cultures of fused cells secreting antibodies of predefined specificity," Nature, 256:495-497 (1975). A spleen cell suspension was prepared. Splenic B cells were fused with murine myeloma cells in the logarithmic growth phase using PEG chemistry, and the fused cells were continuously cultured. The fused hybridoma cells were then plated into 96-well plates and cultured in DMEM medium containing 20% ​​FCS / HAT. Viable hybridoma clones were typically observed under a microscope after 7 to 10 days. Two weeks after cell plating, the hybridoma culture supernatant from each well was collected and screened for hybridomas expressing anti-GPC3 antibodies using ELISA using recombinant human GPC3 protein (Antigen 1 and Antigen 7). The specific screening operation steps are as follows: recombinant human GPC3 protein (Antigen 1 or Antigen 7, 2μg / mL) is coated in a 96-well plate, 50μL / well, and incubated at 4°C overnight. After washing the plate three times with PBST, 200μL of blocking solution (PBS+1% BSA) is added to each well and blocked at 37°C for 1 hour. Each antibody is diluted to 2μg / mL with blocking solution, then diluted 3-fold in series, for a total of eight concentrations, and added to the ELISA plate at 50μL / well and incubated at 37°C for 1 hour. Wash the plate three times with PBST, then add pre-diluted Peroxidase-conjugated affinipure F(ab')2fragment goat anti-mouse IgG (H+L) (Jackson, dilute 5000 times with blocking solution according to the instructions), 50μL / well, and incubate at 37°C for 1 hour. Wash the plate three times with PBST and add 1-Step™ Ultra TMB-ELISA Substrate Development Solution (Thermo) at 50 μL / well. Develop at room temperature in the dark for 3-5 minutes. Terminate the reaction with 2M HCl at 50 μL / well. Measure the OD at 450 nm using a microplate reader. Calculate the EC50 value to identify hybridoma clones that secrete antibodies with high specific binding activity.

[0243] Table 3 EC50 of hybridoma antibodies binding to human GPC3 protein

[0244] According to the results in Table 3, it can be seen that all mouse monoclonal antibodies have high affinity to GPC3 protein. Most of them recognize Antigen7, the C-terminal domain of GPC3 protein, while m2G12 recognizes the domain outside the C-terminus.

[0245] Furthermore, ELISA was used to test the recognition of GPC3 proteins from different species by each antibody. Cynomolgus GPC3 (ACRO Biosystems) and Mouse GPC3 (ACRO Biosystems) were coated in 96-well plates at a concentration of 2 μg / mL, with 50 μL / well. The assay was performed according to the aforementioned ELISA protocol. The results are shown in Table 4.

[0246] Table 4 Recognition of monkey and mouse GPC3 proteins by hybridoma antibodies

[0247] Note: √ represents affinity recognition, and × represents no recognition.

[0248] Furthermore, competitive ELISA was used to determine whether each candidate antibody recognized the same epitope as the GC33 antibody. The specific steps are as follows: dilute each antibody to 2μg / mL, add to a 96-well enzyme-labeled plate, 100μL / well, and coat overnight at 4°C. Wash the plate three times with PBST, add blocking solution (PBS+1% BSA), 200μL / well, and block at 37°C for 1h. Add 2μg / mL Insert 1 antigen to each well, 100μL / well, incubate at 37°C for 1h, and wash the plate three times with PBST. Dilute the GC33 antibody to 2μg / mL with blocking solution, then dilute it 3-fold in series, for a total of eight concentrations, and then add the diluted GC33 to the enzyme-labeled plate, 100μL / well, and incubate at 37°C for 1h. Discard the supernatant, wash the plate three times with PBST, add 100 μL / well of diluted peroxidase-conjugated goat anti-human IgG (Jackson, dilute 5000-fold with blocking buffer according to the instructions), and incubate at 37°C for 1 hour. Discard the supernatant, wash the plate three times with PBST, add 50 μL / well of 1-step Ultra TMB-ELISA developer (Thermo), and incubate in the dark for approximately 4 min 30 s. Then, add 50 μL of stop solution (2 M HCl). Measure the OD value at 450 nm using a microplate reader. The analysis results are summarized in Table 5. m1B12, m1H4, and GC33 have identical or overlapping antigen recognition sites.

[0249] Table 5 Antibodies that recognize different epitopes of GPC3

[0250] 1.3 Sequencing and Identification of Hybridoma Antibodies

[0251] Hybridoma antibody variable region genes were isolated by 5' RLM-RACE. Briefly, total RNA was extracted from each candidate hybridoma, and samples were reverse transcribed using the FirstChoice RLM-RACE Kit (Thermo). The heavy and light chain variable region genes were amplified by PCR. The PCR products were cloned into a vector and sequenced. The sequences of the m1B12, m2D8, m2G12, m1B8, m1H4, m36C12, and m37F4 antibodies were obtained (see Table 6).

[0252] Table 6 Anti-GPC3 hybridoma antibody variable region sequences

[0253] Example 2

[0254] Humanization of mouse anti-GPC3 monoclonal antibody

[0255] Mouse anti-GPC3 monoclonal antibodies were selected for humanization and further research. As described below, the mouse monoclonal antibodies were humanized using a proven CDR transplantation method. The light chain variable region and heavy chain variable region sequences of each mouse monoclonal antibody were BLAST-aligned with the human immunoglobulin gene database to select the acceptor framework regions for the humanization of each mouse antibody. The human germline IGVH and IGVK with the highest homology to each mouse antibody were selected as the acceptor frameworks for humanization. The CDRs of the heavy chain variable region or light chain variable region of each mouse antibody were transplanted into the selected human germline framework regions, and the amino acid residues in the framework regions were back-mutated to obtain more candidate heavy chain variable regions and / or light chain variable regions for humanized antibodies. The heavy chain variable region and light chain variable region of each antibody after humanization, as well as the mutation site design, are summarized in Table 7.

[0256] Table 7 Anti-GPC3 humanized antibody variable region sequences

[0257] Example 3

[0258] Preparation of humanized monoclonal antibodies and bispecific antibodies

[0259] 3.1 Vector construction

[0260] First, the nucleotide sequence encoding the human IgG1 heavy chain constant region (IgG1-CH) was connected to the secretion signal peptide (SP) coding sequence at the front end and the termination codon TAG at the back end. The gene was synthesized and cloned into the pUC57-GW-kan vector (Suzhou Jinweizhi Biotechnology Co., Ltd.), and inserted into the downstream of the CMV promoter of the pCDNA3.1 vector by the In-fusion cloning method. Specifically, by setting specific insertion site primers and high-fidelity PCR enzyme (HiFi PCR Premix, TAKARA), the synthesized gene fragment and the pCDNA3.1 vector plasmid fragment were amplified respectively. After gel recovery, the IgG1 heavy chain gene fragment and the linearized vector fragment were connected (In-fusion Snap Assembly Master Mix, TAKARA) to obtain the heavy chain constant region vector pCDNA3.1-IgG1. According to the above implementation method, the coding sequence of the human immunoglobulin kappa light chain constant region (IgG-CK) was synthesized, with the signal peptide (SP) coding sequence at the front and the termination codon TAG at the rear. After gene synthesis, it was inserted downstream of the CMV promoter in the pCDNA3.1 vector to obtain the light chain constant region vector pCDNA3.1-CK.

[0261] For the construction of each humanized monoclonal antibody expression vector, the coding sequences of the respective heavy chain variable region (VH) and light chain variable region (VL) were gene synthesized and inserted between the SP and constant region of the pCDNA3.1-IgG1 or pCDNA3.1-CK vector according to the above-mentioned in-fusion cloning method to obtain the monoclonal antibody heavy chain expression vector and light chain expression vector.

[0262] To construct a bispecific antibody expression vector, the heavy chain variable region nucleotide sequence of the second antibody was linked to the light chain variable region nucleotide sequence via a (GGGGS)3 or (GGGGS)4 linker coding sequence to generate an scFV nucleotide sequence, which was then gene synthesized. The scFV coding sequence was then inserted between the SP and VH regions of the aforementioned monoclonal antibody heavy chain expression vector to generate the bispecific antibody heavy chain expression vector. A schematic diagram of the bispecific antibody heavy chain vector construction is shown in Figure 1.

[0263] For the construction of mutant expression vector, hYP7-HM was constructed by replacing the 68th residue coding sequence of hYP7-VH from GCC to GGC by gene synthesis, thereby obtaining the heavy chain A68G mutation (alanine to glycine). h1B12-VL(G34X) was constructed by gene synthesis by replacing the coding sequence for residue 34 of h1B12-VL from GGC to CGC, AAG, GCC, GTG, ATG, CTG, TAC, CCG, and TCC, resulting in the light chain G34R, G34K, G34A, G34H, G34M, G34L, G34Y, G34P, and G34S mutations (glycine mutated to arginine, lysine, alanine, histidine, methionine, leucine, tyrosine, proline, and serine). Alternatively, the coding sequence for residue 33 of h1B12-VL was replaced from AAC to CGC and CCG, resulting in the light chain N33R and N33P mutations (asparagine mutated to arginine and proline). Vector construction methods are as described previously.

[0264] 3.2 Cell transfection and expression

[0265] according to Transfection was performed according to the instructions of Transfection Reagent (Mirus). ExpiCHO-S cells (Thermo) were cultured in complete medium ( High Yield Expression System (containing 30 mL / L of 10% Poloxamer 188 solution and 20 mL / L of 200 mM L-glutamine, Mirus) was used for subculture 24 hours before transfection to ensure that the cell density was 4 x 10 on the next day. 6 cells / mL, diluted to 2x10 before transfection 6 cells / mL. Add 25 μg of light and heavy chain plasmids at a ratio of 1:1 to 12.5 mL of complete culture medium and mix well. Then add 50 μL of transfection reagent. Gently invert the Transfection Reagent (Mirus) to mix thoroughly, let it sit for 4 minutes, then add it dropwise to the diluted cells (50 mL) while shaking. Add 1 mL of CHOgro-titer Enhancer (Mirus) dropwise. Immediately after transfection, place the cells in a 32°C, 5% CO2 incubator. Add 5% EfficientFeed C+AGT Supplement (Thermo) every other day for a total of 7 days.

[0266] 3.3 Antibody Purification

[0267] After 7 days of culture in a shake flask, the cell supernatant was collected, centrifuged at 4000 rpm for 20 minutes, and the supernatant was taken and filtered with a 0.22 μm filter (Milipore). The antibody was purified by protein A affinity chromatography. Briefly, a HiTrap Mabselect suRe prepacked column (Cytiva) was equilibrated with 20 mM PB + 0.15 M NaCl buffer for 5 to 10 column volumes, and the filtered supernatant was loaded using an AKTA Avant 150 chromatography system (Cytiva). The purification column was then washed with 3 column volumes of 20 mM PB + 0.15 M NaCl buffer, 1 column volume of 20 mM PB + 1 M NaCl buffer, and then washed with 20 mM PB until the baseline was stable. Finally, the antibody was eluted with 20 mM citric acid (adjusted to pH 3.0 with 20 mM sodium citrate), and the peak 200 mAu-200 mAu was collected. The eluted antibody was immediately neutralized with neutralization buffer (1 M Tris-HCl, pH 9.0), placed in a 1.5 mL tube, and frozen at -80 for later use.

[0268] Example 4

[0269] ELISA affinity testing of humanized monoclonal antibodies and bispecific antibodies

[0270] The relative binding activity of each antibody to human GPC3 protein was determined by ELISA. The specific operation was as follows: 100 μL / well of recombinant human GPC3 protein (Antigen 1, 1 μg / mL) was coated in a 96-well plate and incubated at 4°C overnight. The plates were then blocked with 1% BSA in PBST (containing 0.05% Tween-20) for 2 hours at 37°C (200 μL / well), washed three times with PBST, and serially diluted with 1% BSA in PBST before adding each humanized monoclonal antibody / bispecific antibody to a 96-well plate (100 μL / well). The working concentrations of the bispecific antibodies bsFH1-bsFH6, bsDH1, and bsDH2 were 20 nM, 10 nM, 10 nM, and so on, with eight dilutions down by a factor of 4. The working concentrations of the humanized monoclonal antibodies h1B12, h2D8, hYP7HM, h1B12-N33P-G34P, h1B12-G34Y, and GC33 were 30 nM, 15 nM, and so on, with eight dilutions down by a factor of 4. The plates were incubated at 37°C for 1 hour, and washed three times with PBST. Anti-Human IgG-FC-HRP (Sigma, 1 / 30,000 dilution) was then added at 100 μL / well, incubated at 37°C for 1 hour, washed three times with PBST, and then 50 μL of TMB (SURMOPICS) was added. The reaction was terminated with 1 M H₂SO₄, and the OD value was measured at 450 nm-570 nm on a microplate reader. The EC₅0 values ​​for binding of each bispecific antibody and humanized monoclonal antibody to the GPC3 protein are shown in Table 8, and the results are recorded in Figures 2 and 8A.

[0271] Table 8 EC50 of bispecific antibodies binding to human GPC3 protein

[0272] The results in Table 8, Figure 2, and Figure 8A show that, except for h1B12-N33P-G34P-VL and h1B12-G34Y mAbs, all other humanized mAbs and bispecific antibodies have high affinity (sub-nM level) for GPC3 protein.

[0273] Example 5

[0274] Flow cytometry detection of humanized monoclonal antibodies and bispecific antibodies binding to tumor cells

[0275] Flow cytometry was used to determine the affinity of each anti-GPC3 antibody for HepG2, Hep3B, or Huh-7 cells. The specific procedure was as follows: each humanized monoclonal antibody or bispecific antibody was serially diluted in FACS buffer (PBS + 5% FBS) and added to a 96-well U-shaped plate in sequence: bsFH1-bsFH6, bsDH1, bsDH2, h1B12, h1B12-G34R, h1B12-G34K, h1B12-G34A, h1B12-G34H, h1B12-G34M, h1B12-G34L The working concentrations of h1B12-G34S, h1B12-N33R, h1B12-N33P-G34P, h1B12-G34Y and GC33 were: 300nM, 200nM, 200nM and so on, diluted in multiples of 3 for 8 points; the working concentrations of h2D8 and hYP7HM were: 400nM, 200nM, 200nM and so on, diluted in multiples of 3 for 8 points. HepG2 and Hep3B cells were trypsinized, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended in FACS buffer and plated at 2×10 5 Cells were added to a 96-well plate, gently mixed, and incubated on ice for 90 minutes. Centrifuged at 3500 rpm for 3 minutes at 4°C, the supernatant discarded, and the cells were resuspended in 250 μL of pre-chilled FACS buffer. The centrifugation was repeated three times. 100 μL of diluted PE anti-human IgG FC fluorescent secondary antibody (BioLegend, 1 μL / 2 × 10 5 The cells were prepared using a 400 μL flow cytometer (prepared with 100 cells) and incubated on ice in the dark for 60 minutes. The supernatant was discarded and washed twice, and finally the cells were resuspended in 200 μL FACS Buffer. The MFI value was detected on an Attune NxT flow cytometer (Thermo), and the data were processed using GraphPad Prism software. The binding of each bispecific antibody or humanized monoclonal antibody to the cell surface GPC3 antigen is shown in Figures 3A, 8B (HepG2 cells) and Figures 3B, 8C (Hep3B cells), Figure 8D (Huh-7 cells) and Tables 9 and 10. According to the results of Figures 3A-3B, 8B-8D, Tables 9 and 10, it can be seen that, except for the h1B12-G34Y monoclonal antibody, the remaining monoclonal antibodies or bispecific antibodies of the present invention have good affinity for HepG2, Hep3B or Huh-7 cells.

[0276] Table 9 EC50 of monoclonal antibodies binding to HepG2 cells

[0277] Table 10 EC50 of monoclonal antibodies binding to Hep3B cells

[0278] Example 6

[0279] Flow cytometry to detect the internalization efficiency of humanized monoclonal antibodies / bispecific antibodies

[0280] In HepG2 and Hep3B cells, flow cytometry was used to determine the internalization efficiency of each anti-GPC3 antibody. The specific operation was as follows: each humanized monoclonal antibody or bispecific antibody was diluted to 50nM with FACS Buffer and added to a 96-well U-shaped plate at 50μL / well. The binding of antibodies and cells was carried out in the same manner as in Example 5. Each antibody and cell was incubated for different times: the sample incubated on ice was set as the background 0h, and the remaining samples were incubated at 37°C for 1h, 2h, and 4h, respectively. After incubation, they were immediately transferred to ice. 200μL FACS buffer was added to each well and mixed. After centrifugation at 3500rpm for 3min, the supernatant was discarded and 100μL pre-cooled PE anti-human IgG FC fluorescent secondary antibody (BioLegend, 1μL / 2×10 5 The cells were prepared using a 400 μL FACS buffer (prepared with 100 cells) and incubated on ice in the dark for 60 minutes. The supernatant was discarded and washed twice. Finally, the cells were resuspended in 200 μL FACS Buffer, the MFI value was detected on the machine, and the data were processed using GraphPad Prism software. The cellular internalization efficiency of the bispecific antibody or humanized monoclonal antibody was calculated as follows: (1-antibody 37°C MFI value / antibody 4°C MFI value) × 100%. The specific results are shown in Figures 4A (HepG2 cells) and 4B (Hep3B cells). According to Figures 4A and 4B, it can be seen that the bispecific antibody of the present invention has very good internalization efficiency for HepG2 and Hep3B cells, and the internalization rate of the bispecific antibody of the present invention is also faster.

[0281] Example 7

[0282] Flow cytometry to detect the internalization efficiency of humanized monoclonal antibodies / bispecific antibodies

[0283] In Hep3B cells, flow cytometry was used to determine the internalization efficiency of each anti-GPC3 antibody. The specific operation was as follows: each humanized monoclonal antibody or bispecific antibody was diluted to 200nM with FACS Buffer and added to a 96-well U-shaped plate at 50μL / well. The binding of antibodies and cells was carried out in the same manner as in Example 5. Each antibody and cell was incubated for different times: the sample incubated on ice was set as the background 0h, and the remaining samples were incubated at 37°C for 1h, 2h, and 4h, respectively. After incubation, they were immediately transferred to ice. 200μL FACS buffer was added to each well and mixed. After centrifugation at 3500rpm for 3min, the supernatant was discarded and 100μL pre-cooled PE anti-human IgG FC fluorescent secondary antibody (BioLegend, 1μL / 2×10 5 The cells were prepared using a 400 μL FACS buffer (prepared with 100 cells) and incubated on ice in the dark for 60 minutes. The supernatant was discarded and washed twice. Finally, the cells were resuspended in 200 μL FACS Buffer, and the MFI value was detected on the machine. The data were processed using GraphPad Prism software. The cellular internalization efficiency of the bispecific antibody or humanized monoclonal antibody of Example 2 or 3 was calculated as follows: (1-antibody 37°C MFI value / antibody 4°C MFI value) × 100%. The specific results are shown in Figure 9 (Hep3B cells). According to Figure 9, it can be seen that the monoclonal antibody of the present invention has a very good internalization efficiency for Hep3B cells, and the internalization rate of the monoclonal antibody of the present invention is also significantly better than that of GC33.

[0284] Example 8

[0285] Preparation of Antibody-Drug Conjugates

[0286] Replace the monoclonal antibody or bispecific antibody stock solution with 20mM PBS buffer (pH 7.2) and adjust the concentration to approximately 5mg / mL. Add 250mM EDTA solution at a 50:1 volume ratio (antibody:EDTA) and mix thoroughly. Next, add tris(2-carboxyethyl)phosphine hydrochloride (TCEP) at a 1-12-fold molar excess (relative to the antibody), depending on the monoclonal antibody / bispecific antibody and linker-payload (LP) combination. Mix thoroughly and allow the reduction reaction to proceed at room temperature (25°C) for 3 hours. Add an appropriate amount of DMSO to the reaction solution, followed by a 6-12-fold molar excess (relative to the antibody) of the LP drug (5mM / 10mM pre-dissolved in DMSO), ensuring that the volume fraction of DMSO in the reaction system does not exceed 15%. Mix thoroughly and allow the reaction to react at room temperature for 1.5 hours. Then, add N-acetyl-L-cysteine ​​(NAC) solution and allow the reaction to stand at room temperature for 10 minutes to terminate the reaction.

[0287] Ultrafiltration was used for desalting. The reaction solution was transferred to a 10KD ultrafiltration tube (Millipore), supplemented with PBS buffer (pH 6.0), and concentrated to the desired volume by centrifugation at 3500 g. PBS was added and the centrifugation was repeated five times. The product was filtered through a 0.22 μm filter membrane (Millipore) and stored at -80°C.

[0288] After the coupling reaction, various forms of ADC products were obtained. The purity of the ADC products was analyzed by size exclusion chromatography (SEC), and the drug-antibody coupling ratio (DAR) and naked antibody ratio were analyzed by hydrophobic interaction chromatography (HIC).

[0289] Example 9

[0290] Flow cytometry detection of antibody-drug conjugate binding to tumor cells

[0291] Referring to the method of Example 5, flow cytometry was used to determine the affinity of each antibody-drug conjugate for Hep3B and Huh-7 cells. Cell treatment, antibody-drug conjugate dilution and loading, and flow cytometric analysis were all performed as in Example 5. The binding of each antibody-drug conjugate to the cell surface GPC3 antigen is shown in Figure 5A (Hep3B cells) and Figure 5B (Huh-7 cells). The results in Figures 5A and 5B indicate that the monoclonal antibody ADC or bispecific ADC of the present invention has excellent affinity for Hep3B or Huh-7 cells.

[0292] Example 10

[0293] Antibody-drug conjugates for tumor cell killing assay

[0294] The cell cytotoxicity of each antibody-drug conjugate against HepG2, Hep3B, and Huh-7 liver cancer cells was determined using the Cell Counting Kit-8 (Dojindo). Specifically, HepG2, Hep3B, and Huh-7 cells were cultured in 10% FBS (Gibco) + DMEM medium (Corning). When the cell confluence reached 75% or higher, the cells were digested with trypsin (0.25% Trypsin-EDTA) and counted. 1.5 × 10 4Cells / mL were plated at 160 μL / well (2400 cells / well) in a 96-well plate and placed in a 37°C, 5% CO2 incubator. Each antibody-drug conjugate was diluted to 333.5 nM and 5 nM, respectively, using 10% FBS + DMEM medium. 40 μL / well was added to the 96-well plate, resulting in final concentrations of 66.7 nM (high concentration) and 1 nM (low concentration), respectively. Duplicate wells were plated and cultured in a 37°C, 5% CO2 incubator. Tumor cell viability was assessed using the Cell Counting Kit-8 on days 1, 2, 3, and 4. The specific killing results are shown in Figure 6A (HepG2 cells, high concentration 66.7 nM), Figure 6B (Hep3B cells, high concentration 66.7 nM), Figure 6C (Huh-7 cells, high concentration 66.7 nM), and Figure 6D (HepG2 cells, low concentration 1 nM). According to Figures 6A, 6B, 6C and 6D, it can be seen that the monoclonal antibody ADC or bispecific ADC of the present invention has a very good killing effect on HepG2, Hep3B or Huh-7 cells at high or low concentrations.

[0295] Example 11

[0296] Detection of tumor cell killing by monoclonal or bispecific antibodies

[0297] Cell Counting Kit-8 (Dojindo) was used to determine the cytotoxicity of each anti-GPC3 antibody against HepG2 and Hep3B cells. Specific procedures were as follows: using 10% FBS + DMEM medium, each anti-GPC3 antibody was diluted to 400nM, 80nM, 16nM, 3.2nM, 0.64nM, and 0.13nM, respectively, and HepG2 and Hep3B cells were plated at 1×10 4 Cells / mL, 100 μL / well (10,000 cells / well) were added to a 96-well plate. Then, 100 μL / well of the prepared anti-GPC3 antibody was added to the 96-well plate to final concentrations of 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, and 0.06 nM, respectively. Duplicate wells were placed in a 37°C, 5% CO2 incubator for 96 hours. Tumor cell viability was assessed using the Cell Counting Kit-8. The results are shown in Figures 7A (HepG2 cells) and 7B (Hep3B cells). As can be seen in Figures 7A and 7B, the monoclonal antibody alone or the dual antibody combination had little to no killing effect on HepG2 or Hep3B cells.

[0298] Although the present invention has been described by one or more embodiments, it should be understood that the present invention is not limited to these embodiments, and the present description is intended to cover all substitutions, modifications and variations that fall within the spirit and broad scope of the appended claims. All references cited in the present invention are incorporated into the present invention by reference in their entirety.

Claims

1. An anti-GPC3 monoclonal antibody or an antigen-binding fragment thereof, characterized in that: The anti-GPC3 monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a chain variable region (VL); The heavy chain variable region (VH) comprises a CDR region, wherein the CDR region comprises a CDR region identical to the amino acid sequence of SEQ ID Nos: 14 or SEQ ID Nos: 16, wherein the CDR region includes a CDR1 region, a CDR2 region, and a CDR3 region, wherein the CDR1, CDR2, and CDR3 regions are defined according to IMGT, Kabat, Chothia, AbM, or Contact; The light chain variable region (VL) comprises a CDR region, which comprises a CDR region identical to the amino acid sequence of SEQ ID Nos: 15, SEQ ID Nos: 17, SEQ ID Nos: 44, SEQ ID Nos: 45, SEQ ID Nos: 46, SEQ ID Nos: 56, SEQ ID Nos: 57, SEQ ID Nos: 58, SEQ ID Nos: 59 or SEQ ID Nos: 60, and the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, and the CDR1, CDR2 and CDR3 regions are defined according to IMGT, Kabat, Chothia, AbM or Contact.

2. The anti-GPC3 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein: The CDR region of the heavy chain variable region (VH) is selected from: 1) any group of amino acid residues 26-33, 51-58, and 97-104; or amino acid residues 31-35, 50-66, and 99-104; or amino acid residues 26-32, 52-57, and 99-104; or amino acid residues 26-35, 50-59, and 99-104; or amino acid residues 30-35, 47-59, and 97-103 of SEQ ID Nos: 14; 2) any group of amino acid residues 26-33, 51-58, and 97-104; or amino acid residues 31-35, 50-66, and 99-104; or amino acid residues 26-32, 52-57, and 99-104; or amino acid residues 26-35, 50-59, and 99-104; or amino acid residues 30-35, 47-59, and 97-103 of SEQ ID Nos: 16; The CDR region of the light chain variable region (VL) is selected from: 1) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 15; 2) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 17; 3) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 44; 4) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 45; 5) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 46; 6) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 56; 7) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 57; 8) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 58; 9) amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or any group of amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 59; or 10) amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or any group of amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 60; Preferably, the CDR region of the heavy chain variable region (VH) is selected from: 1) any group of amino acid residues 26-33, 51-58, and 97-104; or amino acid residues 31-35, 50-66, and 99-104; or amino acid residues 26-32, 52-57, and 99-104; or amino acid residues 26-35, 50-59, and 99-104; or amino acid residues 30-35, 47-59, and 97-103 of SEQ ID Nos: 14; Preferably, the CDR region of the light chain variable region (VL) is selected from: 1) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 15; 2) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 44; 3) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 45; 4) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos:

46.

3. The anti-GPC3 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The heavy chain variable region (VH) comprises a sequence identical to the amino acid sequence of SEQ ID Nos: 14 or SEQ ID Nos: 16; The light chain variable region (AVL) comprises an amino acid sequence identical to SEQ ID Nos: 15, SEQ ID Nos: 17, SEQ ID Nos: 44, SEQ ID Nos: 45, or SEQ ID Nos: 46, SEQ ID Nos: 56, SEQ ID Nos: 57, SEQ ID Nos: 58, SEQ ID Nos: 59, or SEQ ID Nos: 60; Preferably, the heavy chain variable region (VH) comprises a sequence identical to the amino acid sequence of SEQ ID Nos: 14; Preferably, the light chain variable region (AVL) comprises a sequence identical to the amino acid sequence of SEQ ID Nos: 15, SEQ ID Nos: 17, SEQ ID Nos: 44, SEQ ID Nos: 45, SEQ ID Nos: 46, SEQ ID Nos: 56, SEQ ID Nos: 57, SEQ ID Nos: 58, SEQ ID Nos: 59 or SEQ ID Nos:

60.

4. The anti-GPC3 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The anti-GPC3 monoclonal antibody or antigen-binding fragment thereof further comprises a heavy chain (H) and a light chain (L), wherein the heavy chain (H) comprises a sequence identical to the amino acid sequence of SEQ ID Nos: 61 or SEQ ID Nos: 71; The light chain (L) comprises a sequence identical to SEQ ID Nos: 62, SEQ ID Nos: 63, SEQ ID Nos: 64, SEQ ID Nos: 65, SEQ ID Nos: 66, SEQ ID Nos: 67, SEQ ID Nos: 68, SEQ ID Nos: 69 or SEQ ID Nos: 70; Preferably, the heavy chain (H) comprises a sequence identical to the amino acid sequence of SEQ ID Nos: 61 or SEQ ID Nos: 71; Preferably, the light chain (L) comprises a sequence identical to SEQ ID Nos: 62, SEQ ID Nos: 63, SEQ ID Nos: 64 or SEQ ID Nos:

65.

5. An anti-GPC3 bispecific antibody or an antigen-binding fragment thereof, characterized in that: The bispecific anti-GPC3 antibody or antigen-binding fragment thereof contains a first GPC3 antigen-binding domain and a second GPC3 antigen-binding domain. The first GPC3 antigen binding domain comprises a first heavy chain variable region (AVH) and a first light chain variable region (AVL); The second GPC3 antigen binding domain comprises a second heavy chain variable region (BVH) and a second light chain variable region (BVL); The CDR region of the first heavy chain variable region (AVH) is selected from the CDR region of any one of SEQ ID Nos: 14, 16, 34, 49 or 51, wherein the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, wherein each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact; The CDR region of the first light chain variable region (AVL) is selected from the CDR region of any one of SEQ ID Nos: 15, 17, 35, 44, 45, 46, 50, 52, 56, 57, 58, 59 or 60, wherein the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, wherein each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact; The CDR region of the second heavy chain variable region (BVH) is selected from the CDR region of any one of SEQ ID Nos: 1, 3, 5, 6, 8, 12, 18, 20, 22, 24, 26, 28, 30, 32 or 47, wherein the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, wherein each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact; The CDR region of the second light chain variable region (BVL) is selected from the CDR region of any one of SEQ ID Nos: 2, 4, 7, 9, 10, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33 or 48, and the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region, wherein each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact.

6. The anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The CDR region of the first heavy chain variable region (AVH) is selected from: 1) any group of amino acid residues 26-33, 51-58, and 97-104; or amino acid residues 31-35, 50-66, and 99-104; or amino acid residues 26-32, 52-57, and 99-104; or amino acid residues 26-35, 50-59, and 99-104; or amino acid residues 30-35, 47-59, and 97-103 of SEQ ID Nos: 14 (h1B12); 2) any group of amino acid residues 26-33, 51-58, and 97-104; or amino acid residues 31-35, 50-66, and 99-104; or amino acid residues 26-32, 52-57, and 99-104; or amino acid residues 26-35, 50-59, and 99-104; or amino acid residues 30-35, 47-59, and 97-103 of SEQ ID Nos: 16(m1B12); The CDR region of the first light chain variable region (AVL) is selected from: 1) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 15 (h1B12); 2) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 17(m1B12); 3) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 44 (h1B12-G34R); 4) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 45 (h1B12-G34K); 5) amino acid residues 27-37, 55-56, and 94-102 of SEQ ID Nos: 46 (h1B12-G34A); or amino acid residues 24-39, 55-61, and 94-102 amino acid residues; or any group of amino acid residues 30-41, 51-60, and 94-101; 6) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 56; 7) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 57; 8) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 58; 9) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 59; 10) amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or any group of amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 60; The CDR region of the second heavy chain variable region (BVH) is selected from: 1) any one of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 1(YP7); 2) any group of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 3 (hYP7); 3) any group of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 5 (hYP7HM); 4) any one of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 6(YP8); 5) any one of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 8(YP9); 6) amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or any group of amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 12 (YP9.1); 7) any group of amino acid residues 26-33, 51-58, and 97-103; or amino acid residues 31-35, 50-66, and 99-103; or amino acid residues 26-32, 52-57, and 99-103; or amino acid residues 26-35, 50-59, and 99-103; or amino acid residues 30-35, 47-59, and 97-102 of SEQ ID Nos: 18 (h2DS); 8) any group of amino acid residues 26-33, 51-58, and 97-103; or amino acid residues 31-35, 50-66, and 99-103; or amino acid residues 26-32, 52-57, and 99-103; or amino acid residues 26-35, 50-59, and 99-103; or amino acid residues 30-35, 47-59, and 97-102 of SEQ ID Nos: 20(m2D8); The CDR region of the second light chain variable region (BVL) is selected from: 1) any one of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 2 (YP7); 2) any group of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 4 (hYP7); 3) any group of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 7(YP8); 4) any group of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 9 (YP9 clone 9); 5) any one of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 10 (YP9 clone 10); 6) any one of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 11 (YP9 clone 1); 7) any one of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 13 (YP9.1); 8) amino acid residues 27-37, 55-56, and 94-102; or any group of amino acid residues 24-39, 55-61, and 94-102, or 30-41, 51-60, and 94-101 of SEQ ID Nos: 19 (h2D8); 9) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 21 (m2D8).

7. The anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 or 6, characterized in that: The CDR region of the first heavy chain variable region (AVH) is selected from the group consisting of amino acid residues 26-33, 51-58, and 97-104; or amino acid residues 31-35, 50-66, and 99-104; or amino acid residues 26-32, 52-57, and 99-104; or amino acid residues 26-35, 50-59, and 99-104; or any one of amino acid residues 30-35, 47-59, and 97-103 of SEQ ID Nos: 14 (h1B12); The CDR region of the first light chain variable region (AVL) is selected from: 1) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 15 (h1B12); 2) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 44 (h1B12-G34R); 3) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 45 (h1B12-G34K); 4) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 46 (h1B12-G34A); The CDR region of the second heavy chain variable region (BVH) is selected from: 1) any one of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 1(YP7); 2) any group of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 3 (hYP7); 3) any group of amino acid residues 26-33, 51-60, and 99-106; or amino acid residues 31-35, 50-68, and 101-106; or amino acid residues 26-32, 52-59, and 101-106; or amino acid residues 26-35, 50-61, and 101-106; or amino acid residues 30-35, 47-61, and 99-105 of SEQ ID Nos: 5 (hYP7HM); 4) any group of amino acid residues 26-33, 51-58, and 97-103; or amino acid residues 31-35, 50-66, and 99-103; or amino acid residues 26-32, 52-57, and 99-103; or amino acid residues 26-35, 50-59, and 99-103; or amino acid residues 30-35, 47-59, and 97-102 of SEQ ID Nos: 18 (h2D8); 5) any group of amino acid residues 26-33, 51-58, and 97-103; or amino acid residues 31-35, 50-66, and 99-103; or amino acid residues 26-32, 52-57, and 99-103; or amino acid residues 26-35, 50-59, and 99-103; or amino acid residues 30-35, 47-59, and 97-102 of SEQ ID Nos: 20(m2D8); The CDR region of the second light chain variable region (BVL) is selected from: 1) any one of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 2 (YP7); 2) any group of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 4 (hYP7); 3) amino acid residues 27-37, 55-56, and 94-102; or any group of amino acid residues 24-39, 55-61, and 94-102, or 30-41, 51-60, and 94-101 of SEQ ID Nos: 19 (h2D8); 4) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 21 (m2D8).

8. The anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 7, characterized in that: The CDR region of the first heavy chain variable region (AVH) is selected from the group consisting of amino acid residues 26-33, 51-58, and 97-104; or amino acid residues 31-35, 50-66, and 99-104; or amino acid residues 26-32, 52-57, and 99-104; or amino acid residues 26-35, 50-59, and 99-104; or any one of amino acid residues 30-35, 47-59, and 97-103 of SEQ ID Nos: 14 (h1B12); The CDR region of the first light chain variable region (AVL) is selected from the group consisting of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or any one of amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 44 (h1B12-G34R); The CDR region of the second heavy chain variable region (BVH) is selected from: 1) amino acid residues 26-33, 51-60, and 99-106 of SEQ ID Nos: 5 (hYP7HM); or amino acid residues 31-35, 50-68, and 101-106 amino acid residues at positions 26-32, 52-59, and 101-106; or amino acid residues at positions 26-35, 50-61, and 101-106; or any group of amino acid residues at positions 30-35, 47-61, and 99-105; 2) any group of amino acid residues 26-33, 51-58, and 97-103; or amino acid residues 31-35, 50-66, and 99-103; or amino acid residues 26-32, 52-57, and 99-103; or amino acid residues 26-35, 50-59, and 99-103; or amino acid residues 30-35, 47-59, and 97-102 of SEQ ID Nos: 18 (h2D8); 3) any group of amino acid residues 26-33, 51-58, and 97-103; or amino acid residues 31-35, 50-66, and 99-103; or amino acid residues 26-32, 52-57, and 99-103; or amino acid residues 26-35, 50-59, and 99-103; or amino acid residues 30-35, 47-59, and 97-102 of SEQ ID Nos: 20(m2D8); The CDR region of the second light chain variable region (BVL) is selected from: 1) any group of amino acid residues 27-38, 56-57, and 95-103; or amino acid residues 24-40, 56-62, and 95-103; or amino acid residues 30-42, 52-61, and 95-102 of SEQ ID Nos: 4 (hYP7); 2) amino acid residues 27-37, 55-56, and 94-102; or any group of amino acid residues 24-39, 55-61, and 94-102, or 30-41, 51-60, and 94-101 of SEQ ID Nos: 19 (h2D8); 3) any group of amino acid residues 27-37, 55-56, and 94-102; or amino acid residues 24-39, 55-61, and 94-102; or amino acid residues 30-41, 51-60, and 94-101 of SEQ ID Nos: 21 (m2D8).

9. The anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 8, characterized in that: The CDR region of the first heavy chain variable region (AVH) is selected from the CDR region of the heavy chain variable region of any one of h1B12 or m1B12, wherein the CDR region includes a CDR1 region, a CDR2 region, and a CDR3 region; The CDR region of the first light chain variable region (AVL) is selected from the CDR region of the light chain variable region of any one of h1B12, h1B12-G34R, h1B12-G34K, h1B12-G34A, h1B12-G34H, h1B12-G34M, h1B12-G34L, h1B12-G34S, h1B12-N33R or m1B12, and the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region; The CDR region of the second heavy chain variable region (BVH) is selected from the CDR region of the heavy chain variable region of any one of YP7, hYP7, hYP7HM, YP8, YP9, YP9.1, h2D8 or m2D8, wherein the CDR region includes a CDR1 region, a CDR2 region and a CDR3 region; The CDR region of the second light chain variable region (BVL) is selected from the CDR region of any one of the light chain variable regions of YP7, hYP7, YP8, YP9, YP9.1, h2D8 or m2D8, and the CDR region includes CDR1, CDR2 and CDR3 regions.

10. The anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 9, characterized in that: The first heavy chain variable region (AVH) is selected from any one of SEQ ID Nos: 14, 16, 34, 49 or 51; The first light chain variable region (AVL) is selected from any one of SEQ ID Nos: 15, 17, 35, 44, 45, 46, 50, 52, 56, 57, 58, 59 or 60; The second heavy chain variable region (BVH) is selected from any one of SEQ ID Nos: 1, 3, 5, 6, 8, 12, 18, 20, 22, 24, 26, 28, 30, 32 or 47; The second light chain variable region (BVL) is selected from any one of SEQ ID Nos: 2, 4, 7, 9, 10, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33 or 48.

11. The anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 10, characterized in that: The connection relationship between the first GPC3 antigen-binding domain and the second GPC3 antigen-binding domain is as follows (a1) or (a2): (a1) the heavy chain variable region and the light chain variable region of the first GPC3 antigen-binding domain are connected via a linker L1 to form an scFv, and the scFv is further connected to the heavy chain or light chain of the second GPC3 antigen-binding domain via a linker L2; (a2) the heavy chain variable region and the light chain variable region of the second GPC3 antigen-binding domain are connected via a linker L1 to form an scFv, and the scFv is further connected to the heavy chain or light chain of the first GPC3 antigen-binding domain via a linker L2; The linkers L1 and L2 are independently selected from (GGGGS)n, wherein n is an integer of 1, 2, 3, 4, 5 or 6; Preferably, the scFv is further connected to the heavy chain or light chain end of the first or second GPC3 antigen binding domain via a linker L2; More preferably, the heavy chain or light chain terminus is selected from the heavy chain or light chain C-terminus or N-terminus.

12. The anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 11, wherein: The bispecific anti-GPC3 antibody comprises a first polypeptide chain and a second polypeptide chain. The structure of the first polypeptide chain (heavy chain) can be selected from any one of the following (b1) to (b8): (b1)[BVH]-[L1]-[BVL]-[L2]-[AVH]-[CH1]-[Fcx]; (b2)[BVL]-[L1]-[BVH]-[L2]-[AVH]-[CH1]-[Fcx]; (b3)[AVH]-[L1]-[AVL]-[L2]-[BVH]-[CH1]-[Fcx]; (b4)[AVL]-[L1]-[AVH]-[L2]-[BVH]-[CH1]-[Fcx]; (b5)[BVH]-[CH1]-[Fcx]-[L2]-[AVH]-[L1]-[AVL]; (b6)[BVH]-[CH1]-[Fcx]-[L2]-[AVL]-[L1]-[AVH]; (b7)[AVH]-[CH1]-[Fcx]-[L2]-[BVH]-[L1]-[BVL]; (b8)[AVH]-[CH1]-[Fcx]-[L2]-[BVL]-[L1]-[BVH]; Wherein, [L1] and [L2] independently represent linkers, and the linkers are selected from (GGGGS)n, wherein n is an integer of 1, 2, 3, 4, 5 or 6; the CH1 is the CH1 of the antibody heavy chain constant region; the [Fcx] represents the Fc domain of the antibody heavy chain constant region, comprising CH2 and CH3; The structure of the second polypeptide chain (light chain) can be selected from the following (c1) or (c2): (c1)[AVL]-[CL]; (c2)[BVL]-[CL]; The [CL] is the antibody light chain constant region.

13. The anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 12, wherein: The first polypeptide chain of the anti-GPC3 bispecific antibody comprises any one of SEQ ID Nos: 36, SEQ ID Nos: 37, SEQ ID Nos: 38, SEQ ID Nos: 39, SEQ ID Nos: 40, SEQ ID Nos: 41, SEQ ID Nos: 42, or SEQ ID Nos:

43.

14. The anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 8 to 13, wherein: The second polypeptide chain of the bispecific anti-GPC3 antibody consists of a light chain variable region (VL) and a light chain constant region (CL), wherein VL comprises any one of SEQ ID Nos: 15, SEQ ID Nos: 17, SEQ ID Nos: 44, SEQ ID Nos: 45, SEQ ID Nos: 46, SEQ ID Nos: 56, SEQ ID Nos: 57, SEQ ID Nos: 58, SEQ ID Nos: 59, or SEQ ID Nos: 60, and CL comprises SEQ ID Nos: 55; More preferably, the VL comprises: any one of SEQ ID Nos: 15; SEQ ID Nos: 17; SEQ ID Nos: 44; SEQ ID Nos: 45 or SEQ ID Nos: 46, and the CL comprises: SEQ ID Nos:

55.

15. The anti-GPC3 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 14, characterized in that: The anti-GPC3 monoclonal antibody or antigen-binding fragment thereof or the anti-GPC3 bispecific antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

16. A pharmaceutical composition, characterized in that The invention comprises the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 14, and a pharmaceutically acceptable carrier.

17. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 14.

18. A polynucleotide, characterized in that The polynucleotide encodes the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 14.

19. An expression vector, characterized in that The expression vector comprises the polynucleotide of claim 18.

20. A host cell, characterized in that The host cell comprises the polynucleotide of claim 18 or the expression vector of claim 19.

21. An antibody-drug conjugate, characterized in that The invention comprises the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 14, and a drug or toxin.

22. The antibody-drug conjugate according to claim 21, wherein The drug or toxin is selected from one or more of SN-38, MMAE, PBD dimer, DX-8951 (DXd) or DUBA.

23. A pharmaceutical composition, characterized in that The invention comprises the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 14, or the antibody-drug conjugate according to claim 21 or 22, and one or more pharmaceutically acceptable carriers.

24. Use of the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 14, the pharmaceutical composition according to claim 16, or the antibody-drug conjugate according to claim 21 or 22 in the preparation of a medicament for treating or preventing cancer, preferably liver cancer.

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