Anti-gpc3 monoclonal antibodies / bispecific antibodies or antigen-binding fragments thereof and uses thereof

Abstract

This invention provides a monoclonal antibody (particularly a mouse, chimeric, or humanized monoclonal antibody) / bispecific antibody or an antigen-binding fragment thereof, which specifically binds to human GPC3 with high affinity and functional activity. The invention also provides a nucleotide molecule encoding the antibody, an expression vector and host cell for expressing the antibody, and a method for preparing the antibody. Furthermore, the invention provides pharmaceutical compositions comprising the antibody, and the use of the antibody in the preparation of a medicament for treating cancer.

Description

Technical Field

[0001] This invention relates to a monoclonal antibody (particularly mouse, chimeric, or humanized monoclonal antibodies) / bispecific antibody or an antigen-binding fragment thereof, which specifically binds to human GPC3 with high affinity and functional activity. The invention also provides nucleic acid molecules encoding the antibody or antigen-binding fragment, expression vectors, host cells, and methods for expressing the antibody or antigen-binding fragment. Furthermore, the invention 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 of the invention. Background Technology

[0002] GPC3 is a heparan sulfate proteoglycan expressed on the surface of various malignant cells, such as hepatocellular carcinoma (HCC) cells. Glypican-3 is attached to the cell surface via a glycosylphosphatidylinositol anchor (GPI). GPC3 has been shown to be highly expressed in over 70% of HCC biopsy tissues, but not in adjacent non-tumor tissues. Patients with GPC3-positive HCC have significantly lower disease-free survival than those with GPC3-negative HCC.

[0003] Antibodies binding to GPC3 have been found to possess cell growth-inhibitory activity through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (International Patent Application WO 2003 / 000883). Furthermore, it has been shown that GPC3 cleaves in vivo and is secreted into the bloodstream in its secretory form, and that antibodies capable of detecting the secretory form of GPC3 can be used for tumor diagnosis (International Patent Applications WO2004 / 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 compared with traditional antibodies, but its binding ability to GPC3 epitopes is still relatively weak.

[0005] Patent CN104520331B discloses a high-affinity antibody specifically targeting phosphatidylinositol proteoglycan 3 (GPC3). Although its binding ability to GPC3 epitopes has been improved, its cytotoxicity still needs to be improved.

[0006] Nevertheless, due to drug resistance in primary or secondary tumors and tumor heterogeneity, it is becoming increasingly difficult to inhibit tumor growth by relying on single epitope antibodies and the physiological effects of the antibodies themselves. However, bispecific antibodies that recognize two GPC3 epitopes can have a stronger binding ability to tumor cells, thus enabling the development of drug compositions with stronger killing power.

[0007] Antibody-drug conjugates (ADCs) are a novel targeted drug therapy method. They consist of antibodies conjugated to small-molecule drugs with strong cytotoxicity, combining the potent killing power of small-molecule drugs with the high targeting specificity of monoclonal antibodies. Therefore, they have become a hot topic in the research and development of targeted cancer therapy. ADCs generally consist of three parts linked in a specific way: an antibody or antibody-like ligand, a linker, and a small-molecule drug. The targeting specificity of ADCs comes from the antibody portion, while the toxicity mainly comes from the small-molecule drug; the antibody portion can also be toxic. After the antibody portion binds to the surface antigens of tumor cells, it is internalized into the cell. The ADC drug is then broken down in lysosomes, releasing the active toxic drug, which damages DNA or inhibits tumor cell division, ultimately killing the tumor cells. Compared to other treatment methods, ADCs have the following characteristics: strong therapeutic efficacy; high tumor cell specificity, low false positive rate, and a wider therapeutic safety window; weak immunogenicity, making it less likely to develop drug resistance; long circulation time in serum (shorter than naked antibodies); and weak cytotoxicity to non-target cells.

[0008] Therefore, there is an urgent need to develop an anti-GPC3 monoclonal / bispecific antibody or its antigen-binding fragment that has both strong binding ability to GPC3 epitopes and good cytotoxicity, as well as an ADC drug containing such an antibody or antigen-binding fragment. Summary of the Invention

[0009] This invention provides an anti-GPC3 monoclonal / bispecific antibody or its antigen-binding fragment, such as a mouse, human, chimeric or humanized monoclonal antibody or its antigen-binding fragment, and a bispecific antibody containing two antigen-binding domains, wherein the antibody or its antigen-binding fragment binds to the GPC3 protein and the antibody or its antigen-binding fragment has good GPC3 protein binding affinity.

[0010] 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.

[0011] 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);

[0012] The heavy chain variable region (VH) includes a CDR region, which contains a CDR region identical to any one of the amino acid sequences SEQ ID Nos: 14 or SEQ ID Nos: 16. The CDR region includes CDR1, CDR2, and CDR3 regions, which are defined according to IMGT, Kabat, Chothia, AbM, or Contact.

[0013] The light chain variable region (VL) includes a CDR region, which contains a CDR region identical to any one of the amino acid sequences 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. The CDR region includes CDR1, CDR2, and CDR3 regions, which are defined according to IMGT, Kabat, Chothia, AbM, or Contact.

[0014] In one aspect, as a preferred embodiment of the present invention, the CDR region of the heavy chain variable region (VH) is selected from:

[0015] 1) Any group of amino acid residues at positions 26-33, 51-58, and 97-104 of SEQ ID Nos: 14; or amino acid residues at positions 31-35, 50-66, and 99-104; or amino acid residues at positions 26-32, 52-57, and 99-104; or amino acid residues at positions 26-35, 50-59, and 99-104; or any group of amino acid residues at positions 30-35, 47-59, and 97-103;

[0016] 2) SEQ ID Nos: 16, amino acid residues at positions 26-33, 51-58, and 97-104; or amino acid residues at positions 31-35, 50-66, and 99-104; or amino acid residues at positions 26-32, 52-57, and 99-104; or amino acid residues at positions 26-35, 50-59, and 99-104; or any group of amino acid residues at positions 30-35, 47-59, and 97-103;

[0017] The CDR region of the light chain variable region (VL) is selected from:

[0018] 1) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 15; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0019] 2) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 17; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0020] 3) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 44; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0021] 4) SEQ ID Nos: amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0022] 5) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 46; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0023] 6) SEQ ID Nos: amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0024] 7) SEQ ID Nos: amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0025] 8) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 58; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0026] 9) SEQ ID Nos: amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos 59; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0027] 10) SEQ ID Nos: 60, amino acid residues at positions 27-37, 55-56 and 94-102; or amino acid residues at positions 24-39, 55-61 and 94-102; or any group of amino acid residues at positions 30-41, 51-60 and 94-101.

[0028] In one aspect, as a preferred embodiment of the present invention, the CDR region of the heavy chain variable region (VH) is selected from:

[0029] 1) Any group of amino acid residues at positions 26-33, 51-58, and 97-104 of SEQ ID Nos: 14; or amino acid residues at positions 31-35, 50-66, and 99-104; or amino acid residues at positions 26-32, 52-57, and 99-104; or amino acid residues at positions 26-35, 50-59, and 99-104; or any group of amino acid residues at positions 30-35, 47-59, and 97-103;

[0030] The CDR region of the light chain variable region (VL) is selected from:

[0031] 5) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 15; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0032] 6) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 44; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0033] 7) SEQ ID Nos: amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0034] 8) Any group of amino acid residues at positions 27-37, 55-56 and 94-102 of SEQ ID Nos: 46; or amino acid residues at positions 24-39, 55-61 and 94-102; or any group of amino acid residues at positions 30-41, 51-60 and 94-101.

[0035] In one aspect, as a preferred embodiment of the present invention, the heavy chain variable region (VH) contains the same sequence as the amino acid sequence SEQ ID Nos: 14 or SEQ ID Nos: 16;

[0036] The light chain variable region (AVL) contains the same sequence as the amino acid sequences 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.

[0037] In one aspect, as a preferred technical solution of the present invention, the anti-GPC3 monoclonal antibody or its antigen-binding fragment further includes a heavy chain (H) and a light chain (L), wherein the heavy chain (H) contains a sequence identical to the amino acid sequence SEQ ID Nos: 61 or SEQ ID Nos: 71;

[0038] The light chain (L) contains 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.

[0039] In one aspect, as a preferred technical solution of the present invention, the anti-GPC3 monoclonal antibody or its antigen-binding fragment further includes a heavy chain (H) and a light chain (L), wherein the heavy chain (H) contains a sequence identical to the amino acid sequence SEQ ID Nos: 61;

[0040] The light chain (L) contains the same sequence as SEQ ID Nos: 62, SEQ ID Nos: 63, SEQ ID Nos: 64 or SEQ ID Nos: 65.

[0041] In another aspect, the present invention also provides an anti-GPC3 bispecific antibody (e.g., mouse, chimeric, or humanized antibody) or an antigen-binding fragment thereof, said antibody or antigen-binding fragment binding to the GPC3 protein, said antibody or antigen-binding fragment containing a first GPC3 antigen-binding domain and a second GPC3 antigen-binding domain.

[0042] Furthermore, both the first and second GPC3 antigen-binding domains may contain 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 bisomatic antibody; (d) a small antibody; (e) F(ab')2; (f) F(ab); or other known antibody derivatives in this field.

[0043] Further, both the first GPC3 antigen-binding domain and the second GPC3 antigen-binding domain can be any of the following or composed of: animal-derived antibodies, humanized antibodies, fully human antibodies, chimeric antibodies, or affinity-optimized antibodies. More preferably, they are humanized antibodies, human-animal chimeric antibodies (such as human-mouse chimeric antibodies), and even more preferably, fully human antibodies. The antibody can be any one or more of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD antibodies or other subtypes of antibodies. Preferred are IgG1, IgG2, or IgG4 subtypes. More preferably, the IgG1 antibody is an hIgG1 antibody or a variant thereof, and in some embodiments, the amino acid sequence of 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.

[0044] Furthermore, the first GPC3 antigen-binding domain and the second GPC3 antigen-binding domain specifically bind to different epitopes of GPC3.

[0045] 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):

[0046] (a1) The heavy chain variable region and the light chain variable region of the first GPC3 antigen-binding domain are connected by a connector 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 by a connector L2.

[0047] (a2) The heavy chain variable region and the light chain variable region of the second GPC3 antigen-binding domain are connected by a connector 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 by a connector L2.

[0048] The connectors L1 and L2 are each independently selected from (GGGGS)n, where n is an integer of 1, 2, 3, 4, 5 or 6;

[0049] Preferably, the scFv is further connected to the heavy or light chain end of the first or second GPC3 antigen-binding domain via a connector L2;

[0050] More preferably, the end of the heavy chain or light chain is selected from the C-end or N-end of the heavy chain or light chain.

[0051] Furthermore, the anti-GPC3 bispecific antibody comprises a first polypeptide chain and a second polypeptide chain;

[0052] The structure of the first polypeptide chain can be selected from any one of the following (b1)-(b8):

[0053] (b1)[BVH]-[L1]-[BVL]-[L2]-[AVH]-[CH1]-[Fcx];

[0054] (b2)[BVL]-[L1]-[BVH]-[L2]-[AVH]-[CH1]-[Fcx];

[0055] (b3)[AVH]-[L1]-[AVL]-[L2]-[BVH]-[CH1]-[Fcx];

[0056] (b4)[AVL]-[L1]-[AVH]-[L2]-[BVH]-[CH1]-[Fcx];

[0057] (b5)[BVH]-[CH1]-[Fcx]-[L2]-[AVH]-[L1]-[AVL];

[0058] (b6)[BVH]-[CH1]-[Fcx]-[L2]-[AVL]-[L1]-[AVH];

[0059] (b7)[AVH]-[CH1]-[Fcx]-[L2]-[BVH]-[L1]-[BVL];

[0060] (b8)[AVH]-[CH1]-[Fcx]-[L2]-[BVL]-[L1]-[BVH];

[0061] Wherein, [L1] and [L2] independently represent linkers, which are selected from (GGGGS)n, where n is an integer of 1, 2, 3, 4, 5 or 6; CH1 is the antibody heavy chain constant region 1; and [Fcx] represents the Fc domain.

[0062] [AVH] and [AVL] represent the heavy chain variable region (AVH) and light chain variable region (AVL) of the first GPC3 antigen-binding domain, respectively;

[0063] [BVH] and [BVL] represent the heavy chain variable region (BVH) and light chain variable region (BVL) of the second GPC3 antigen-binding domain, respectively;

[0064] [Fcx] represents the Fc domain, which contains CH2 and CH3;

[0065] The structure of the second polypeptide chain may be selected from either (c1) or (c2):

[0066] (c1)[AVL]-[CL];

[0067] (c2)[BVL]-[CL];

[0068] The [CL] is the constant region of the antibody light chain;

[0069] [AVL]-[CL] represent the light chain variable region (AVL) and light chain constant region (CL) of the first GPC3 antigen-binding domain;

[0070] [BVL]-[CL] represents the light chain variable region (BVL) and light chain constant region (CL) of the second GPC3 antigen-binding domain.

[0071] The first polypeptide chain (heavy chain) and the second polypeptide chain (light chain) are connected by disulfide bonds.

[0072] 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, and the CDR region includes CDR1 region, CDR2 region and CDR3 region, wherein each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact.

[0073] Further, the CDR region of 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, and the CDR region includes CDR1 region, CDR2 region and 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 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, and the CDR region includes CDR1 region, CDR2 region and 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 light chain variable region (BVL) is selected from any one of the CDR regions 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 CDR1 region, CDR2 region and CDR3 region, wherein each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact.

[0076] Furthermore, the CDR region of the first heavy chain variable region (AVH) is selected from:

[0077] 1) Any group of amino acid residues at positions 26-33, 51-58, and 97-104 of SEQ ID Nos: 14(h1B12); or amino acid residues at positions 31-35, 50-66, and 99-104; or amino acid residues at positions 26-32, 52-57, and 99-104; or amino acid residues at positions 26-35, 50-59, and 99-104; or any group of amino acid residues at positions 30-35, 47-59, and 97-103;

[0078] 2) SEQ ID Nos: amino acid residues at positions 26-33, 51-58, and 97-104 of SEQ ID Nos: 16(m1B12); or amino acid residues at positions 31-35, 50-66, and 99-104; or amino acid residues at positions 26-32, 52-57, and 99-104; or amino acid residues at positions 26-35, 50-59, and 99-104; or any group of amino acid residues at positions 30-35, 47-59, and 97-103;

[0079] Furthermore, the CDR region of the first light chain variable region (AVL) is selected from:

[0080] 1) SEQ ID Nos: amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 15(h1B12); or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101;

[0081] 2) SEQ ID Nos: amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 17(m1B12); or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101;

[0082] 3) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 44(h1B12-G34R); or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0083] 4) SEQ ID Nos: amino acid residues at positions 27-37, 55-56, and 94-102 of 45(h1B12-G34K); or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101;

[0084] 5) SEQ ID Nos: 46(h1B12-G34A) amino acid residues at positions 27-37, 55-56, and 94-102; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101;

[0085] 6) SEQ ID Nos: amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0086] 7) SEQ ID Nos: amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0087] 8) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 58; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0088] 9) SEQ ID Nos: amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos 59; or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0089] 10) SEQ ID Nos: 60, amino acid residues at positions 27-37, 55-56 and 94-102; or amino acid residues at positions 24-39, 55-61 and 94-102; or any group of amino acid residues at positions 30-41, 51-60 and 94-101;

[0090] Furthermore, the CDR region of the second heavy chain variable region (BVH) is selected from:

[0091] 1) Any group of amino acid residues at positions 26-33, 51-60, and 99-106 of SEQ ID Nos: 1 (YP7); or amino acid residues at positions 31-35, 50-68, and 101-106; or 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;

[0092] 2) SEQ ID Nos: amino acid residues at positions 26-33, 51-60, and 99-106 of 3(hYP7); or amino acid residues at positions 31-35, 50-68, and 101-106; or 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;

[0093] 3) Any group of amino acid residues at positions 26-33, 51-60, and 99-106 of SEQ ID Nos: 5(hYP7HM); or amino acid residues at positions 31-35, 50-68, and 101-106; or 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.

[0094] 4) Any group of amino acid residues at positions 26-33, 51-60, and 99-106 of SEQ ID Nos: 6(YP8); or amino acid residues at positions 31-35, 50-68, and 101-106; or 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.

[0095] 5) Any group of amino acid residues at positions 26-33, 51-60, and 99-106 of SEQ ID Nos: 8(YP9); or amino acid residues at positions 31-35, 50-68, and 101-106; or 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;

[0096] 6) Any group of amino acid residues at positions 26-33, 51-60, and 99-106 of SEQ ID Nos: 12(YP9.1); or amino acid residues at positions 31-35, 50-68, and 101-106; or 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;

[0097] 7) SEQ ID Nos: amino acid residues at positions 26-33, 51-58, and 97-103 of SEQ ID Nos: 18(h2D8); or amino acid residues at positions 31-35, 50-66, and 99-103; or amino acid residues at positions 26-32, 52-57, and 99-103; or any group of amino acid residues at positions 26-35, 50-59, and 99-103; or amino acid residues at positions 30-35, 47-59, and 97-102.

[0098] 8) SEQ ID Nos: amino acid residues at positions 26-33, 51-58, and 97-103 of 20(m2D8); or amino acid residues at positions 31-35, 50-66, and 99-103; or amino acid residues at positions 26-32, 52-57, and 99-103; or amino acid residues at positions 26-35, 50-59, and 99-103; or any group of amino acid residues at positions 30-35, 47-59, and 97-102;

[0099] Furthermore, the CDR region of the second light chain variable region (BVL) is selected from:

[0100] 1) Any group of amino acid residues at positions 27-38, 56-57, and 95-103 of SEQ ID Nos: 2(YP7); or amino acid residues at positions 24-40, 56-62, and 95-103; or any group of amino acid residues at positions 30-42, 52-61, and 95-102.

[0101] 2) Any group of amino acid residues at positions 27-38, 56-57, and 95-103 of SEQ ID Nos: 4(hYP7); or amino acid residues at positions 24-40, 56-62, and 95-103; or any group of amino acid residues at positions 30-42, 52-61, and 95-102.

[0102] 3) Any group of amino acid residues at positions 27-38, 56-57, and 95-103 of SEQ ID Nos: 7(YP8); or amino acid residues at positions 24-40, 56-62, and 95-103; or any group of amino acid residues at positions 30-42, 52-61, and 95-102.

[0103] 4) Any group of amino acid residues at positions 27-38, 56-57, and 95-103 of SEQ ID Nos: 9 (YP9 clone 9); or amino acid residues at positions 24-40, 56-62, and 95-103; or any group of amino acid residues at positions 30-42, 52-61, and 95-102.

[0104] 5) Amino acid residues at positions 27-38, 56-57, and 95-103 of SEQ ID Nos: 10 (YP9 clone 10); or amino acid residues at positions 24-40, 56-62, and 95-103; or any group of amino acid residues at positions 30-42, 52-61, and 95-102;

[0105] 6) Any group of amino acid residues at positions 27-38, 56-57, and 95-103 of SEQ ID Nos: 11 (YP9 clone 1); or amino acid residues at positions 24-40, 56-62, and 95-103; or any group of amino acid residues at positions 30-42, 52-61, and 95-102;

[0106] 7) Any group of amino acid residues at positions 27-38, 56-57, and 95-103 of SEQ ID Nos: 13(YP9.1); or amino acid residues at positions 24-40, 56-62, and 95-103; or any group of amino acid residues at positions 30-42, 52-61, and 95-102;

[0107] 8) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 19(h2D8); or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0108] 9) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 21(m2D8); or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0109] Preferably, the CDR region of the first heavy chain variable region (AVH) is selected from any group of the following: amino acid residues at positions 26-33, 51-58, and 97-104 of SEQ ID Nos: 14 (h1B12); or amino acid residues at positions 31-35, 50-66, and 99-104; or amino acid residues at positions 26-32, 52-57, and 99-104; or amino acid residues at positions 26-35, 50-59, and 99-104; or amino acid residues at positions 30-35, 47-59, and 97-103.

[0110] The CDR region of the first light chain variable region (AVL) is selected from:

[0111] 1) SEQ ID Nos: amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 15(h1B12); or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101;

[0112] 2) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 44(h1B12-G34R); or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0113] 3) SEQ ID Nos: amino acid residues at positions 27-37, 55-56, and 94-102 of 45(h1B12-G34K); or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101;

[0114] 4) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 46(h1B12-G34A); or amino acid residues at positions 24-39, 55-61, and 94-102; or amino acid residues at positions 30-41, 51-60, and 94-101.

[0115] Preferably, the CDR region of the second heavy chain variable region (BVH) is selected from:

[0116] 1) Any group of amino acid residues at positions 26-33, 51-60, and 99-106 of SEQ ID Nos: 1 (YP7); or amino acid residues at positions 31-35, 50-68, and 101-106; or 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.

[0117] 2) SEQ ID Nos: amino acid residues at positions 26-33, 51-60, and 99-106 of 3(hYP7); or amino acid residues at positions 31-35, 50-68, and 101-106; or 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;

[0118] 3) Any group of amino acid residues at positions 26-33, 51-60, and 99-106 of SEQ ID Nos: 5(hYP7HM); or amino acid residues at positions 31-35, 50-68, and 101-106; or 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.

[0119] 4) SEQ ID Nos: amino acid residues at positions 26-33, 51-58, and 97-103 of SEQ ID Nos: 18(h2D8); or amino acid residues at positions 31-35, 50-66, and 99-103; or amino acid residues at positions 26-32, 52-57, and 99-103; or any group of amino acid residues at positions 26-35, 50-59, and 99-103; or amino acid residues at positions 30-35, 47-59, and 97-102.

[0120] 5) SEQ ID Nos: amino acid residues at positions 26-33, 51-58, and 97-103 of 20(m2D8); or amino acid residues at positions 31-35, 50-66, and 99-103; or amino acid residues at positions 26-32, 52-57, and 99-103; or amino acid residues at positions 26-35, 50-59, and 99-103; or any group of amino acid residues at positions 30-35, 47-59, and 97-102;

[0121] Preferably, the CDR region of the second light chain variable region (BVL) is selected from:

[0122] 1) Any group of amino acid residues at positions 27-38, 56-57, and 95-103 of SEQ ID Nos: 2(YP7); or amino acid residues at positions 24-40, 56-62, and 95-103; or any group of amino acid residues at positions 30-42, 52-61, and 95-102.

[0123] 2) Any group of amino acid residues at positions 27-38, 56-57, and 95-103 of SEQ ID Nos: 4(hYP7); or amino acid residues at positions 24-40, 56-62, and 95-103; or amino acid residues at positions 30-42, 52-61, and 95-102.

[0124] 3) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 19(h2D8); or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0125] 4) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 21(m2D8); or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0126] Preferably, the CDR region of the first heavy chain variable region (AVH) is selected from: amino acid residues at positions 26-33, 51-58, and 97-104 of SEQ ID Nos: 14 (h1B12); or amino acid residues at positions 31-35, 50-66, and 99-104; or amino acid residues at positions 26-32, 52-57, and 99-104; or amino acid residues at positions 26-35, 50-59, and 99-104; or any group of amino acid residues at positions 30-35, 47-59, and 97-103.

[0127] The CDR region of the first light chain variable region (AVL) is selected from: amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 44 (h1B12-G34R); or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0128] Preferably, the CDR region of the second heavy chain variable region (BVH) is selected from:

[0129] 1) Any group of amino acid residues at positions 26-33, 51-60, and 99-106 of SEQ ID Nos: 5(hYP7HM); or amino acid residues at positions 31-35, 50-68, and 101-106; or 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;

[0130] 2) SEQ ID Nos: amino acid residues at positions 26-33, 51-58, and 97-103 of SEQ ID Nos: 18(h2D8); or amino acid residues at positions 31-35, 50-66, and 99-103; or amino acid residues at positions 26-32, 52-57, and 99-103; or any group of amino acid residues at positions 26-35, 50-59, and 99-103; or amino acid residues at positions 30-35, 47-59, and 97-102.

[0131] 3) SEQ ID Nos: amino acid residues at positions 26-33, 51-58, and 97-103 of 20(m2D8); or amino acid residues at positions 31-35, 50-66, and 99-103; or amino acid residues at positions 26-32, 52-57, and 99-103; or amino acid residues at positions 26-35, 50-59, and 99-103; or any group of amino acid residues at positions 30-35, 47-59, and 97-102;

[0132] The CDR region of the second light chain variable region (BVL) is selected from:

[0133] 1) Any group of amino acid residues at positions 27-38, 56-57, and 95-103 of SEQ ID Nos: 4(hYP7); or amino acid residues at positions 24-40, 56-62, and 95-103; or any group of amino acid residues at positions 30-42, 52-61, and 95-102;

[0134] 2) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 19(h2D8); or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0135] 3) Any group of amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID Nos: 21(m2D8); or amino acid residues at positions 24-39, 55-61, and 94-102; or any group of amino acid residues at positions 30-41, 51-60, and 94-101.

[0136] Preferably, the CDR region of the first heavy chain variable region (AVH) is selected from the CDR region of any one of the heavy chain variable regions h1B12 or m1B12, and the CDR region includes CDR1 region, CDR2 region and CDR3 region, each CDR region is defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact.

[0137] Preferably, the CDR region of the first light chain variable region (AVL) is selected from the CDR region of any one of h1B12, h1B12-G34R, h1B12-G34K, h1B12-G34A or m1B12, and the CDR region includes CDR1 region, CDR2 region and 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 second heavy chain variable region (BVH) is selected from the CDR region of any one of the heavy chain variable regions of YP7, hYP7, hYP7HM, YP8, YP9, YP9.1, h2D8 or m2D8, and the CDR region includes CDR1 region, CDR2 region and 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 light chain variable region (BVL) is selected from the CDR regions 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 region, CDR2 region and CDR3 region, each CDR region being defined according to the scheme defined by IMGT, Kabat, Chothia, AbM or Contact.

[0140] Preferably, the first heavy chain variable region (AVH) is selected from any one of SEQ ID Nos: 14, 16, 34, 49 or 51;

[0141] Preferably, 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;

[0142] 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;

[0143] 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.

[0144] 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.

[0145] Preferably, the second polypeptide chain of the bispecific anti-GPC3 antibody is composed of a light chain variable region (VL) and a light chain constant region (CL), wherein the 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 the CL comprises SEQ ID Nos: 55;

[0146] More preferably, the second polypeptide chain of the bispecific anti-GPC3 antibody is composed of a light chain variable region (VL) and a light chain constant region (CL), wherein 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;

[0147] On the other hand, the present invention also provides a chimeric antigen receptor, characterized in that the chimeric antigen receptor comprises the anti-GPC3 monoclonal antibody or its antigen-binding fragment described in the present invention, or the anti-GPC3 bispecific antibody or its antigen-binding fragment.

[0148] On the other hand, the present invention also provides a polynucleotide, characterized in that the polynucleotide encodes the anti-GPC3 monoclonal antibody or its antigen-binding fragment thereof, or the anti-GPC3 bispecific antibody or its antigen-binding fragment thereof, as described in the present invention. The polynucleotide of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the polynucleotide is a cDNA molecule.

[0149] On the other hand, the present invention also provides an expression vector, characterized in that the expression vector comprises the polynucleotides described in the present invention. The expression vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0150] On the other hand, the present invention also provides a host cell, characterized in that the host cell contains the polynucleotide or expression vector described in the present invention. The host cell contains prokaryotic cells, yeast cells, or mammalian cells, such as CHO cells, NSO cells, or other mammalian cells, preferably CHO cells.

[0151] On the other hand, the present invention also provides an antibody-drug conjugate, characterized in that it comprises the anti-GPC3 monoclonal antibody or its antigen-binding fragment as described in the present invention, or the anti-GPC3 bispecific antibody or its antigen-binding fragment as described in the present invention, and a drug or toxin.

[0152] 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.

[0153] The anti-GPC3 bispecific antibody can be prepared by a method including the following steps:

[0154] 1) Construct an expression vector, wherein the expression vector is obtained by ligating a nucleotide encoding any of the anti-GPC3 bispecific antibodies as described above to a base vector;

[0155] 2) Transfect or transform the expression vector constructed in step 1) into host cells and culture the host cells;

[0156] 3) Isolate and purify anti-GPC3 bispecific antibody.

[0157] The DNA sequence of the anti-GPC3 bispecific antibody or its antigen-binding fragment described in this invention can be obtained using conventional techniques, such as hybridoma PCR amplification or phage display library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form a single-chain antibody (e.g., scFV).

[0158] Once the relevant sequence is obtained, it can be cloned into a vector, then transferred into host bacteria, and the vector can then be extracted from the host bacteria using conventional methods.

[0159] Furthermore, the relevant sequences can be synthesized artificially, especially when the fragment length is short. Currently, the DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. In addition, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0160] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0161] 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 cells). Preferred animal cells include (but are not limited to): CHO and HEK-293 cells.

[0162] The anti-GPC3 bispecific antibody or its antigen-binding fragment 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, host cells transformed with the present invention are cultured under conditions suitable for antibody expression, and then purified using conventional immunoglobulin purification steps, 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 combinations thereof, to obtain the anti-GPC3 bispecific antibody or its antigen-binding fragment of the present invention.

[0163] As a preferred embodiment of the preparation method of the anti-GPC3 bispecific antibody or its antigen-binding fragment according to the present invention, the method for separating and purifying the anti-GPC3 bispecific antibody or its antigen-binding fragment is protein A affinity chromatography, cation exchange, or anion exchange.

[0164] The resulting monoclonal or bispecific antibodies can be identified using conventional methods. For example, the binding specificity of an 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 an antibody can be determined, for example, by the Scatchard assay described by Munson et al., Anal. Biochem., 107:220 (1980).

[0165] This invention provides an immunoconjugate comprising:

[0166] 1) Anti-GPC3 bispecific antibodies, monoclonal antibodies, or antigen-binding fragments thereof as described in this invention; and

[0167] 2) The conjugate portion selected from the following group: drug, toxin, detectable marker, cytokine, radionuclide, enzyme, gold nanoparticles / nanoran, magnetic nanoparticles, viral capsid protein or VLP, or a combination thereof.

[0168] Preferably, the radionuclides include:

[0169] (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 combinations thereof; and / or

[0170] (b) A therapeutic isotope 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 combinations thereof.

[0171] Preferably, the coupling portion is a drug or toxin.

[0172] Preferably, the drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In an embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0173] 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 combinations thereof.

[0174] As preferred, particularly useful examples of 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.

[0175] Preferably, the toxin is selected from the group consisting of: ostatins (e.g., ostatin E, ostatin F, MMAE, and MMAF), chlortetracycline, methemosiderin, pyrethroids, pyrethroid A-chain, cobustatin, docalimicin, dolalastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, and dihydroxybenzene. Anthraxindione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE)A, PE40, abrin, abrin A chain, saccharin A chain, α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, curicin, croton toxin, chachomycin, Sapaonaria officinalis inhibitor, glucocorticoids, or combinations thereof.

[0176] Preferably, the drug or toxin is selected from one or more of the following: 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).

[0177] The antibody can be conjugated to the drug to form an antibody-drug conjugate (ADC). Typically, an ADC comprises an anti-GPC3 bispecific antibody, monoclonal antibody, or antigen-binding fragment as described in this invention, linked to a drug or toxin via a linker. The linker can be degradable or non-degradable. Degradable linkers typically degrade readily in an intracellular environment, thereby releasing the therapeutic agent from the antibody. Suitable degradable linkers include enzymatically degradable linkers, such as peptide-containing linkers that can be degraded by intracellular lysosomal proteases, or glycolinkers, such as glucuronidase-containing linkers. 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 pH less than 5.5) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0178] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through this reactive group. Thiol-specific reactive groups are preferred, such as maleimide compounds, haloamides, haloesters, halomethyl ketones, benzyl halides, vinyl sulfones, pyridyl disulfides, mercury derivatives, and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0179] Preferably, the drug or toxin connected to the connector is selected from: 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:

[0180]

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

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

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

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

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

[0186] In this invention, the antibody-drug conjugate is prepared by a method comprising the following steps:

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

[0188] Drug-connector compounds crosslink with reduced antibody thiol groups to generate corresponding antibody-drug conjugates;

[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 thereof, or an anti-GPC3 bispecific antibody or its antigen-binding fragment thereof, or an antibody-drug conjugate, or an oncolytic virus, and a pharmaceutically acceptable carrier. When the composition comprises more than one antibody (or its antigen-binding fragment thereof, or an antibody-drug conjugate, or an oncolytic virus), the antibody (or its antigen-binding fragment thereof, or the antibody-drug conjugate, or the oncolytic virus) can be administered in batches. The composition may optionally comprise one or more other pharmaceutically active ingredients, such as another antibody or drug, such as an antitumor drug.

[0191] Pharmaceutical compositions may contain any number of excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, or combinations thereof. The selection and use of appropriate excipients is taught in the following, Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins 2003), the contents of which are incorporated herein by reference.

[0192] On the other hand, the present invention provides a nucleotide molecule encoding the anti-GPC3 bispecific antibody of the present invention or an antigen-binding fragment thereof. The nucleic acid molecule may be present in intact cells or cell lysates, or in a partially purified or substantially pure form. The nucleic acid is considered "isolated" or "substantially pure" when separated from other cellular components or other contaminants (such as other intracellular nucleic acids or proteins) 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 may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0193] On one hand, the present invention provides a vector comprising the nucleotides described herein. The vector includes: 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 carrier or antibody; said host cell comprises prokaryotic cells, yeast or mammalian cells, such as CHO cells, NSO cells or other mammalian cells, preferably CHO cells.

[0195] The anti-GPC3 bispecific antibody or its antigen-binding fragment disclosed in this invention can also be used to prepare chimeric antigen receptors (CARs; also known as chimeric T-cell receptors, artificial T-cell receptors, or chimeric immune receptors).

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

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

[0198] In another aspect, the present invention provides a method for inhibiting tumor growth in a subject, the method comprising administering to the subject an effective dose of a drug or a composition thereof comprising the anti-GPC3 bispecific antibody or an antigen-binding fragment thereof described in this invention. The tumor may 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 the composition of the present invention, the anti-GPC3 bispecific antibody or an antigen-binding fragment thereof, an antibody-drug conjugate, or an oncolytic virus encoding or carrying an antibody, or a nucleic acid molecule capable of expressing the aforementioned substances in the subject. In some embodiments, at least one additional antitumor antibody may be administered in combination with the anti-GPC3 bispecific antibody or its antigen-binding fragment of the present invention. The at least one additional antitumor antibody may be selected from anti-VISTA antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, anti-CD40 antibody, anti-TIM-3 antibody, anti-STAT3 antibody, and / or anti-ROR1 antibody. In another embodiment, the antibody or its antigen-binding fragment of the present invention may be administered in combination with a chemotherapeutic agent, such as epirubicin, oxaliplatin, and / or 5-fluorouracil (5-FU). The antibody of the present invention may be mouse, human, chimeric, or humanized antibody.

[0199] To ensure a clearer understanding of the invention, certain terms are first defined, with additional definitions provided throughout the detailed description:

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

[0201] As used in this article, an antibody that "specifically binds to human GPC3" refers to an antibody that binds to human GPC3 protein (and may be derived from one or more non-human species) but does not actually bind to non-GPC3 proteins.

[0202] The term "antibody" as used herein includes complete antibodies and any antigen-binding fragment (i.e., "antigen-binding moiety") or its single chain. A complete antibody is a glycoprotein consisting of two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH herein) 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 as VL herein) 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 in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0203] The term "bispecific antibody" refers to an antibody molecule that can bind to two independent antigens or has binding specificity to 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, and the other antigen-binding fragment binds to the second antigen-binding domain of GPC3, thereby further enhancing the affinity for GPC3 antigens on the cell surface.

[0204] As used herein, the term “antigen-binding fragment” (or simply “antibody portion”) of an antibody refers to one or more fragments of an antibody that specifically bind to an antigen (e.g., the GPC3 protein). Antigen-binding functionality of antibodies has been demonstrated to be achieved through fragments of full-length antibodies. Examples of binding fragments covered by the term “antigen-binding fragment” of an antibody include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by disulfide bonds in a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains in a single arm of an antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) separated complementarity-determining regions (CDRs); and (vii) nanobodies, heavy-chain variable regions containing a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the FV fragment are encoded by separate genes, they can be linked together via adapters using recombination methods, thus forming a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a 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 within the scope of the term "antigen-binding fragment" in the term antibody. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragment screening performed for use is the same as that for intact antibodies.

[0205] The term "scFv" refers to a genetically modified molecule containing the light chain variable region and the heavy chain variable region of an immunoglobulin, linked by a suitable polypeptide linker to form a single-chain antibody fused to a gene.

[0206] The term "Fc" refers to the Fc fragment region of an antibody molecule, which contains a portion of the hinge region, CH2 and CH3 domains, and has a molecular weight of approximately 50,000 Da. In human IgG heavy chain Fc regions, the region extends from threonine at position 225 to the C-terminus when the antibody is digested with papain.

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

[0208] The term "Fab" refers to a fragment containing a monovalent antigen-binding segment of an antibody molecule, which can be produced by digesting an intact antibody with papain to generate a complete light chain and a portion of a heavy chain.

[0209] The term "Fab′" refers to an antibody molecule fragment obtained by treating an intact antibody with pepsin and then reducing it to produce a portion of the complete light chain and heavy chain; each antibody molecule yields two Fab′ fragments.

[0210] The term "(Fab′)2" refers to an antibody fragment obtained by treating an intact antibody with pepsin but 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 segment containing a light chain variable region and a heavy chain variable region expressed as two strands.

[0212] The terms "h1B12-G34R, h1B12-G34K, h1B12-G34A, h1B12-G34H, h1B12-G34M, h1B12-G34L, h1B12-G34S, or h1B12-N33R" refer to antibodies or antigen-binding fragments obtained by mutating glycine (G) to R, K, A, H, M, L, or S at position 34 of the light chain of h1B12, or by mutating asparagine (N) to R at position 33 of the light chain of h1B12.

[0213] As used herein, "isolated antibody" means an antibody that substantially does not contain other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to the GPC3 protein substantially does not contain antibodies that specifically bind to antigens other than GPC3. However, for example, in some embodiments, an isolated antibody that specifically binds to the human GPC3 protein may be cross-reactive with other antigens (e.g., GPC3 proteins from other species). Furthermore, the isolated antibody may substantially contain no other cellular material and / or chemicals.

[0214] As used herein, the term "monoclonal antibody" refers to a formulation of an antibody molecule having a single molecular composition. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope.

[0215] As used herein, the term "mouse antibody" is intended to include antibodies having variable regions, wherein both the frame region and the CDR region are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from mouse germline immunoglobulin sequences. The mouse antibodies of the present invention may include amino acid residues not encoded by mouse germline immunoglobulin sequences, such as mutations introduced in vitro by random or site-directed mutagenesis or in vivo by somatic mutations. However, as used herein, the term "mouse antibody" is not intended to include antibodies in which a CDR sequence from another mammalian germline has been grafted onto a mouse frame sequence.

[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 derived from a non-human species whose protein sequence has been modified to increase its similarity to naturally occurring human antibody variants.

[0218] The term "antibody-drug conjugate" refers to a drug-antibody conjugate (ADC) that utilizes the specific recognition of tumor cell surface antigens by antibodies to precisely deliver antitumor therapeutic agents (such as cytotoxins or cell inhibitors, radioisotopes, small molecule chemotherapeutic agents, etc.) to tumor target cells, causing intracellular accumulation and release, thereby achieving precise tumor killing. ADCs are considered among the most promising antitumor drugs due to their suitable molecular weight, high stability, strong targeting, and low toxicity. In addition to monoclonal antibodies, bispecific antibodies can also be conjugated to therapeutic agents. In some embodiments, the portion conjugated with the antibody or bispecific antibody of the present invention to form the antibody-drug conjugate is a cytotoxin, which refers to a substance that inhibits or prevents cell function and / or causes cell damage, including 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 "therapeutic effective amount" refers to the amount of the anti-GPC3 bispecific antibody or its antigen-binding fragment of the present invention sufficient to prevent or improve symptoms associated with a disease or condition (e.g., cancer) and / or reduce the severity of the disease or condition. Therapeutic effective amount should be understood in the context of the condition being treated, where the actual effective amount can be readily identified by those skilled in the art.

[0221] The antibodies of the present invention are monoclonal antibodies characterized structurally and chemically as described below and in the following examples. The amino acid sequence IDs of the heavy / 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 may be the human IgG1 heavy chain constant region having, for example, the amino acid sequence shown in SEQ ID NO: 54, and the light chain constant region may be the human immunoglobulin κ light chain constant region having, for example, the amino acid sequence shown in SEQ ID NO: 55.

[0222] The heavy chain variable region CDR and light chain variable region CDR in Table 1 are defined by the Kabat, Chothia, IMGT, AbM, or Contact numbering system / method. However, as is known in the art, the CDR regions can also be based on other numbering systems such as those used for the heavy chain / light chain variable region sequences. See Table 2 for details of the CDR regions of the antibodies of this invention.

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

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231] Table 2. Numbering system / method definition of heavy chain variable regions and light chain variable regions (CDRs) of anti-GPC3 bispecific antibodies.

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239] Other features and advantages of the present invention will become apparent from the following detailed description and examples, and should not be construed as limiting. All references, Genbank entries, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. Attached Figure Description

[0240] 1) Figure 1 A schematic diagram of the construction of a bispecific antibody (heavy chain). The heavy chain (1B12-VH) of the h1B12 antibody is linked to different scFV forms of a secondary antibody (such as hYP7, h2D8) via two GGGGS linkers. The linkers between the VH and VL variable regions of the secondary antibody can be 3 or 4 GGGGS links. YP7-HM refers to YP7-VH containing the A68G mutation, and IgG1-CH refers to the constant region of the heavy chain of human IgG1.

[0241] 2) Figure 2 The ELISA binding curves of bispecific antibodies and humanized monoclonal antibodies with human GPC3 protein are shown. All antibodies have high affinity for human GPC3 (EC50 is at the sub-nM level).

[0242] 3) Figure 3A and Figure 3B The binding of bispecific antibodies and humanized monoclonal antibodies to human hepatocellular carcinoma cells HepG2 or Hep3B was demonstrated. Compared with the bsDH series bispecific antibodies (based on h1B12+h2D8) or various monoclonal antibodies, the bs-FH series bispecific antibodies (based on h1B12+hYP7) showed higher cell affinity (EC50 values ​​of around 2 nM for both HepG2 and Hep3B cells).

[0243] 4) Figure 4A and Figure 4BThe study demonstrated the endocytosis of bispecific antibodies and humanized monoclonal antibodies in human hepatocellular carcinoma cells HepG2 or Hep3B. Compared to the bsDH series bispecific antibodies (based on h1B12+h2D8) or various monoclonal antibodies, the bs-FH series bispecific antibodies (based on h1B12+hYP7) exhibited better antibody endocytosis.

[0244] 5) Figure 5A and Figure 5B The binding of the antibody-drug conjugate to human hepatocellular carcinoma cells Hep3B or Huh-7 was demonstrated. Compared with the monoclonal antibody-drug conjugate, the bispecific antibody-drug conjugate showed higher cell affinity in both the GPC3 antigen-overexpressing cell line (Hep3B) and the low-expressing cell line (Huh-7).

[0245] 6) Figures 6A-6D The antibody-drug conjugate was shown to kill human hepatocellular carcinoma cells HepG2, Hep3B, or Huh-7. At high concentrations ( Figures 6A-6C 66.7 nM) and at low concentrations ( Figure 6D Compared to monoclonal antibody-drug conjugates, bispecific antibody-drug conjugates (1 nM) exhibit better killing effects on tumor cells.

[0246] 7) Figure 7A and Figure 7B The study demonstrated the effectiveness of unconjugated bispecific antibodies and monoclonal antibodies against HepG2 human liver cancer cells. Figure 7A ) or Hep3B ( Figure 7B It has no direct killing effect.

[0247] 8) Figure 8A The ELISA binding curves of humanized monoclonal antibodies of 1B12 and its variants to human GPC3 protein are shown.

[0248] 9) Figure 8B , 8C The results from 8D show the binding of humanized monoclonal antibodies of 1B12 and its variants to human hepatocellular carcinoma cells HepG2, Hep3B, or Huh-7.

[0249] 10) Figure 9 The endocytosis of the 1B12 humanized monoclonal antibody in human hepatocellular carcinoma cells Hep3B was shown. Specific Implementation

[0250] The present invention will now be explained with reference to the embodiments and accompanying drawings, but the present invention is not limited thereto.

[0251] Example 1

[0252] Mouse anti-GPC3 monoclonal antibodies were prepared using hybridoma technology.

[0253] 1.1 Immunizing mice

[0254] 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 extracellular full-length domain amino acid residues 25 (Gln) to 559 (His) as immunogen (Antigen 1). Serum titers were tested after the second and third immunizations, and the two mice showing the highest antibody titers were electrofused. Antigen 1 immunogen, or the extracellular C-terminal domain amino acid residues 359 (Ser) to 559 (His) polypeptide antigen (Antigen 7), was used to determine antiserum titers and screen hybridoma cells secreting antigen-specific antibodies.

[0255] 1.2 Hybridoma cell fusion and screening

[0256] Prior to cell fusion, mouse myeloma cell lines (SP2 / 0-Ag14, ATCC#CRL-1581) were cultured to the logarithmic growth phase. Following the method described by Kohler G and Milstein C, “Continuous cultures of fused cells secreting antibody of predefined specificity,” Nature, 256:495-497 (1975), immunized mice were sacrificed, and their spleens were aseptically removed and prepared into a spleen cell suspension. Splenic B cells and logarithmic growth phase mouse myeloma cells were fused using a PEG chemical method, 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 seeding, the culture supernatant from each hybridoma was collected. Hybridomas expressing anti-GPC3 antibodies were screened using ELISA based on recombinant human GPC3 protein (Antigen 1 and Antigen 7). The specific screening procedure was as follows: Recombinant human GPC3 protein (Antigen 1 or Antigen 7, 2 μg / mL) was coated onto 96-well plates at 50 μL / well and incubated overnight at 4°C. After washing the plates three times with PBST, 200 μL of blocking buffer (PBS + 1% BSA) was added to each well and the plates were blocked at 37°C for 1 hour. Each antibody was diluted to 2 μg / mL with blocking buffer, and then serially diluted 3-fold to a total of eight concentrations. Each concentration was added to the ELISA plate at 50 μL / well and incubated at 37°C for 1 hour. Wash the plate three times with PBST, then add 50 μL / well of prediluted Peroxidase-conjugated affinipure F(ab')2fragment goat anti-mouse IgG (H+L) (Jackson, diluted 5000 times with blocking buffer according to the manufacturer's instructions), and incubate at 37°C for 1 hour. Wash the plate three times with PBST, then add 50 μL / well of 1-Step™ Ultra TMB-ELISA substrate development solution (Thermo), and incubate at room temperature in the dark for 3-5 minutes. Stop the reaction with 50 μL / well of 2M HCl. Measure the OD value at 450 nm using a microplate reader. Calculate the EC50 value and screen for hybridoma clones that secrete antibodies with high specific binding activity.

[0257] Table 3 EC50 of hybridoma antibody binding to human GPC3 protein

[0258]

[0259] As shown in Table 3, all mouse monoclonal antibodies have high affinity for GPC3 protein. Most of them recognize the C-terminal domain Antigen7 of GPC3 protein, while m2G12 recognizes domains other than the C-terminus.

[0260] Furthermore, the recognition of different species' GPC3 proteins by various antibodies was detected using the ELISA method. Cynomolgus GPC3 (ACRO Biosystems) and Mouse GPC3 (ACRO Biosystems) were coated into 96-well plates at a concentration of 2 μg / mL, 50 μL / well, respectively. The ELISA method described above was followed for detection, and the results are shown in Table 4.

[0261] Table 4. Recognition of GPC3 protein in monkeys and mice by hybridoma antibodies.

[0262]

[0263] Note: √ indicates affinity recognition, × indicates no recognition.

[0264] Furthermore, a competitive ELISA was used to determine whether each candidate antibody recognized the same epitope as the GC33 antibody. The specific steps were as follows: Each antibody was diluted to 2 μg / mL and added to a 96-well ELISA plate (100 μL / well), and incubated overnight at 4°C. The plate was washed three times with PBST, and blocking buffer (PBS + 1% BSA) was added (200 μL / well), and the plate was blocked at 37°C for 1 hour. 2 μg / mL Insert 1 antigen was added to each well (100 μL / well), and the plate was incubated at 37°C for 1 hour. The plate was washed three times with PBST. The GC33 antibody was diluted to 2 μg / mL with blocking buffer, and then serially diluted 3-fold to a total of eight concentrations. The diluted GC33 was then added to the ELISA plate (100 μL / well), and the plate was incubated at 37°C for 1 hour. Discard the supernatant, wash the plate three times with PBST, add 100 μL of diluted Peroxidase-conjugated goat anti-human IgG (Jackson, diluted 5000 times with blocking buffer according to the instructions) to each well, and incubate at 37°C for 1 h. Discard the supernatant, wash the plate three times with PBST, add 50 μL of 1-step Ultra TMB-ELISA substrate (Thermo) to each well, and incubate in the dark for approximately 4 min 30 s. Then add 50 μL of stop solution (2M HCl). Measure the OD value at 450 nm using a microplate reader. The analytical results are summarized in Table 5, where m1B12, m1H4, and GC33 have the same or overlapping antigen recognition sites.

[0265] Table 5. Antibodies that identify different epitopes of GPC3

[0266]

[0267] 1.3 Sequencing and Identification of Hybridoma Antibodies

[0268] The variable region genes of hybridoma antibodies were extracted using 5' RLM-RACE. In short, total RNA was extracted from each candidate hybridoma, and cDNA was obtained by reverse transcription using the FirstChoice RLM-RACE Kit (Thermo). The variable region genes of the heavy and light chains were amplified by PCR. The PCR products were cloned into vectors and sequenced to obtain the antibody sequences m1B12, m2D8, m2G12, m1B8, m1H4, m36C12, and m37F4, as shown in Table 6.

[0269] Table 6. Variable region sequence of anti-GPC3 hybridoma antibody

[0270]

[0271] Example 2

[0272] Humanization of mouse anti-GPC3 monoclonal antibody

[0273] Mouse anti-GPC3 monoclonal antibodies were selected for humanization and further research. As described below, the mouse monoclonal antibodies were humanized using a mature CDR transplantation method. The light chain and heavy chain variable region sequences of each mouse monoclonal antibody were BLAST-aligned with the human immunoglobulin gene database to select the receptor framework regions for humanization. The human lineages IGVH and IGVK, which showed the highest homology with each mouse antibody, were selected as the receptor frameworks for humanization. The CDRs of the heavy chain or light chain variable regions of each mouse antibody were transplanted into the selected human framework regions, and the amino acid residues in the framework regions were reverse-mutated to obtain more candidate heavy chain and / or light chain variable regions for humanized antibodies. The heavy chain and light chain variable regions of each humanized antibody, and the mutation site design, are summarized in Table 7.

[0274] Table 7. Variable region sequence of anti-GPC3 humanized antibody

[0275]

[0276]

[0277] Example 3

[0278] Preparation of humanized monoclonal antibodies and bispecific antibodies

[0279] 3.1 Carrier Construction

[0280] First, the nucleotide sequence encoding the human IgG1 heavy chain constant region (IgG1-CH) was coupled with a secretion signal peptide (SP) coding sequence at the front end and a stop codon (TAG) at the back end. The gene was synthesized and cloned into the pUC57-GW-kan vector (Suzhou Genewiz Biotechnology Co., Ltd.). It was then inserted downstream of the CMV promoter in the pCDNA3.1 vector using an in-fusion cloning method. Specifically, the synthesized gene fragment and the pCDNA3.1 vector plasmid fragment were amplified using primers with specific insertion sites and a high-fidelity PCR enzyme (HiFi PCR Premix, TAKARA). After gel recovery, the IgG1 heavy chain gene fragment and the linearized vector fragment were ligated (using an in-fusion snap assembly master mix, TAKARA) to obtain the heavy chain constant region vector pCDNA3.1-IgG1. Following the above implementation method, the coding sequence of the human immunoglobulin κ light chain constant region (IgG-CK) is modified by adding a signal peptide (SP) coding sequence to the front end and a stop codon TAG to the back end. After gene synthesis, the sequence is inserted downstream of the CMV promoter of the pCDNA3.1 vector to obtain the light chain constant region vector pCDNA3.1-CK.

[0281] For the construction of each humanized monoclonal antibody expression vector, the coding sequences of their respective heavy chain variable region (VH) and light chain variable region (VL) were synthesized and inserted between the SP and constant regions of the pCDNA3.1-IgG1 or pCDNA3.1-CK vectors using the in-fusion cloning method described above, to obtain the heavy chain expression vector and light chain expression vector of the monoclonal antibody.

[0282] For the construction of bispecific antibody expression vectors, the nucleotide sequence of the heavy chain variable region of the second antibody is linked to the nucleotide sequence of the light chain variable region through a (GGGGS)3 or (GGGGS)4 linker to obtain the scFV nucleotide sequence, and the gene is synthesized. Then, the scFV coding sequence is inserted between SP and VH of the above-mentioned monoclonal antibody heavy chain expression vector to obtain the bispecific antibody heavy chain expression vector. A schematic diagram of the construction of the bispecific antibody heavy chain vector is shown below. Figure 1 .

[0283] For the construction of the mutant expression vector, hYP7-HM is obtained by replacing the coding sequence of the 68th residue of hYP7-VH with GGC through gene synthesis, resulting in the heavy chain A68G mutation (alanine is mutated to glycine). h1B12-VL(G34X) is synthesized by replacing the coding sequence of residue 34 of h1B12-VL with CGC, AAG, GCC, GTG, ATG, CTG, TAC, CCG, or TCC, resulting in a series of light chain mutations: G34R, G34K, G34A, G34H, G34M, G34L, G34Y, G34P, and G34S (glycine mutated to arginine, lysine, alanine, histidine, methionine, leucine, tyrosine, proline, and serine); or replacing the coding sequence of residue 33 of h1B12-VL with AAC and CGC or CCG, resulting in light chain mutations: N33R and N33P (asparagine mutated to arginine and proline). The vector construction method is as described above.

[0284] 3.2 Cell transfection expression

[0285] according to Transfection was performed according to the Transfection Reagent (Mirus) instructions. As described below, ExpiCHO-S cells (Thermo) were cultured in complete medium (…). High Yield Expression System, containing 30 mL / L Poloxamer 188 solution 10% and 20 mL / L L-glutamine 200 mM (Mirus), was used for subculturing 24 hours before transfection to ensure a cell density of 4 x 10⁻⁶ cells on the second day. 6 Cells / mL, diluted to 2x10⁻⁶ cells / mL before transfection 6 Cells / mL. Add 25 μg each of the light and heavy chain plasmids at a 1:1 ratio to 12.5 mL of complete culture medium and mix well. Then add 50 μL of transfection reagent. After gently inverting and mixing the Transfection Reagent (Mirus), let it stand for 4 minutes. Then, while shaking, add it dropwise to 50 mL of diluted cells, and add 1 mL of CHOgro-titer Enhancer (Mirus) dropwise. Immediately after transfection, incubate at 32°C and 5% CO2. Add 5% EfficientFeed C+AGTSupplement (Thermo) every other day for a total of 7 days.

[0286] 3.3 Antibody purification

[0287] After culturing in shake flasks for 7 days, the cell supernatant was collected, centrifuged at 4000 rpm for 20 minutes, and the supernatant was filtered through a 0.22 μm filter (Milipore) and purified by protein A affinity chromatography. In short, a HiTrap Mabselect suRe pre-packed column (Cytiva) was equilibrated with 5 to 10 column volumes of 20 mM PB + 0.15 M NaCl buffer. Using an AKTA Avant 150 chromatography system (Cytiva), the filtered supernatant was loaded onto the column, followed by washing with 3 column volumes of 20 mM PB + 0.15 M NaCl buffer, then 1 column volume of 20 mM PB + 1 M NaCl buffer, and finally 20 mM PB until the baseline stabilized. Finally, the antibody was eluted with 20 mM citric acid (adjusted to pH 3.0 with 20 mM sodium citrate), and the peak shape of 200 mAu-200 mAu was collected. The eluted antibody was immediately neutralized with neutralization buffer (1 M Tris-HCl, pH 9.0) and placed in a 1.5 mL tube for freezing at -80°C for later use.

[0288] Example 4

[0289] ELISA Affinity Detection of Humanized Monoclonal Antibodies and Bispecific Antibodies

[0290] The relative binding activity of each antibody to human GPC3 protein was determined using ELISA. Specifically, 100 μL of recombinant human GPC3 protein (Antigen 1, 1 μg / mL) was coated into each well of a 96-well plate and incubated overnight at 4°C. Next, the plates were blocked at 37°C for 2 hours with PBST containing 1% BSA (containing 0.05% Tween-20) (200 μL / well). After washing three times with PBST, each humanized monoclonal antibody / bispecific antibody was serially diluted with PBST containing 1% BSA and added sequentially to 96-well plates (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, diluted sequentially in 4-fold increments for 8 spots. The working concentrations of the humanized monoclonal antibodies h1B12, h2D8, hYP7HM, h1B12-N33P-G34P, h1B12-G34Y, and GC33 were 30 nM, 15 nM, 15 nM, and so on, diluted sequentially in 4-fold increments for 8 spots. The plates were incubated at 37°C for 1 hour and washed three times with PBST. Then, 100 μL of Anti-Human IgG-FC-HRP (Sigma, 1 / 30000 dilution) was added to each well, and the mixture was incubated at 37°C for 1 h. After washing three times with PBST, 50 μL of TMB (SURMOPICS) was added, and the reaction was terminated with 1 M H₂SO₄. The OD values ​​were measured at 450 nm-570 nm using a microplate reader. The EC50 values ​​of each bispecific antibody and humanized monoclonal antibody binding to GPC3 protein are shown in Table 8, and the results are recorded in [the table / document / etc.]. Figure 2 and Figure 8A middle.

[0291] Table 8. EC50 of bispecific antibody binding to human GPC3 protein

[0292]

[0293] According to Table 8, Figure 2 and Figure 8A The results showed that, except for h1B12-N33P-G34P-VL and h1B12-G34Y monoclonal antibodies, all other humanized monoclonal antibodies and bispecific antibodies had high affinity for GPC3 protein (sub-nM level).

[0294] Example 5

[0295] Flow cytometry detection of the binding of humanized monoclonal antibodies and bispecific antibodies to tumor cells

[0296] Flow cytometry was used to determine the affinity of each anti-GPC3 antibody for HepG2, Hep3B, or Huh-7 cells. Specifically, each humanized monoclonal antibody or bispecific antibody was serially diluted with FACS Buffer (PBS + 5% FBS) and added sequentially to 96-well U-shaped plates as follows: 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: 300 nM, 200 nM, 200 nM, and so on, diluted in 3-fold increments for 8 spots. The working concentrations of h2D8 and hYP7HM were: 400 nM, 200 nM, 200 nM, and so on, diluted in 3-fold increments for 8 spots. HepG2 and Hep3B cells were digested with trypsin, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in FACS Buffer at a concentration of 2 × 10⁻⁶. 5 Add cells per well to a 96-well plate, mix gently, and incubate on ice for 90 min. Centrifuge at 3500 rpm for 3 min at 4°C, discard the supernatant, and resuspend the cells in 250 μL of pre-chilled FACS Buffer. Repeat centrifugation and washing three times. Add 100 μL of diluted PE anti-human IgG FC fluorescent secondary antibody (BioLegend, 1 μL / 2×10⁻⁶) to each well. 5 Cells were prepared individually and incubated on ice in the dark for 60 min. The supernatant was discarded, and the cells were washed twice. The cells were then resuspended in 200 μL of FACS Buffer. MFI values ​​were measured using an Attune NxT flow cytometer (Thermo), and the data were processed using GraphPad Prism software. The binding of various bispecific antibodies or humanized monoclonal antibodies to the cell surface GPC3 antigen is shown in the figure. Figure 3A , Figure 8B (HepG2 cells) and Figure 3B , Figure 8C (Hep3B cells) Figure 8D (Huh-7 cells) and as shown in Tables 9 and 10, according to Figures 3A-3B , Figure 8B-8D The results in Tables 9 and 10 show that, except for the h1B12-G34Y monoclonal antibody, the other monoclonal antibodies or biclonal antibodies of the present invention have good affinity for HepG2, Hep3B or Huh-7 cells.

[0297] Table 9 EC50 of monoclonal antibody binding to HepG2 cells

[0298]

[0299] Table 10 EC50 of monoclonal antibody binding to Hep3B cells

[0300]

[0301] Example 6

[0302] Flow cytometry detection of internalization efficiency of humanized monoclonal antibodies / bispecific antibodies

[0303] In HepG2 and Hep3B cells, the internalization efficiency of each anti-GPC3 antibody was determined by flow cytometry. Specifically, each humanized monoclonal antibody or bispecific antibody was diluted to 50 nM with FACS Buffer and added at 50 μL / well to a 96-well U-shaped plate. Antibody-cell binding was performed according to the method described in Example 5. The antibodies and cells were incubated for different times: samples incubated on ice were set as background at 0 h, and the remaining samples were incubated at 37°C for 1 h, 2 h, and 4 h, respectively. Immediately after incubation, the cells were transferred to ice. 200 μL of FACS buffer was added to each well, mixed, and centrifuged at 3500 rpm for 3 min. The supernatant was discarded, and 100 μL of pre-chilled PE anti-human IgG FC fluorescent secondary antibody (BioLegend, 1 μL / 2 × 10⁻⁶) was added. 5 (Prepared per cell type), incubated on ice in the dark for 60 min. The supernatant was discarded, and the cells were washed twice. Finally, the cells were resuspended in 200 μL of FACS Buffer, and the MFI value was measured. Data were processed using GraphPadPrism software. The cell internalization efficiency of bispecific antibodies or humanized monoclonal antibodies was calculated using the formula: (1 - antibody MFI value at 37℃ / antibody MFI value at 4℃) × 100%. Specific results are shown below. Figure 4A (HepG2 cells) and Figure 4B As shown in (Hep3B cells), according to Figure 4A and Figure 4B It can be seen that the bispecific antibody of the present invention has very good internalization efficiency in HepG2 and Hep3B cells, and the internalization rate of the bispecific antibody of the present invention is also faster.

[0304] Example 7

[0305] Flow cytometry detection of internalization efficiency of humanized monoclonal antibodies / bispecific antibodies

[0306] In Hep3B cells, the internalization efficiency of each anti-GPC3 antibody was determined by flow cytometry. Specifically, each humanized monoclonal antibody or bispecific antibody was diluted to 200 nM with FACS Buffer and added at 50 μL / well to a 96-well U-shaped plate. Antibody-cell binding was performed according to the method described in Example 5. The antibodies and cells were incubated for different times: samples incubated on ice were set as background at 0 h, and the remaining samples were incubated at 37°C for 1 h, 2 h, and 4 h, respectively. Immediately after incubation, the cells were transferred to ice. 200 μL of FACS buffer was added to each well, mixed, and centrifuged at 3500 rpm for 3 min. The supernatant was discarded, and 100 μL of pre-chilled PE anti-human IgG FC fluorescent secondary antibody (BioLegend, 1 μL / 2 × 10⁻⁶) was added. 5 (Prepared by individual cells), incubated on ice in the dark for 60 min. The supernatant was discarded, and the cells were washed twice. Finally, the cells were resuspended in 200 μL of FACS Buffer, and the MFI value was measured. Data were processed using GraphPad Prism software. The cell internalization efficiency of the bispecific antibody or humanized monoclonal antibody in Example 2 or 3 was calculated using the formula: (1 - antibody MFI value at 37℃ / antibody MFI value at 4℃) × 100%. Specific results are shown below. Figure 9 As shown in (Hep3B cells), according to Figure 9 It can be seen that the monoclonal antibody of the present invention has a very good internalization efficiency on Hep3B cells, and the internalization rate of the monoclonal antibody of the present invention is also significantly better than that of GC33.

[0307] Example 8

[0308] Preparation of antibody-drug conjugates

[0309] Replace the monoclonal antibody or bispecific antibody stock solution with 20 mM PBS buffer (pH = 7.2) and adjust the concentration to approximately 5 mg / mL. Add 250 mM EDTA solution at a 50:1 volume ratio (antibody: EDTA) and mix thoroughly. Then, depending on the combination of different monoclonal antibody / bispecific antibody and linker-payload (LP), add 1-12 times the excess molar ratio (relative to antibody) of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), mix thoroughly, and incubate at room temperature (25°C) for 3 hours for reduction. Add an appropriate amount of DMSO to the above reaction solution, followed by 6-12 times the excess molar ratio (relative to antibody) of LP drug (5 mM / 10 mM pre-dissolved in DMSO), ensuring that the volume percentage of DMSO in the reaction system does not exceed 15%. Mix thoroughly and react at room temperature for 1.5 hours. Then add N-acetyl-L-cysteine ​​(NAC) solution and incubate at room temperature for 10 minutes to terminate the reaction.

[0310] Ultrafiltration desalting was performed. The reaction solution was transferred to a 10KD ultrafiltration tube (Millipore), PBS buffer (pH 6.0) was added, and the solution was concentrated to the desired volume by centrifugation at 3500g. PBS was added again, and the concentration was repeated 5 times. The product was then filtered through a 0.22μm filter membrane (Millipore) and stored at -80℃.

[0311] 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 conjugation ratio (DAR) and naked antibody ratio were analyzed by hydrophobic interaction chromatography (HIC).

[0312] Example 9

[0313] Flow cytometry detection of antibody-drug conjugates binding to tumor cells

[0314] Following the method described in Example 5, flow cytometry was used to determine the affinity of each antibody-drug conjugate for Hep3B and Huh-7 cells. Cell processing, antibody-drug conjugate dilution and loading, and flow cytometry analysis were all performed as described in Example 5. The binding of each antibody-drug conjugate to the GPC3 antigen on the cell surface is shown below. Figure 5A (Hep3B cells) and Figure 5B As shown in (Huh-7 cells), according to Figure 5A and Figure 5B The results show that the monoclonal antibody ADC or biclonal antibody ADC of the present invention has very good affinity for Hep3B or Huh-7 cells.

[0315] Example 10

[0316] Detection of the killing effect of antibody-drug conjugates on tumor cells

[0317] The killing effects of various antibody-drug conjugates on HepG2, Hep3B, and Huh-7 hepatocellular carcinoma cells were determined using the Cell Counting Kit-8 (Dojindo). Specifically, HepG2, Hep3B, and Huh-7 cells were cultured in 10% FBS (Gibco) + DMEM (Corning) medium. When cell confluence reached 75% or higher, cells were digested with trypsin (0.25% Trypsin-EDTA) and counted at 1.5 × 10⁻⁶ cells / cells. 4Cells / mL, 160 μL / well (2400 cells / well) were seeded into 96-well plates and incubated at 37°C with 5% CO2. Then, using 10% FBS + DMEM medium, each antibody-drug conjugate was diluted to 333.5 nM and 5 nM, respectively, and added to the 96-well plates at 40 μL / well, resulting in final concentrations of 66.7 nM (high concentration) and 1 nM (low concentration), respectively. Duplicate wells were prepared, and the plates were then incubated at 37°C with 5% CO2. Tumor cell viability was assessed using a Cell Counting Kit-8 on days 1, 2, 3, and 4. Specific killing results are shown below. 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) as shown, according to Figure 6A , 6B As can be seen from 6C and 6D, the monoclonal antibody ADC or biclonal antibody ADC of the present invention has a very good killing effect on HepG2, Hep3B or Huh-7 cells at both high and low concentrations.

[0318] Example 11

[0319] Detection of tumor cell killing by monoclonal or bispecific antibodies

[0320] The killing effects of various anti-GPC3 antibodies on HepG2 and Hep3B cells were determined using the Cell Counting Kit-8 (Dojindo). Specifically, the anti-GPC3 antibodies were diluted to 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, and 0.13 nM respectively using 10% FBS + DMEM medium. HepG2 and Hep3B cells were then inoculated at a concentration of 1×10⁻⁶ cells / mL. 4 100 μL / well (10,000 cells / well) of the prepared anti-GPC3 antibody was added to each well of a 96-well plate. Then, 100 μL / well of the prepared anti-GPC3 antibody was added to each well, resulting in final concentrations of 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, and 0.06 nM, with duplicate wells. The plates were incubated at 37°C with 5% CO2 for 96 hours. The viability of the tumor cells was detected using a Cell Counting Kit-8. Specific results are shown below. Figure 7A (HepG2 cells) Figure 7B As shown in (Hep3B cells), according to Figure 7A and Figure 7B It can be seen that single or double antibodies alone have almost no killing effect on HepG2 or Hep3B cells.

[0321] Although the invention has been described through one or more embodiments, it should be understood that the invention is not limited to these embodiments, and the specification is intended to cover all alternatives, modifications, and variations falling within the spirit and broad scope of the appended claims. All references cited in this invention are incorporated herein 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 its antigen-binding fragment includes a heavy chain variable region (VH) and a light chain variable region (VL); The heavy chain variable region (VH) includes a CDR region, which includes CDR1, CDR2 and CDR3 regions, and the CDR1, CDR2 and CDR3 regions are defined according to IMGT, Kabat or Contact. The light chain variable region (VL) includes a CDR region, which includes CDR1, CDR2 and CDR3 regions, and the CDR1, CDR2 and CDR3 regions are defined according to IMGT, Kabat or Contact. According to the IMGT definition, the CDR1-3 regions of the heavy chain variable region (VH) are amino acid residues at positions 26-33, 51-58, and 97-104 of SEQ ID No: 14, respectively, and the CDR1-3 regions of the light chain variable region (VL) are amino acid residues at positions 27-37, 55-56, and 94-102 of any of the sequences shown in SEQ ID Nos: 15 and 56-59, respectively. According to the Kabat definition, the CDR1-3 regions of the heavy chain variable region (VH) are amino acid residues at positions 31-35, 50-66, and 99-104 of SEQ ID No: 14, respectively, and the CDR1-3 regions of the light chain variable region (VL) are amino acid residues at positions 24-39, 55-61, and 94-102 of any of the sequences shown in SEQ ID Nos: 15 and 56-59, respectively. According to the Contact definition, the CDR1-3 regions of the heavy chain variable region (VH) are amino acid residues at positions 30-35, 47-59, and 97-103 of SEQ ID No: 14, respectively, and the CDR1-3 regions of the light chain variable region (VL) are amino acid residues at positions 30-41, 51-60, and 94-101 of any of the sequences shown in SEQ ID Nos: 15 and 56-59, respectively.

2. The anti-GPC3 monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, According to the IMGT definition, the CDR1-3 regions of the heavy chain variable region (VH) are sequentially the amino acid sequences GYTFTDYE, IHPGSGGT, and TRFYSFAY, and the CDR1-3 regions of the light chain variable region (VL) are sequentially the amino acid sequences QSLVHSNXNTY, KV, and YQSRHVPYT, where X is selected from any one of the amino acids G, H, M, L, and S. According to the Kabat definition, the CDR1-3 regions of the heavy chain variable region (VH) are, in order, the amino acid sequences DYEIH, AIHPGSGGTAYNQKFKG, and FYSFAY, and the CDR1-3 regions of the light chain variable region (VL) are, in order, the amino acid sequences RSTQSLVHSNXNTYLH, KVSNRFS, and YQSRHVPYT, where X is selected from any one of the amino acids G, H, M, L, and S.

3. The anti-GPC3 monoclonal antibody or its antigen-binding fragment according to any one of claims 1-2, characterized in that, The heavy chain variable region (VH) contains the same sequence as the amino acid sequence SEQ ID No: 14; The light chain variable region (VL) contains the same sequence as the amino acid sequences SEQ ID No: 15, SEQ ID No: 56, SEQ ID No: 57, SEQ ID No: 58 or SEQ ID No:

59.

4. The anti-GPC3 monoclonal antibody or its antigen-binding fragment according to claim 3, characterized in that, The anti-GPC3 monoclonal antibody or its antigen-binding fragment comprises a heavy chain (H) and a light chain (L), wherein the heavy chain (H) contains a sequence identical to the amino acid sequence SEQ ID No: 61; The light chain (L) contains the same sequence as SEQ ID No: 62, SEQ ID No: 66, SEQ ID No: 67, SEQ ID No: 68 or SEQ ID No:

69.

5. An anti-GPC3 bispecific antibody or its antigen-binding fragment, characterized in that: The anti-GPC3 bispecific antibody or its antigen-binding fragment contains a first GPC3 antigen-binding domain and a second GPC3 antigen-binding domain. The first GPC3 antigen-binding domain includes a first heavy chain variable region (AVH) and a first light chain variable region (AVL); The second GPC3 antigen-binding domain includes a second heavy chain variable region (BVH) and a second light chain variable region (BVL). The CDR1-3 regions of the first heavy chain variable region (AVH) are, in sequence, the CDR1-3 regions of the heavy chain variable region (VH) as described in any one of claims 1-2, and the CDR1-3 regions of the first light chain variable region (AVL) are, in sequence, the CDR1-3 regions of the light chain variable region (VL) as described in any one of claims 1-2.

6. The anti-GPC3 bispecific antibody or its antigen-binding fragment according to claim 5, characterized in that: According to the IMGT definition, the CDR1-3 regions of the second heavy chain variable region (BVH) are, in order, amino acid residues at positions 26-33, 51-60, and 99-106 of SEQ ID No: 3 or 5, and the CDR1-3 regions of the second light chain variable region (BVL) are, in order, amino acid residues at positions 27-38, 56-57, and 95-103 of SEQ ID No: 4; According to Kabat's definition, the CDR1-3 regions of the second heavy chain variable region (BVH) are, in order: amino acid residues at positions 31-35, 50-68, and 101-106 of SEQ ID No. 3 or 5; and the CDR1-3 regions of the second light chain variable region (BVL) are, in order: amino acid residues at positions 24-40, 56-62, and 95-103 of SEQ ID No. 4; or, According to the definition of Contact, the CDR1-3 regions of the second heavy chain variable region (BVH) are, in order: amino acid residues at positions 30-35, 47-61, and 99-105 of SEQ ID No: 3 or 5, and the CDR1-3 regions of the second light chain variable region (BVL) are, in order: amino acid residues at positions 30-42, 52-61, and 95-102 of SEQ ID No:

4.

7. The anti-GPC3 bispecific antibody or its antigen-binding fragment according to claim 5, characterized in that, According to the IMGT definition, the CDR1-3 regions of the second heavy chain variable region (BVH) are, in order: amino acid residues at positions 26-33, 51-58, and 97-103 of SEQ ID No: 18, and the CDR1-3 regions of the second light chain variable region (BVL) are, in order: amino acid residues at positions 27-37, 55-56, and 94-102 of SEQ ID No: 19; According to Kabat's definition, the CDR1-3 regions of the second heavy chain variable region (BVH) are, in order, amino acid residues 31-35, 50-66, and 99-103 of SEQ ID No: 18, and the CDR1-3 regions of the second light chain variable region (BVL) are, in order, amino acid residues 24-39, 55-61, and 94-102 of SEQ ID No: 19; or, According to the definition of Contact, the CDR1-3 regions of the second heavy chain variable region (BVH) are, in order, amino acid residues at positions 30-35, 47-59, and 97-102 of SEQ ID No: 18, and the CDR1-3 regions of the second light chain variable region (BVL) are, in order, amino acid residues at positions 30-41, 51-60, and 94-101 of SEQ ID No:

19.

8. The anti-GPC3 bispecific antibody or its antigen-binding fragment according to any one of claims 6 or 7, characterized in that, The first heavy chain variable region (AVH) is selected from SEQ ID No: 14; The first light chain variable region (AVL) is selected from any one of SEQ ID Nos: 15, 56-59; The second heavy chain variable region (BVH) is selected from any one of SEQ ID Nos: 3 or 5; The second light chain variable region (BVL) is selected from SEQ ID No: 4; or, The first heavy chain variable region (AVH) is selected from SEQ ID No: 14; The first light chain variable region (AVL) is selected from any one of SEQ ID Nos: 15, 56-59; The second heavy chain variable region (BVH) is selected from SEQ ID No: 18; The second light chain variable region (BVL) is selected from SEQ ID No:

19.

9. The anti-GPC3 bispecific antibody or its antigen-binding fragment according to claim 8, characterized in that, The first heavy chain variable region (AVH) is selected from SEQ ID No: 14; The first light chain variable region (AVL) is selected from SEQ ID No: 15; The second heavy chain variable region (BVH) is selected from any one of SEQ ID Nos: 3 or 5; The second light chain variable region (BVL) is selected from SEQ ID No:

4.

10. The anti-GPC3 bispecific antibody or its antigen-binding fragment according to any one of claims 5-7 or 9, 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 by a connector L1 to form an scFv, and the scFv is further connected to the heavy chain or light chain end of the second GPC3 antigen-binding domain by a connector L2. (a2) The heavy chain variable region and the light chain variable region of the second GPC3 antigen-binding domain are connected by a connector L1 to form an scFv, and the scFv is further connected to the heavy chain or light chain end of the first GPC3 antigen-binding domain by a connector L2. The connectors L1 and L2 are each independently selected from (GGGGS)n, where n is an integer of 1, 2, 3, 4, 5 or 6.

11. The anti-GPC3 bispecific antibody or its antigen-binding fragment according to claim 10, characterized in that, The anti-GPC3 bispecific antibody comprises a first polypeptide chain and a second polypeptide chain; Wherein, the structure of the first polypeptide chain is selected from any one of the following (b1)-(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, which are selected from (GGGGS)n, where n is an integer of 1, 2, 3, 4, 5 or 6; CH1 is the CH1 of the antibody heavy chain constant region; and [Fcx] represents the Fc domain of the antibody heavy chain constant region, which contains CH2 and CH3. The structure of the second polypeptide chain is selected from either (c1) or (c2): (c1)[AVL]-[CL]; (c2)[BVL]-[CL]; [CL] refers to the constant region of the antibody light chain.

12. The anti-GPC3 bispecific antibody or its antigen-binding fragment according to claim 11, characterized in that, The structure of the first polypeptide chain is selected from (b1) or (b2), and the structure of the second polypeptide chain is selected from (c1).

13. The anti-GPC3 bispecific antibody or its antigen-binding fragment according to claim 12, characterized in that, The first polypeptide chain of the anti-GPC3 bispecific antibody comprises any one of SEQ ID Nos: 36-43.

14. The anti-GPC3 bispecific antibody or its antigen-binding fragment according to claim 12 or 13, characterized in that, The second polypeptide chain of the anti-GPC3 bispecific antibody is composed of a light chain variable region (VL) and a light chain constant region (CL), wherein the VL includes any one of SEQ ID Nos: 15, 56-59, and the CL includes SEQ ID No:

55.

15. The anti-GPC3 bispecific antibody or its antigen-binding fragment according to claim 14, characterized in that, The second polypeptide chain of the anti-GPC3 bispecific antibody consists of a light chain variable region (VL) and a light chain constant region (CL), wherein the VL contains: SEQ ID Nos: 15, and the CL contains: SEQ ID Nos:

55.

16. The anti-GPC3 monoclonal antibody or its antigen-binding fragment according to any one of claims 1-2, or the anti-GPC3 bispecific antibody or its antigen-binding fragment according to any one of claims 5-7, 9, 11-13, 15, characterized in that, The anti-GPC3 monoclonal antibody or its antigen-binding fragment, or the anti-GPC3 bispecific antibody or its antigen-binding fragment, is a humanized antibody or its antigen-binding fragment.

17. A pharmaceutical composition, characterized in that, It comprises the anti-GPC3 monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-4, 16, or the anti-GPC3 bispecific antibody or its antigen-binding fragment as described in any one of claims 5-16, and a pharmaceutically acceptable carrier.

18. A polynucleotide, characterized in that, The polynucleotide encodes the anti-GPC3 monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-4 and 16, or the anti-GPC3 bispecific antibody or its antigen-binding fragment as described in any one of claims 5-16.

19. An expression carrier, 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, It comprises an anti-GPC3 monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-4, 16, or an anti-GPC3 bispecific antibody or its antigen-binding fragment as described in any one of claims 5-16, and a drug or toxin, wherein the drug or toxin is selected from: PBD or DUBA, wherein, The anti-GPC3 monoclonal antibody or its antigen-binding fragment, or the anti-GPC3 bispecific antibody or its antigen-binding fragment, is coupled to PBD via a maleimide-dPEG8-VA-PABA linker. The anti-GPC3 monoclonal antibody or its antigen-binding fragment, or the anti-GPC3 bispecific antibody or its antigen-binding fragment, is coupled to DUBA via a Vc-seco linker.

22. A pharmaceutical composition, characterized in that, It comprises the anti-GPC3 monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-4, 16, or the anti-GPC3 bispecific antibody or its antigen-binding fragment as described in any one of claims 5-16, or the antibody-drug conjugate as described in claim 21, and one or more pharmaceutically acceptable carriers.

23. Use of the anti-GPC3 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4, 16, or the anti-GPC3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5-16, or the pharmaceutical composition according to claim 17 or 22, or the antibody-drug conjugate according to claim 21, in the preparation of a medicament for the treatment or prevention of cancer, wherein the cancer is a cancer expressing GPC3, and the cancer is selected from liver cancer, gastric cancer, colorectal cancer, lung cancer, or ovarian cancer.

24. The use according to claim 23, characterized in that, The cancer in question is liver cancer.

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