Bispecific antibody or antigen binding fragment targeting GPC3 and application of bispecific antibody or antigen binding fragment

Abstract

The invention relates to a bispecific antibody targeting GPC-3 and an antigen binding fragment thereof, and also provides a nucleotide molecule for coding the antibody, an expression vector and a host cell for expressing the antibody, and a preparation method of the antibody. In addition, the invention also provides a pharmaceutical composition containing the antibody, and an application of the antibody in preparing a medicine for treating cancer.

Description

A bispecific antibody or antigen-binding fragment targeting GPC3 and its use Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to a bispecific antibody targeting GPC3 or an antigen-binding fragment thereof and uses thereof. Background Art

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

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

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

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

[0006] Despite this, due to primary or secondary tumor resistance and tumor heterogeneity, it has become increasingly difficult to inhibit tumor growth by relying on single-epitope antibodies and the physiological effects of the antibodies themselves. Bispecific antibodies that simultaneously recognize two GPC3 epitopes can have stronger binding ability to tumor cells, thereby enabling the development of more lethal pharmaceutical compositions (International Patent Application WO2024067764).

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

[0008] In summary, the antibodies that specifically bind to the GPC3 target in the prior art have problems such as insufficient binding ability and insufficient cell killing. Therefore, there is an urgent need to develop a GPC3-targeting bispecific antibody or its antigen-binding fragment that has both strong binding ability to the GPC3 target and good cytotoxicity, as well as an ADC drug containing the bispecific antibody or antigen-binding fragment.

[0009] Summary of the Invention

[0010] The purpose of the present invention is to provide a bispecific antibody or antigen-binding fragment thereof targeting GPC3, in order to solve the problems existing in the prior art of insufficient binding ability and cell cytotoxicity of antibodies targeting GPC3.

[0011] In one aspect, the present invention provides a bispecific antibody or an antigen-binding fragment thereof targeting GPC3, wherein the bispecific antibody or the antigen-binding fragment thereof targeting GPC3 comprises a first GPC3 antigen-binding domain and a second GPC3 antigen-binding domain.

[0012] The first GPC3 antigen-binding domain comprises a first heavy chain variable region (AVH) and a first light chain variable region (AVL);

[0013] The second GPC3 antigen binding domain comprises a second heavy chain variable region (BVH) and a second light chain variable region (BVL);

[0014] The first heavy chain variable region (AVH) comprises a CDR region comprising a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR region of any one of the amino acid sequences of SEQ ID Nos: 1-6; the first light chain variable region (AVL) comprises a CDR region comprising a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR region of SEQ ID No: 7;

[0015] the second heavy chain variable region (BVH) comprising a CDR region comprising a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR region of the amino acid sequence of SEQ ID Nos: 8 or 9; the second light chain variable region (BVL) comprising a CDR region comprising a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR region of any one of the amino acid sequences of SEQ ID Nos: 10-19;

[0016] The CDR region comprises CDR1, CDR2 and CDR3 regions; the CDR1, CDR2 and CDR3 regions are defined according to IMGT, Kabat, Chothia, AbM or Contact.

[0017] In some preferred technical embodiments, the AVH comprises a CDR region, wherein the CDR region comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR region of any one of the amino acid sequences SEQ ID Nos: 1-6; the AVL comprises a CDR region, wherein the CDR region comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR region of the amino acid sequence SEQ ID No: 7; and

[0018] The BVH comprises a CDR region, which comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR region of the amino acid sequence of SEQ ID Nos: 8 or 9; the BVL comprises a CDR region, which comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR region of any one of the amino acid sequences of SEQ ID Nos: 10 or 19.

[0019] On the other hand, as a preferred technical solution of the present invention, the first heavy chain variable region (AVH) comprises a CDR region, and the CDR region comprises a sequence identical to the CDR region of any one of the amino acid sequences of SEQ ID Nos: 1-6 and 38-43; the first light chain variable region (AVL) comprises a CDR region, and the CDR region comprises a sequence identical to the CDR region of the amino acid sequence of SEQ ID No: 7;

[0020] The second heavy chain variable region (BVH) comprises a CDR region comprising a sequence identical to the CDR region of the amino acid sequence of SEQ ID Nos: 8 or 9; the second light chain variable region (BVL) comprises a CDR region comprising a sequence identical to the CDR region of any one of the amino acid sequences of SEQ ID Nos: 10-19;

[0021] The CDR region comprises CDR1, CDR2 and CDR3 regions, and the CDR1, CDR2 and CDR3 regions are defined according to IMGT, Kabat, Chothia, AbM or Contact.

[0022] On the other hand, as a preferred technical solution of the present invention, the AVH comprises a CDR region, and the CDR region comprises a sequence identical to the CDR region of the amino acid sequence SEQ ID Nos: 1 or 6; the AVL comprises a CDR region, and the CDR region comprises a sequence identical to the CDR region of the amino acid sequence SEQ ID No: 7;

[0023] The BVH comprises a CDR region comprising a sequence identical to that of the CDR region of the amino acid sequence SEQ ID No: 8; the BVL comprises a CDR region comprising a sequence identical to that of the CDR region of the amino acid sequence SEQ ID Nos: 10 or 11.

[0024] On the other hand, as a preferred technical solution of the present invention, the CDR regions of AVH and AVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: 1 and 7, or consist of the said sequences; the CDR regions of AVH and AVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: 6 and 7, or consist of the said sequences; the CDR regions of AVH and AVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: 38 and 7, or consist of the said sequences; the CDR regions of AVH and AVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: 39 and 7, or consist of the said sequences; the CDR regions of AVH and AVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: 40 and 7, or consist of the said sequences; the CDR regions of AVH and AVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: 41 and 7, or consist of the said sequences; the CDR regions of AVH and AVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: NO: 42 and 7 CDR region identical sequences, or consisting of said sequences; or, the CDR regions of AVH and AVL comprise or consist of sequences identical sequences to the CDR regions of SEQ ID NO: 43 and 7, respectively;

[0025] The CDR regions of the BVH and BVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: 9 and 19, or consist of the said sequences; the CDR regions of the BVH and BVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: 8 and 10, or consist of the said sequences; the CDR regions of the BVH and BVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: 8 and 11, or consist of the said sequences; the CDR regions of the BVH and BVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: 8 and 14, or consist of the said sequences; the CDR regions of the BVH and BVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: 8 and 15, or consist of the said sequences; the CDR regions of the BVH and BVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: 8 and 16, or consist of the said sequences; the CDR regions of the BVH and BVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: The CDR regions of BVH and BVL comprise the same sequences as the CDR regions of SEQ ID NOs: 8 and 17, or consist of the said sequences; the CDR regions of BVH and BVL comprise the same sequences as the CDR regions of SEQ ID NOs: 8 and 18, respectively, or consist of the said sequences.

[0026] On the other hand, as a preferred technical solution of the present invention, the AVH and AVL as well as the BVH and BVL are selected from the following groups:

[0027] (1) The CDR regions of AVH and AVL comprise the same sequences as the CDR regions of SEQ ID NOs: 1 and 7, respectively, or consist of the said sequences; and the CDR regions of BVH and BVL comprise the same sequences as the CDR regions of SEQ ID NOs: 8 and 11, respectively, or consist of the said sequences;

[0028] (2) the CDR regions of the AVH and AVL comprise the same sequences as the CDR regions of SEQ ID NOs: 6 and 7, respectively, or consist of the said sequences; and the CDR regions of the BVH and BVL comprise the same sequences as the CDR regions of SEQ ID NOs: 8 and 11, respectively, or consist of the said sequences;

[0029] (3) the CDR regions of the AVH and AVL comprise the same sequences as the CDR regions of SEQ ID NOs: 1 and 7, respectively, or consist of the said sequences; and the CDR regions of the BVH and BVL comprise the same sequences as the CDR regions of SEQ ID NOs: 8 and 10, respectively, or consist of the said sequences; or

[0030] (4) The CDR regions of the AVH and AVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: 6 and 7, or consist of the said sequences; and the CDR regions of the BVH and BVL respectively comprise the same sequences as the CDR regions of SEQ ID NOs: 8 and 10, or consist of the said sequences.

[0031] Furthermore, the first and second GPC3 antigen-binding domains can each be or consist of an animal-derived antibody, a humanized antibody, a fully human antibody, a chimeric antibody, or an affinity-optimized antibody. More preferably, they are humanized antibodies, human-animal chimeric antibodies (e.g., 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, or other subtypes. IgG1, IgG2, or IgG4 subtypes are preferred.

[0032] In some embodiments, the first or second GPC3 antigen-binding domain further comprises a human immunoglobulin Fc region, for example, a human IgG1, IgG2, or IgG4 Fc region. Amino acid modifications in the Fc region alter effector function activity relative to the activity of the parent Fc region, for example, altering (i.e., increasing or decreasing) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding. In one or more embodiments, the Fc region comprises one or more of the following amino acid mutations: L234A, L235A, P329G, S239C, M428L, or N434S. In some embodiments, the Fc region comprises an S239C mutation. In some embodiments, the Fc region comprises L234A, L235A, P329G, and S239C mutations. In some embodiments, the Fc region comprises L234A, L235A, and P329G mutations. In some embodiments, the Fc region comprises an S239C mutation. In some embodiments, the Fc region comprises an L234A, L235A, P329G, S239C, M428L, and N434S mutation. In some embodiments, the Fc region comprises an L234A, L235A, P329G, M428L, and N434S mutation. In some embodiments, the Fc region comprises an M428L and N434S mutation. The above mutations in the Fc region are numbered according to EU numbering conventions.

[0033] In some embodiments, the first or second GPC3 antigen-binding domain comprises a heavy chain constant region selected from the sequence represented by amino acid residues 120-449 of SEQ ID NO: 30 or amino acid residues 122-451 of any of SEQ ID NOs: 21, 31-32, or a sequence having 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 thereto.

[0034] In some embodiments, the first or second GPC3 antigen-binding domain comprises a light chain constant region selected from the sequence set forth at amino acid residues 108-214 of SEQ ID NO: 25, or a sequence having 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 thereto.

[0035] Furthermore, the first GPC3 antigen binding domain and the second GPC3 antigen binding domain specifically bind to different epitopes of GPC3.

[0036] In one aspect, the present invention also provides a bispecific antibody or antigen-binding fragment thereof targeting GPC3, wherein the first heavy chain variable region (AVH) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID Nos: 1-6, 38-43; the first light chain variable region (AVL) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No: 7;

[0037] The second heavy chain variable region (BVH) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID Nos: 8 or 9; the second light chain variable region (BVL) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID Nos: 10-19.

[0038] On the other hand, as a preferred technical solution of the present invention, the first heavy chain variable region (AVH) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID Nos: 1 or 6; the first light chain variable region (AVL) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No: 7;

[0039] The second heavy chain variable region (BVH) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No: 8; the second light chain variable region (BVL) comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID Nos: 10 or 11.

[0040] On the other hand, as a preferred technical solution of the present invention, the AVH and AVL respectively comprise sequences that are at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences of SEQ ID No: 1 and 7; and the BVH and BVL respectively comprise sequences that are at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences of SEQ ID No: 8 and 11.

[0041] On the other hand, as a preferred technical solution of the present invention, the AVH and AVL respectively comprise sequences that are at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences of SEQ ID No: 6 and 7; and the BVH and BVL respectively comprise sequences that are at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences of SEQ ID No: 8 and 11.

[0042] On the other hand, as a preferred technical solution of the present invention, the first heavy chain variable region (AVH) comprises a sequence identical to any one of the amino acid sequences SEQ ID Nos: 1-6, 38-43; the first light chain variable region (AVL) comprises a sequence identical to the amino acid sequence SEQ ID No: 7; the second heavy chain variable region (BVH) comprises a sequence identical to the amino acid sequence SEQ ID Nos: 8 or 9; the second light chain variable region (BVL) comprises a sequence identical to any one of the amino acid sequences SEQ ID Nos: 10-19.

[0043] On the other hand, as a preferred technical solution of the present invention, the first heavy chain variable region (AVH) comprises a sequence identical to the amino acid sequence of SEQ ID Nos: 1 or 6; the first light chain variable region (AVL) comprises a sequence identical to the amino acid sequence of SEQ ID No: 7;

[0044] The second heavy chain variable region (BVH) comprises the same sequence as the amino acid sequence of SEQ ID No: 8; the second light chain variable region (BVL) comprises the same sequence as the amino acid sequence of SEQ ID No: 10 or 11.

[0045] On the other hand, as a preferred technical solution of the present invention, the AVH and AVL respectively comprise SEQ ID NOs: 1 and 7; the AVH and AVL respectively comprise SEQ ID NOs: 6 and 7; the AVH and AVL respectively comprise SEQ ID NOs: 38 and 7; the AVH and AVL respectively comprise SEQ ID NOs: 39 and 7; the AVH and AVL respectively comprise SEQ ID NOs: 40 and 7; the AVH and AVL respectively comprise SEQ ID NOs: 41 and 7; the AVH and AVL respectively comprise SEQ ID NOs: 42 and 7; or the AVH and AVL respectively comprise SEQ ID NOs: 43 and 7;

[0046] The BVH and BVL contain SEQ ID NOs: 9 and 19, respectively; the BVH and BVL contain SEQ ID NOs: 8 and 10, respectively; the BVH and BVL contain SEQ ID NOs: 8 and 11, respectively; the BVH and BVL contain SEQ ID NOs: 8 and 14, respectively; the BVH and BVL contain SEQ ID NOs: 8 and 15, respectively; the BVH and BVL contain SEQ ID NOs: 8 and 16, respectively; the BVH and BVL contain SEQ ID NOs: 8 and 17, respectively; the BVH and BVL contain SEQ ID NOs: 8 and 18, respectively.

[0047] On the other hand, as a preferred technical solution of the present invention, the AVH and AVL as well as the BVH and BVL are selected from the following groups:

[0048] (1) the AVH and AVL comprise SEQ ID NOs: 1 and 7, respectively, and the BVH and BVL comprise SEQ ID NOs: 8 and 11, respectively;

[0049] (2) the AVH and AVL comprise SEQ ID NOs: 6 and 7, respectively, and the BVH and BVL comprise SEQ ID NOs: 8 and 11, respectively;

[0050] (3) the AVH and AVL comprise SEQ ID NOs: 1 and 7, respectively, and the BVH and BVL comprise SEQ ID NOs: 8 and 10, respectively; or

[0051] (4) The AVH and AVL comprise SEQ ID NOs: 6 and 7, respectively, and the BVH and BVL comprise SEQ ID NOs: 8 and 10, respectively.

[0052] In one aspect, the present invention further provides a bispecific antibody or antigen-binding fragment thereof targeting GPC3, wherein the connection relationship between the first GPC3 antigen-binding domain and the second GPC3 antigen-binding domain is as follows (a1) or (a2):

[0053] (a1) the heavy chain variable region and the light chain variable region of the first GPC3 antigen-binding domain are connected via a linker L1 to form an scFv, and the scFv is further connected to the heavy chain or light chain of the second GPC3 antigen-binding domain via a linker L2;

[0054] (a2) the heavy chain variable region and the light chain variable region of the second GPC3 antigen-binding domain are connected via a linker L1 to form an scFv, and the scFv is further connected to the heavy chain or light chain of the first GPC3 antigen-binding domain via a linker L2;

[0055] The linkers L1 and L2 are independently selected from (GGGGS)n, wherein n is an integer of 1, 2, 3, 4, 5 or 6;

[0056] Preferably, the scFv is further connected to the heavy chain or light chain end of the first or second GPC3 antigen binding domain via a linker L2;

[0057] More preferably, the heavy chain or light chain terminus is selected from the C-terminus or N-terminus of the heavy chain or light chain.

[0058] In some specific embodiments, the connection relationship between the first GPC3 antigen binding domain and the second GPC3 antigen binding domain is as follows (a1) or (a2):

[0059] (a1) the heavy chain variable region and the light chain variable region of the first GPC3 antigen-binding domain are connected via a linker L1 to form an scFv, the second GPC3 antigen-binding domain is in the form of IgG, and the scFv is further connected to the heavy chain or light chain of the second GPC3 antigen-binding domain via a linker L2;

[0060] (a2) the heavy chain variable region and the light chain variable region of the second GPC3 antigen-binding domain are connected via a linker L1 to form an scFv, the first GPC3 antigen-binding domain is in the form of IgG, and the scFv is further connected to the heavy chain or light chain of the first GPC3 antigen-binding domain via a linker L2;

[0061] The linkers L1 and L2 are independently selected from (GGGGS)n, wherein n is an integer of 1, 2, 3, 4, 5 or 6;

[0062] Preferably, the scFv is further connected to the heavy chain or light chain end of the first or second GPC3 antigen binding domain via a linker L2;

[0063] More preferably, the heavy chain or light chain terminus is selected from the C-terminus or N-terminus of the heavy chain or light chain.

[0064] In some specific embodiments, the first GPC3 antigen-binding domain is in the form of IgG, and the second GPC3 antigen-binding domain is in the form of ScFv; the ScFv is composed of a heavy chain variable region, a linker L1, and a light chain variable region in order from N-terminus to C-terminus; the light chain variable region of the ScFv is connected to the N-terminus of the IgG via a linker L2, preferably to the N-terminus of the light chain of the IgG.

[0065] In some specific embodiments, the second GPC3 antigen-binding domain is in the form of IgG, and the first GPC3 antigen-binding domain is in the form of ScFv. The ScFv consists of a heavy chain variable region, a linker L1, and a light chain variable region in order from N-terminus to C-terminus. The light chain variable region of the ScFv is connected to the N-terminus of the IgG via a linker L2, preferably to the N-terminus of the light chain of the IgG.

[0066] In another aspect, the present invention further provides a bispecific anti-antibody targeting GPC3 or an antigen-binding fragment thereof, wherein the bispecific anti-antibody targeting GPC3 comprises a first polypeptide chain and a second polypeptide chain.

[0067] The structure of the first polypeptide chain can be selected from any one of the following (b1) to (b10):

[0068] (b1)[BVH]-[L1]-[BVL]-[L2]-[AVH]-[CH];

[0069] (b2)[BVL]-[L1]-[BVH]-[L2]-[AVH]-[CH];

[0070] (b3)[AVH]-[L1]-[AVL]-[L2]-[BVH]-[CH];

[0071] (b4)[AVL]-[L1]-[AVH]-[L2]-[BVH]-[CH];

[0072] (b5)[AVH]-[CH]-[L2]-[BVH]-[L1]-[BVL];

[0073] (b6)[AVH]-[CH]-[L2]-[BVL]-[L1]-[BVH];

[0074] (b7)[AVH]-[CH];

[0075] (b8)[BVH]-[CH]-[L2]-[AVH]-[L1]-[AVL];

[0076] (b9)[BVH]-[CH]-[L2]-[AVL]-[L1]-[AVH];

[0077] (b10)[BVH]-[CH]

[0078] wherein [L1] and [L2] independently represent linkers, the linkers being selected from (GGGGS)n, wherein n is an integer of 1, 2, 3, 4, 5 or 6; the [CH] represents a heavy chain constant region comprising CH1, CH2 and CH3; the AVH represents a first heavy chain variable region; the AVL represents a first light chain variable region; the BVH represents a second heavy chain variable region; and the BVL represents a second light chain variable region.

[0079] And / or, the structure of the second polypeptide chain may be selected from any one of the following (c1)-(c10):

[0080] (c1)[AVL]-[CL];

[0081] (c2)[BVL]-[CL];

[0082] (c3)[BVH]-[L1]-[BVL]-[L2]-[AVL]-[CL];

[0083] (c4)[BVL]-[L1]-[BVH]-[L2]-[AVL]-[CL];

[0084] (c5)[AVL]-[CL]-[L2]-[BVL]-[L1]-[BVH];

[0085] (c6)[AVL]-[CL]-[L2]-[BVH]-[L1]-[BVL];

[0086] (c7)[AVH]-[L1]-[AVL]-[L2]-[BVL]-[CL];

[0087] (c8)[AVL]-[L1]-[AVH]-[L2]-[BVL]-[CL];

[0088] (c9)[BVL]-[CL]-[L2]-[AVL]-[L1]-[AVH];

[0089] (c10)[BVL]-[CL]-[L2]-[AVH]-[L1]-[AVL]

[0090] The [CL] is the antibody light chain constant region.

[0091] Furthermore, the first polypeptide chain (heavy chain) and the second polypeptide chain (light chain) are connected via a disulfide bond.

[0092] In some specific embodiments, the structure of the first polypeptide chain is selected from (b1), (b2), (b5), or (b6), and the structure of the second polypeptide chain is selected from (c1). The structure of the first polypeptide chain is selected from (b3), (b4), (b8), or (b9), and the structure of the second polypeptide chain is selected from (c2). The structure of the first polypeptide chain is selected from (b7), and the structure of the second polypeptide chain is selected from (c3), (c4), (c5), or (c6). The structure of the first polypeptide chain is selected from (b10), and the structure of the second polypeptide chain is selected from (c7), (c8), (c9), or (c10).

[0093] In some preferred embodiments, the structures of the first and second polypeptide chains are selected from (b1) and (c1); (b7) and (c3); (b3) and (c2); or, (b10) and (c7).

[0094] On the other hand, as a preferred technical solution of the present invention, the first polypeptide chain of the bispecific antibody targeting GPC3 or the antigen-binding fragment thereof comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID Nos: 20-24, 44-47.

[0095] On the other hand, as a preferred technical solution of the present invention, the first polypeptide chain of the bispecific antibody targeting GPC3 or the antigen-binding fragment thereof comprises an amino acid sequence identical to any one of SEQ ID Nos: 20-24, 44-47.

[0096] On the other hand, as a preferred technical solution of the present invention, the second polypeptide chain of the bispecific antibody targeting GPC3 or the antigen-binding fragment thereof comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID Nos: 25-29, 48-51.

[0097] On the other hand, as a preferred technical solution of the present invention, the second polypeptide chain of the bispecific antibody targeting GPC3 or the antigen-binding fragment thereof comprises an amino acid sequence identical to any one of SEQ ID Nos: 25-29, 48-51.

[0098] In another aspect, as a preferred technical solution of the present invention, the first and second polypeptide chains of the bispecific antibody targeting GPC3 or its antigen-binding fragment are selected from one of the following combinations:

[0099] 1) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 20, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 25;

[0100] 2) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 21, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 26;

[0101] 3) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 21, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 27;

[0102] 4) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 22, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 25;

[0103] 5) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 23, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 25;

[0104] 6) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 21, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 28;

[0105] 7) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 21, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 29;

[0106] 8) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 24, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 25;

[0107] 9) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 44, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 48;

[0108] 10) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 45, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 49;

[0109] 11) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 45, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 50;

[0110] 12) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 45, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 51;

[0111] 13) The first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 46, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 25; or

[0112] 14) The first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 47, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 25.

[0113] In another aspect, as a preferred technical solution of the present invention, the first and second polypeptide chains of the bispecific antibody targeting GPC3 or its antigen-binding fragment are selected from one of the following combinations:

[0114] 1) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 23, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 25;

[0115] 2) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 21, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 28; or

[0116] 3) The first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 45, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 50.

[0117] In one aspect, the present invention provides a bispecific antibody targeting GPC3 or an antigen-binding fragment thereof, wherein the bispecific antibody targeting GPC3 or the antigen-binding fragment thereof comprises a first GPC3 antigen-binding domain and a second GPC3 antigen-binding domain, wherein the first GPC3 antigen-binding domain and the second GPC3 antigen-binding domain specifically bind to different epitopes of GPC3.

[0118] In some preferred embodiments, the first GPC3 antigen-binding domain recognizes the same GPC3 epitope as antibody A, which comprises a heavy chain variable region set forth in SEQ ID NO: 1 or 6 and a light chain variable region set forth in SEQ ID NO: 7; and the second GPC3 antigen-binding domain recognizes the same GPC3 epitope as antibody B, which comprises a heavy chain variable region set forth in SEQ ID NO: 8 and a light chain variable region set forth in SEQ ID NO: 10 or 11.

[0119] In a preferred embodiment, the antibody A comprises the heavy chain variable region set forth in SEQ ID NO: 1 and the light chain variable region set forth in SEQ ID NO: 7, and the antibody B comprises the heavy chain variable region set forth in SEQ ID NO: 8 and the light chain variable region set forth in SEQ ID NO: 11. Alternatively, the antibody A comprises the heavy chain variable region set forth in SEQ ID NO: 6 and the light chain variable region set forth in SEQ ID NO: 7, and the antibody B comprises the heavy chain variable region set forth in SEQ ID NO: 8 and the light chain variable region set forth in SEQ ID NO: 11.

[0120] In a preferred embodiment, the epitope recognized by the first GPC3 antigen binding domain comprises at least amino acid residue 487 (N) and amino acid residue 493 (F) of the human GPC3 protein. The amino acid sequence of the human GPC3 protein is shown in NCBI reference sequence NP_004475.1.

[0121] In a preferred embodiment, the epitope recognized by the first GPC3 antigen binding domain comprises the amino acid sequence DKNLDEEGFESG.

[0122] In another preferred embodiment, the first GPC3 antigen binding domain is capable of specifically binding to the GPC3-B peptide but not to the GPC3-A peptide; wherein the amino acid sequence of the GPC3-B peptide is MPKGRVLDKNLDEEGFESGDCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDLD, and the amino acid sequence of the GPC3-A peptide is GDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHS.

[0123] On the other hand, as a preferred technical solution of the present invention, the bispecific antibody or antigen-binding fragment thereof targeting GPC3 is a humanized bispecific antibody or antigen-binding fragment thereof.

[0124] In one aspect, the present invention further provides a chimeric antigen receptor, comprising the bispecific antibody targeting GPC3 or an antigen-binding fragment thereof according to the present invention.

[0125] In one aspect, the present invention also provides a polynucleotide encoding the bispecific antibody or antigen-binding fragment thereof targeting GPC3 described herein. The polynucleotide of the present invention can be, for example, DNA or RNA and may or may not contain intronic sequences. In a preferred embodiment, the polynucleotide is a cDNA molecule. The polynucleotide of the present invention can be prepared or obtained by known means based on the amino acid sequence information of the present invention, such as by automated DNA synthesis and / or recombinant DNA technology.

[0126] As is well known in the art, multiple codons can encode the same amino acid. Therefore, nucleic acids encoding protein sequences include nucleic acids with codon degeneracy. The amino acid sequence of the present invention can be encoded by a variety of nucleic acids. The genetic code is universal and well known. The nucleic acid encoding any amino acid sequence of the present invention can be easily conceived based on the common knowledge in the art, and can be optimized for production. Although the possible number of nucleic acid sequences encoding a given amino acid is very large, given the standard table of the genetic code, and with the assistance of a calculator, those skilled in the art can easily produce every possible combination of nucleic acid sequences encoding a given amino acid.

[0127] In one aspect, the present invention further provides an expression vector comprising the polynucleotide of the present invention, including bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0128] In one aspect, the present invention further provides a host cell comprising the polynucleotide of the present invention or the expression vector of the present invention. The host cell comprises a prokaryotic cell, a yeast cell, or a mammalian cell, such as a CHO cell, a NSO cell, or other mammalian cell, preferably a CHO cell.

[0129] In one aspect, the present invention also provides an antibody-drug conjugate comprising a bispecific antibody or antigen-binding fragment thereof targeting GPC3 described herein and a drug or toxin. The bispecific antibody or antigen-binding fragment thereof and the drug can be coupled via a linker to form an antibody-drug conjugate (ADC). Typically, the ADC comprises a bispecific antibody or antigen-binding fragment thereof targeting GPC3 described herein, linked to a drug or toxin via a linker. The linker can be degradable or non-degradable. Degradable linkers typically readily degrade in the intracellular environment, thereby releasing the therapeutic agent from the antibody. Suitable degradable linkers include enzymatically degradable linkers, such as peptidyl linkers that can be degraded by intracellular lysosomal proteases, or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidases. Peptidyl 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 below 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.

[0130] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Preferred are sulfhydryl-specific active reactive groups, such as maleimides, halogenated amides, halogenated esters, halogenated methyl ketones, benzyl halides, vinyl sulfones, pyridyl disulfides, mercury derivatives, and polymethylene dimethyl sulfide thiosulfonates. The linker can include, for example, a maleimide attached to the antibody via thiosuccinimide.

[0131] Preferably, the linker-connected drug or toxin is selected from the group consisting of: CL2A-SN-38 (CAS No.: 1279680-68-0), mc-vc-PAB-MMAE (CAS No.: 646502-53-6), Tesirine (SG3249, CAS No.: 1595275-62-9), Deruxtecan (CAS No.: 1599440-13-7), and Vc-seco-DUBA (SYD985, CAS No.: 1345681-58-4). The molecular structure is shown in the figure below:

[0132] In the present invention, the bispecific antibody or antigen-binding fragment thereof targeting GPC3 is coupled to SN-38 via a CL2A linker.

[0133] In the present invention, the bispecific antibody targeting GPC3 or the antigen-binding fragment thereof is coupled to MMAE via a mc-VC-PAB linker.

[0134] In the present invention, the bispecific antibody or antigen-binding fragment thereof targeting GPC3 is coupled to the PBD dimer via a maleimide-dPEG8-VA-PABA linker.

[0135] In the present invention, the bispecific antibody targeting GPC3 or the antigen-binding fragment thereof is coupled to DX-8951 (DXd) via a maleimide-GGFG linker.

[0136] In the present invention, the bispecific antibody or antigen-binding fragment thereof targeting GPC3 is coupled to DUBA via a Vc-seco linker.

[0137] In some embodiments, the bispecific antibody targeting GPC3 or its antigen-binding fragment is randomly conjugated to a drug or toxin. In a specific embodiment, the bispecific antibody targeting GPC3 or its antigen-binding fragment is randomly conjugated to DXD.

[0138] In some embodiments, the bispecific antibody targeting GPC3 or its antigen-binding fragment is site-specifically conjugated to a drug or toxin, preferably, the drug or toxin is site-specifically conjugated to the cysteine ​​residue 239 of the antibody heavy chain constant region (S239C, EU numbering). In a specific embodiment, the bispecific antibody targeting GPC3 or its antigen-binding fragment is site-specifically conjugated to PBD or DUBA, preferably, PBD or DUBA is site-specifically conjugated to the cysteine ​​residue 239 of the antibody heavy chain constant region (S239C, EU numbering).

[0139] Preferably, the drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In one embodiment, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In the case where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0140] Preferably, the cytotoxic drug is selected from the group consisting of anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, DNA alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0141] Preferably, examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, and 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 a combination thereof.

[0142] Preferably, the toxin is selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansinoids, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxybenzoate, daptomycin, acetaminophen ... anthracnose dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, a Sapaonaria officinalis inhibitor, a glucocorticoid, or a combination thereof.

[0143] Preferably, the drug or toxin is selected from one or more of SN-38 (NK012, CAS No.: 86639-52-3), MMAE (Monomethyl auristatin E, CAS No.: 474645-27-7), PBD dimer (SG3199, CAS No.: 1595275-71-0), DX-8951 (Exatecan, CAS No.: 171335-80-1) or DUBA (duocarmycin-hydroxybenzamide-azaindole).

[0144] In some specific embodiments, the antibody-drug conjugate is selected from the group consisting of:

[0145] (1) The bispecific antibody comprises a heavy chain as shown in SEQ ID NO: 23 and a light chain as shown in SEQ ID NO: 25, which is coupled to PBD via a maleimide-dPEG8-VA-PABA linker;

[0146] (2) The bispecific antibody comprises a heavy chain as shown in SEQ ID NO: 21 and a light chain as shown in SEQ ID NO: 28, which is coupled to PBD via a maleimide-dPEG8-VA-PABA linker;

[0147] (3) The bispecific antibody comprises a heavy chain as shown in SEQ ID NO: 23 and a light chain as shown in SEQ ID NO: 25, which is coupled to DUBA via a Vc-seco linker;

[0148] (4) The bispecific antibody comprises a heavy chain as shown in SEQ ID NO: 21 and a light chain as shown in SEQ ID NO: 28, which is coupled to DUBA via a Vc-seco linker;

[0149] (5) The bispecific antibody comprises a heavy chain as shown in SEQ ID NO: 45 and a light chain as shown in SEQ ID NO: 50, which is coupled to DXd via a maleimide-GGFG linker;

[0150] (6) The bispecific antibody comprises a heavy chain as shown in SEQ ID NO: 45 and a light chain as shown in SEQ ID NO: 50, which is coupled to DUBA via a Vc-seco linker; or

[0151] (7) The bispecific antibody comprises a heavy chain represented by SEQ ID NO: 45 and a light chain represented by SEQ ID NO: 50, which is coupled to PBD via a maleimide-dPEG8-VA-PABA linker.

[0152] In another aspect, the present invention also provides a use of an antibody or antigen-binding fragment thereof targeting GPC3, a pharmaceutical composition, or an antibody-drug conjugate of the present invention in the preparation of a medicament for treating or preventing cancer, preferably liver cancer.

[0153] Furthermore, the present invention also provides a method for preparing the antibody or antigen-binding fragment thereof targeting GPC3 of the present invention:

[0154] The DNA sequence of the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention can be obtained using conventional techniques, such as hybridoma PCR amplification or phage display library screening. In addition, the coding sequences of the light chain and heavy chain can be fused together to form a single-chain antibody (e.g., scFV).

[0155] Once the relevant sequence is obtained, it can be cloned into a vector, then transferred into host bacteria, and then the relevant vector can be extracted from the host bacteria by conventional methods.

[0156] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is relatively short. Currently, DNA sequences encoding the antibodies of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely through chemical synthesis. In addition, mutations can also be introduced into the protein sequences of the present invention through chemical synthesis.

[0157] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0158] The GPC3-targeting antibodies or antigen-binding fragments thereof of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified using various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose affinity chromatography, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and other conventional separation and purification methods, as well as combinations of these methods, to obtain the GPC3-targeting antibodies or antigen-binding fragments thereof of the present invention.

[0159] As a preferred embodiment of the method for preparing the antibody targeting GPC3 or its antigen-binding fragment of the present invention, the method for separating and purifying the antibody targeting GPC3 or its antigen-binding fragment is protein A affinity chromatography, cation exchange method or anion exchange method.

[0160] The resulting monoclonal antibodies or bispecific antibodies can be characterized by conventional means. For example, the binding specificity of the antibody can be determined by immunoprecipitation or in vitro binding assays such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). The binding affinity of the antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0161] 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.

[0162] In the present invention, the antibody-drug conjugate is prepared according to a method comprising the following steps:

[0163] The interchain disulfide bonds of the bispecific antibody targeting GPC3 or antigen-binding fragment thereof of the present invention are reduced to generate 2n (e.g., 2, 4, 6, 8) thiol groups;

[0164] The drug-linker compound cross-links with the reduced antibody sulfhydryl group to generate the corresponding antibody-drug conjugate;

[0165] The product was further purified by ultrafiltration and desalting.

[0166] In one aspect, the present invention also provides a pharmaceutical composition comprising the bispecific antibody targeting GPC3 described herein, or its antigen-binding fragment, or the antibody-drug conjugate described herein, and one or more pharmaceutically acceptable carriers. When the pharmaceutical composition comprises more than one antibody (or its antigen-binding fragment, or antibody conjugate), the antibodies (or its antigen-binding fragment, or antibody conjugate) can be administered in batches. The pharmaceutical composition may optionally comprise one or more additional pharmaceutically active ingredients, such as another antibody or drug, such as an anti-tumor drug.

[0167] The pharmaceutical composition can include any number of excipients. Operable excipients include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, coloring agents, flavorings, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, or combinations thereof. Selection and use of suitable excipients are taught in the following, Gennaro writes, Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0168] In one aspect, the present invention provides a kit comprising the bispecific antibody targeting GPC3 or an antigen-binding fragment thereof according to the present invention or the antibody-drug conjugate according to the present invention.

[0169] In one aspect, the present invention also provides a use of the bispecific antibody or antigen-binding fragment thereof targeting GPC3 according to the present invention, the pharmaceutical composition according to the present invention, the antibody-drug conjugate according to the present invention, or the kit according to the present invention in the preparation of a medicament for treating or preventing cancer, preferably liver cancer.

[0170] To ensure that the present invention may be more readily understood, certain terms are first defined, with additional definitions set forth throughout the detailed description:

[0171] The term "GPC3" (also known as glypican 3) refers to a member of the heparan sulfate proteoglycan family, anchored to the cell membrane by glycosylphosphatidylinositol (GPI). The human GPC3 gene is located on chromosome X (Xp26) and encodes a 70 kDa protein containing 580 amino acids. This protein is cleaved endonucleolytically by a furin-like convertase between Arg358 and Ser359 to produce a 40 kDa N-terminal subunit and a 30 kDa C-terminal subunit, which also contains two heparan sulfate (HS) chains.

[0172] The term "antibody" as used herein includes intact antibodies and any antigen-binding fragments thereof (i.e., "antigen-binding portions") or single chains thereof. A complete antibody is a glycoprotein comprising two heavy (H) chains and two light (L) chains connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), separated by more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of antibodies mediate the binding of the immunoglobulin 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.

[0173] The term "bispecific antibody" refers to an antibody molecule that can bind to two separate antigens or has binding specificities for different epitopes within the same antigen. For example, in some embodiments, one antigen-binding fragment of a bispecific antibody molecule binds to the first antigen-binding domain of GPC3, while the other antigen-binding fragment binds to the second antigen-binding domain of GPC3, thereby further enhancing affinity for the cell surface GPC3 antigen.

[0174] As used herein, the term "antigen-binding fragment" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that specifically binds to an antigen (e.g., a GPC3 protein). It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity-determining region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains VL and VH of the FV fragment are encoded by separate genes, they can be connected by a linker using recombinant methods to form a single protein chain, wherein the VL region and the VH region are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also included in the term "antigen-binding fragment" of an antibody. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragment screening for use is the same as that for intact antibodies.

[0175] The term "scFv" refers to a genetically engineered molecule comprising the variable regions of the light and heavy chains of an immunoglobulin, connected by a suitable polypeptide linker as a genetically fused single-chain antibody.

[0176] The term "Fc": The Fc region refers to the Fc fragment region of an antibody molecule, which includes the hinge region, part of the CH2 and CH3 domains, and has a molecular weight of approximately 50,000 Da. When the antibody is digested with papain, the human IgG heavy chain Fc region is from Threonine 225 to the C-terminus.

[0177] The term "Fab" refers to a fragment comprising a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digesting an intact antibody with the enzyme papain to produce an intact light chain and a portion of one heavy chain.

[0178] The term "Fab'" refers to a fragment of an antibody molecule that can be obtained by treating an intact antibody with pepsin followed by reduction to produce an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule.

[0179] The term "(Fab')2" refers to an antibody fragment that can be obtained by treating an intact antibody with pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments linked together by two disulfide bonds.

[0180] The term "Fv" refers to a gene fragment containing the light chain variable region and the heavy chain variable region expressed as two chains.

[0181] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities. For example, an isolated antibody that specifically binds to a GPC3 protein is substantially free of antibodies that specifically bind to antigens other than GPC3. However, for example, in some embodiments, an isolated antibody that specifically binds to a human GPC3 protein may have cross-reactivity with other antigens (e.g., GPC3 proteins from other species). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemical substances.

[0182] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0183] As used herein, the term "mouse antibody" is intended to include antibodies with variable regions, wherein the framework region and CDR regions are all derived from the immunoglobulin sequences of mouse germline. In addition, if the antibody comprises a constant region, the constant region is also derived from the immunoglobulin sequences of mouse germline. Mouse antibodies of the present invention can include amino acid residues that are not encoded by mouse germline immunoglobulin sequences, such as mutations introduced in vitro by random or site-directed mutagenesis or in vivo by somatic mutation. However, as used herein, the term "mouse antibody" is not intended to include antibodies whose CDR sequences from another mammalian germline have been transplanted onto the mouse framework sequences.

[0184] 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.

[0185] As used herein, the term "humanized antibody" refers to an antibody from a non-human species whose protein sequence has been modified to increase its similarity to naturally occurring antibody variants in humans.

[0186] The term "antibody-drug conjugate" refers to the use of antibodies to specifically recognize specific antigens on the surface of tumor cells, thereby achieving accurate delivery of anti-tumor therapeutic agents (such as cytotoxins or cytostatics, radioactive isotopes, small molecule chemotherapy drugs, etc.) to tumor target cells, resulting in intracellular accumulation and release, to achieve the purpose of accurately killing tumors. ADC is also considered to be the most promising anti-tumor drug because of its suitable molecular weight, high stability, strong targeting, and low toxic side effects. In addition to monoclonal antibodies, bispecific antibodies can also be coupled to therapeutic agents. In some embodiments, the part coupled to the antibody or bispecific antibody of the present invention to form an antibody conjugate is a cytotoxin, which refers to a substance that inhibits or prevents cell function and / or causes cell destruction, and includes small molecule cytotoxins. In some embodiments, the cytotoxin is selected from SN-38, MMAE, PBD dimer, DX-8951 (DXd) or DUBA.

[0187] The terms "identity" and "sequence ... identity" are used interchangeably herein and are calculated as follows: To determine the percent "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0188] 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.

[0189] The term "therapeutically effective amount" refers to an amount of the bispecific antibody targeting GPC3 of the present invention or its antigen-binding fragment sufficient to prevent or ameliorate symptoms associated with a disease or condition (e.g., cancer) and / or lessen the severity of the disease or condition. A therapeutically effective amount should be understood in the context of the condition being treated, wherein those skilled in the art can readily identify the actual effective amount.

[0190] As used herein, the term "epitope" refers to the site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope" is also referred to as an "antigenic determinant" in the art. An epitope or antigenic determinant is typically composed of chemically active surface groups of a molecule, such as amino acids or carbohydrates or sugar side chains, and typically has specific three-dimensional structural characteristics and specific charge characteristics. For example, an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or non-continuous amino acids in a unique spatial conformation, which can be "linear" or "conformational". See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all interacting points between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interacting points exist across separate protein amino acid residues.

[0191] The GPC3-targeting bispecific antibodies or antigen-binding fragments thereof of the present invention are structurally and chemically characterized monoclonal antibodies as described below and in the Examples below. The amino acid sequence ID numbers of the heavy and light chain variable regions of the antibodies are summarized in Table 1. Some antibodies have the same VH or VL. For example, h1B12 and its mutants h1B12-G34R, h1B12-G34K, h1B12-G34A, h1B12-G34H, h1B12-G34M, h1B12-G34L, h1B12-G34S, or h1B12-N33R have the same light chain (h1B12-L).

[0192] The heavy chain variable region CDRs and light chain variable region CDRs in Table 1 are defined by the Kabat, Chothia, IMGT, AbM, Contact numbering systems / methods, or a combination thereof. However, as is well known in the art, CDR regions may also be defined by other, such as different, numbering systems based on heavy chain / light chain variable region sequences. The CDR regions of exemplary antibodies of the present invention are detailed in Table 2.

[0193] Table 1 Amino acid sequences of bispecific antibodies targeting GPC3

[0194] Note: The underlined part is the CDR region defined by Kabat

[0195] Table 2 CDR region sequences of exemplary antibodies

[0196] The control antibodies used in the present invention, such as hYP7HM and FH4, were prepared with reference to patent document WO2024067764, which is incorporated herein by reference in its entirety. The light and heavy chain variable regions of hYP7HM are set forth in SEQ ID NOs: 4 and 5 of WO2024067764, and the light / heavy chain constant regions are consistent with those of GC90-6mu of the present invention. FH4 is bs-FH4 described in WO2024067764, and its light / heavy chains are set forth in SEQ ID NOs: 62 and 39 of WO2024067764.

[0197] By the following detailed description and examples, other features and advantages disclosed by the present invention will become apparent, which should not be construed as limiting. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0198] Figure 1 is a schematic diagram of the construction of a GPC-3-targeting bispecific antibody and its antigen-binding fragment (heavy chain or light chain). The heavy or light chain of the first antibody (e.g., GC90 or h1B12) is linked to different scFv formats of the second antibody (e.g., h1B12 or GC90) via a (GGGGS)n linker. GC90CK or 1B12CK represents the constant region kappa chain of the antibody light chain, and IgG1-Fc-6mu refers to the constant region portion of the IgG1 antibody heavy chain (including CH1, CH2, and CH3). 6mu indicates that six functional amino acid point mutations have been designed into the Fc region.

[0199] Figures 2A to 2C show the binding of bispecific antibodies targeting GPC-3 to human liver cancer cells HepG2, Hep3B, or Huh-7.

[0200] Figures 3A to 3D show the internalization of GPC-3-targeting monoclonal antibodies or bispecific antibodies in human hepatoma cells HepG2 or Hep3B.

[0201] Figures 4A to 4F show the cytotoxicity of antibody-drug conjugates against human hepatocellular carcinoma cells (HepG2, Hep3B, or Huh-7), as well as GPC3-low-expressing cells. Bispecific antibody-drug conjugates demonstrated superior tumor cell cytotoxicity compared to monoclonal antibody-drug conjugates. In Figure 4E, blank cells indicate that accurate IC50 values ​​could not be fitted.

[0202] Figures 5A and 5B show the affinity of GC90 and its mutant monoclonal antibodies to GPC3 protein.

[0203] Figures 6A to 6F show the binding of GC90 and its mutant monoclonal antibodies and bispecific antibodies targeting GPC-3 to human liver cancer cells HepG2, Hep3B or Huh7.

[0204] Figure 7 shows the cell binding of antibody-drug conjugates.

[0205] Figures 8A, 8B, 9, and 10 show the in vivo tumor inhibition of the antibody-drug conjugate.

[0206] FIG11 shows the binding strength results between GC90 and overlapping peptides detected by ELISA, wherein the underlined amino acid sequences correspond to the specific sequences of the overlapping peptides.

[0207] FIG12 shows an amino acid sequence alignment analysis between human GPC3 protein and mouse GPC3 protein.

[0208] FIG13 shows the distribution of antigen recognition regions of GC90 and other reported anti-GPC3 antibodies on the GPC3 protein.

[0209] The hIgG1 or IgG1 mentioned in the figures refers to the IgG1 isotype negative control antibody. Specific embodiments

[0210] Example 1

[0211] Generation of mouse anti-GPC3 monoclonal antibodies using hybridoma technology

[0212] 1.1 Immunization of mice

[0213] Mice were immunized according to the method described in E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988. BALB / c mice were immunized with the human GPC3 protein (Antigen 1), which contains amino acid residues Gln at position 25 to His at position 559 of the extracellular full-length domain. Serum titers were measured after the second and third immunizations, and the two mice with the highest antibody titers were electrofused. Antigen 1 or a peptide antigen containing amino acid residues Ser at position 359 to His at position 559 of the extracellular C-terminal domain (Antigen 7) was used to determine antibody serum titers and screen for hybridoma cells secreting antigen-specific antibodies.

[0214] 1.2 Hybridoma cell fusion and screening

[0215] Prior to cell fusion, cells of a murine myeloma cell line (SP2 / 0-Ag14, ATCC #CRL-1581) were cultured to the logarithmic growth phase. Immunized mice were sacrificed, and spleens were removed under sterile conditions according to the method described in Kohler G and Milstein C, "Continuous cultures of fused cells secreting antibodies of predefined specificity," Nature, 256:495-497 (1975). A spleen cell suspension was prepared. Splenic B cells were fused with murine myeloma cells in the logarithmic growth phase using PEG chemistry, and the fused cells were continuously cultured. The fused hybridoma cells were then plated into 96-well plates and cultured in DMEM medium containing 20% ​​FCS / HAT. Viable hybridoma clones were typically observed under a microscope after 7 to 10 days. Two weeks after cell plating, the hybridoma culture supernatant from each well was collected and screened for hybridomas expressing anti-GPC3 antibodies using ELISA using recombinant human GPC3 protein (Antigen 1 and Antigen 7). The specific screening operation steps are as follows: recombinant human GPC3 protein (Antigen 1 or Antigen 7, 2μg / mL) is coated in a 96-well plate, 50μL / well, and incubated at 4°C overnight. After washing the plate three times with PBST, 200μL of blocking solution (PBS+1% BSA) is added to each well and blocked at 37°C for 1 hour. Each antibody is diluted to 2μg / mL with blocking solution, then diluted 3-fold in series, for a total of eight concentrations, and added to the ELISA plate at 50μL / well and incubated at 37°C for 1 hour. Wash the plate three times with PBST, then add pre-diluted Peroxidase-conjugated affinipure F(ab')2fragment goat anti-mouse IgG (H+L) (Jackson, dilute 5000 times with blocking solution according to the instructions), 50μL / well, and incubate at 37°C for 1 hour. Wash the plate three times with PBST and add 1-Step™ Ultra TMB-ELISA Substrate Development Solution (Thermo) at 50 μL / well. Develop at room temperature in the dark for 3-5 minutes. Terminate the reaction with 2M HCl at 50 μL / well. Measure the OD at 450 nm using a microplate reader. Calculate the EC50 value to identify hybridoma clones that secrete antibodies with high specific binding activity.

[0216] Table 3 EC50 of hybridoma antibodies binding to human GPC3 protein

[0217] According to the results in Table 3, it can be seen that all mouse monoclonal antibodies have high affinity to GPC3 protein. Most of them recognize Antigen 7, the C-terminal domain of GPC3 protein, while m2G12 recognizes the domain outside the C-terminus.

[0218] Furthermore, ELISA was used to test the recognition of GPC3 proteins from different species by each antibody. Cynomolgus GPC3 (ACRO Biosystems) and Mouse GPC3 (ACRO Biosystems) were coated in 96-well plates at a concentration of 2 μg / mL, with 50 μL / well. The assay was performed according to the aforementioned ELISA protocol. The results are shown in Table 4.

[0219] Table 4 Recognition of monkey and mouse GPC3 proteins by hybridoma antibodies

[0220] Note: √ represents affinity recognition, and × represents no recognition.

[0221] Furthermore, competitive ELISA was used to determine whether each candidate antibody recognized the same epitope as the GC33 antibody. The specific steps are as follows: dilute each antibody to 2 μg / mL, add to a 96-well enzyme-labeled plate, 100 μL / well, and coat overnight at 4°C. Wash the plate three times with PBST, add blocking solution (PBS + 1% BSA), 200 μL / well, and block at 37°C for 1 hour. Add 2 μg / mL Antigen 1 antigen to each well, 100 μL / well, incubate at 37°C for 1 hour, and wash the plate three times with PBST. Dilute the GC33 antibody to 2 μg / mL with blocking solution, then dilute it 3-fold in series, for a total of eight concentrations, and then add the diluted GC33 to the enzyme-labeled plate, 100 μL / well, and incubate at 37°C for 1 hour. Discard the supernatant, wash the plate three times with PBST, add 100 μL / well of diluted peroxidase-conjugated goat anti-human IgG (Jackson, dilute 5000-fold with blocking buffer according to the instructions), and incubate at 37°C for 1 hour. Discard the supernatant, wash the plate three times with PBST, add 50 μL / well of 1-step Ultra TMB-ELISA developer (Thermo), and incubate in the dark for approximately 4 min 30 s. Then, add 50 μL of stop solution (2 M HCl). Measure the OD value at 450 nm using a microplate reader. The analysis results are summarized in Table 5. m1B12, m1H4, and GC33 have identical or overlapping antigen recognition sites.

[0222] Table 5 Antibodies that recognize different epitopes of GPC3

[0223] 1.3 Sequencing and Identification of Hybridoma Antibodies

[0224] Hybridoma antibody variable region genes were isolated by 5' RLM-RACE. Briefly, total RNA was extracted from each candidate hybridoma, and samples were reverse transcribed using the FirstChoice RLM-RACE Kit (Thermo). The heavy and light chain variable region genes were amplified by PCR. The PCR products were cloned into a vector and sequenced. The sequences of the m1B12, m2D8, m2G12, m1B8, m1H4, m36C12, and m37F4 antibodies were obtained (see Table 6).

[0225] Table 6 Anti-GPC3 hybridoma antibody variable region sequences

[0226] Example 2

[0227] Humanization of mouse anti-GPC3 monoclonal antibody

[0228] Mouse anti-GPC3 monoclonal antibodies were selected for humanization and further research. As described below, the mouse monoclonal antibodies were humanized using a proven CDR transplantation method. The light chain variable region and heavy chain variable region sequences of each mouse monoclonal antibody were BLAST-aligned with the human immunoglobulin gene database to select the antibody framework regions for humanization of each mouse antibody. The human germline IGVH and IGVK with the highest homology to each mouse antibody were selected as the antibody frameworks for humanization. The CDRs of the heavy chain variable region or light chain variable region of each mouse antibody were transplanted into the selected human germline framework regions, and the amino acid residues in the framework regions were back-mutated to obtain more candidate heavy chain variable regions and / or light chain variable regions for humanized antibodies. The heavy chain variable region and light chain variable region of each antibody after humanization, as well as the mutation site design, are summarized in Table 7.

[0229] Table 7 Anti-GPC3 humanized antibody variable region sequences

[0230] Example 3:

[0231] Obtaining a fully human antibody-positive clone targeting GPC3

[0232] Three rounds of panning were performed using a natural fully human phage display library with the GPC3-A peptide (Gly from amino acid residue 510 to Ser from amino acid residue 560 in the extracellular domain of the GPC3 protein, i.e., GDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHS, used as an antigen to screen the humanized recombinant antibody library) and the GPC3-B peptide (Met from amino acid residue 478 to Asp from amino acid residue 531 in the extracellular domain of the GPC3 protein, i.e., MPKGRVLDKNLDEEGFESGDCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDLD, used as an antigen to screen the humanized recombinant antibody library). The phage library eluate was obtained. The neutralized phage panning eluate was added to the prepared TG1 bacterial suspension, mixed, and incubated at 37°C to infect the TG1 host bacteria for 45 minutes. After full infection, the bacterial solution was gradiently diluted and spread on an agar plate with relevant resistance, and inverted to culture overnight at 37°C. The next day, a 96-well sterile deep-well plate was prepared with 0.5mL of 2YT medium (with 0.2% w / v glucose and 0.1mg / mL ampicillin antibiotics), and the monoclonal colonies cultured in the plate were picked up with a sterile pipette tip and placed in the corresponding well plate, and cultured overnight at 37°C with shaking at 220rpm (16-18 hours). The next day, 0.05-0.1mL of the overnight cultured monoclonal bacterial solution was transferred to a newly prepared sterile deep-well plate (filled with 0.5mL of 2YT medium containing a final concentration of 0.1mg / mL ampicillin antibiotics) and cultured to OD 600 The value is approximately 0.6-0.8. After adding a certain proportion of helper phage and shaking to mix, the mixture is allowed to infect at 37°C for 45 minutes. Then, 0.25mL of 2YT medium (containing 0.1mg / mL ampicillin and kanamycin with a final concentration of 0.05mg / mL after addition) is added and cultured overnight at 30°C and 220rpm for 16-18 hours. The overnight expressed bacterial solution is centrifuged at 4000rpm for 10 minutes to obtain the phage display expression supernatant for ELISA binding detection of GPC3-A peptide and GPC3-B peptide and FACS binding detection of the corresponding cells to obtain positive fully human antibody clones.

[0233] The positive clones screened in this example were sequenced, and the amino acid sequences of the candidate fully human anti-GPC3 antibody GC90 and the optimized mutants designed therefor are shown in Table 8.

[0234] Table 8 Light chain and heavy chain sequences of humanized monoclonal antibodies

[0235] Example 4

[0236] Preparation of humanized monoclonal antibodies and bispecific antibodies

[0237] 4.1 Vector Construction

[0238] First, the nucleotide sequence encoding the human IgG1 heavy chain constant region (IgG1-CH) was connected to the secretion signal peptide (SP) coding sequence at the front end and the termination codon TAG at the back end. The gene was synthesized and cloned into the pUC57-GW-kan vector (Suzhou Jinweizhi Biotechnology Co., Ltd.), and inserted into the downstream of the CMV promoter of the pCDNA3.1 vector by the In-fusion cloning method. Specifically, by setting specific insertion site primers and high-fidelity PCR enzyme (HiFi PCR Premix, TAKARA), the synthesized gene fragment and the pCDNA3.1 vector plasmid fragment were amplified respectively. After gel recovery, the IgG1 heavy chain gene fragment and the linearized vector fragment were connected (In-fusion Snap Assembly Master Mix, TAKARA) to obtain the heavy chain constant region vector pCDNA3.1-IgG1. According to the above implementation method, the coding sequence of the human immunoglobulin kappa light chain constant region (IgG-CK) was synthesized, with the signal peptide (SP) coding sequence at the front and the termination codon TAG at the rear. After gene synthesis, it was inserted downstream of the CMV promoter in the pCDNA3.1 vector to obtain the light chain constant region vector pCDNA3.1-CK.

[0239] For the construction of each humanized monoclonal antibody expression vector, the coding sequences of the respective heavy chain variable region (VH) and light chain variable region (VL) were gene synthesized and inserted between the SP and constant region of the pCDNA3.1-IgG1 or pCDNA3.1-CK vector according to the above-mentioned in-fusion cloning method to obtain the monoclonal antibody heavy chain expression vector and light chain expression vector.

[0240] To construct a bispecific antibody expression vector, the heavy chain variable region nucleotide sequence and the light chain variable region nucleotide sequence of the second antibody were linked via a (GGGGS)n linker coding sequence (n = 3, 4, 5, or 6) to generate an scFV nucleotide sequence and gene synthesis. The scFV coding sequence was then linked via a (GGGGS)n linker coding sequence (n = 2, 3, or 4) and inserted into the corresponding positions of the monoclonal antibody heavy chain or light chain expression vector described above according to the structure shown in Figure 1, thereby generating a bispecific antibody heavy chain or light chain expression vector. A schematic diagram of the construction of a bispecific antibody heavy chain or light chain vector is shown in Figure 1.

[0241] For the construction of mutant expression vectors, h1B12-VL (G34X) was constructed by replacing the coding sequence for residue 34 of h1B12-VL from GGC to CGC, AAG, GCC, GTG, ATG, CTG, and TCC via gene synthesis, resulting in the light chain G34R, G34K, G34A, G34H, G34M, G34L, and G34S series of mutations (glycine mutated to arginine, lysine, alanine, histidine, methionine, leucine, and serine); or replacing the coding sequence for residue 33 of h1B12-VL from AAC to CGC, resulting in the light chain N33R mutation (asparagine mutated to arginine). Humanized point mutations in hGC90-VH were synthesized by replacing the coding sequence of residue 1 of hGC90-VH from CAG to GAG, resulting in the heavy chain Q1E mutation (glutamine to glutamate); the coding sequence of residue 20 was replaced from GTG to CTG, resulting in the heavy chain V20L mutation (glutamine to glutamate); the coding sequence of residue 82 was replaced from GAG to CAG, resulting in the heavy chain E82Q mutation (glutamine to glutamate); and the coding sequence of residue 116 was replaced from CCT to ATG, resulting in the heavy chain P116M mutation (glutamine to glutamate); the vector construction method was as described above.

[0242] 4.2 Cell transfection and expression

[0243] according to Transfection was performed according to the instructions of Transfection Reagent (Mirus). ExpiCHO-S cells (Thermo) were cultured in complete medium ( High Yield Expression System (containing 30 mL / L of Poloxamer 188 solution 10% and 20 mL / L of L-glutamine 200 mM, Mirus) was used for subculture 24 hours before transfection to ensure that the cell density was 4 x 10 on the next day. 6 cells / mL, diluted to 2x10 before transfection 6 cells / mL. Add 25 μg of light and heavy chain plasmids at a ratio of 1:1 to 12.5 mL of complete culture medium and mix well. Then add 50 μL of transfection reagent. Gently invert the Transfection Reagent (Mirus) to mix thoroughly, let it sit for 4 minutes, then add it dropwise to the diluted cells (50 mL) while shaking. Add 1 mL of CHOgro-titer Enhancer (Mirus) dropwise. Immediately after transfection, place the cells in a 32°C, 5% CO2 incubator. Add 5% EfficientFeed C+AGT Supplement (Thermo) every other day for a total of 7 days.

[0244] 4.3 Antibody Purification

[0245] After 7 days of culture in a shake flask, the cell supernatant was collected, centrifuged at 4000 rpm for 20 minutes, and the supernatant was taken and filtered with a 0.22 μm filter (Milipore). The antibody was purified by protein A affinity chromatography. Briefly, a HiTrap Mabselect suRe prepacked column (Cytiva) was equilibrated with 20 mM PB + 0.15 M NaCl buffer for 5 to 10 column volumes, and the filtered supernatant was loaded using an AKTA Avant 150 chromatography system (Cytiva). The purification column was then washed with 3 column volumes of 20 mM PB + 0.15 M NaCl buffer, 1 column volume of 20 mM PB + 1 M NaCl buffer, and then washed with 20 mM PB until the baseline was stable. Finally, the antibody was eluted with 20 mM citric acid (adjusted to pH 3.0 with 20 mM sodium citrate), and the peak of 200 mAu-200 mAu was collected. The eluted antibody was immediately neutralized with neutralization buffer (1 M Tris-HCl, pH 9.0), placed in a 1.5 mL tube, and frozen at -80 until use. The sequences of the prepared bispecific antibodies are shown in Table 9.

[0246] Table 9 Light chain and heavy chain sequences of humanized bispecific antibodies

[0247] Example 5

[0248] 5.1 ELISA affinity testing of humanized monoclonal antibodies and bispecific antibodies

[0249] The relative binding activity of each antibody against human GPC3 protein was determined by ELISA. Recombinant human GPC3 protein (1 μg / mL) was coated onto a 96-well plate at 100 μL / well and incubated overnight at 4°C. The plates were then blocked with 1% BSA in PBST (containing 0.05% Tween-20) for 2 hours at 37°C (200 μL / well). Washed three times with PBST, each humanized monoclonal antibody / bispecific antibody was serially diluted in PBST containing 1% BSA and added to the 96-well plate (100 μL / well). The working concentrations of humanized monoclonal antibodies GC90, GC90-4mu, GC90LH1, GC90LH2, GC90LH3, GC90LH4, and GC90LH5 were 15 nM, 3.75 nM, and then diluted 4 times from 3.75 nM to 8 dilutions. The cells were incubated at 37°C for 1 hour and washed three times with PBST. Anti-Human IgG-FC-HRP (Sigma, 1 / 30,000 dilution) was then added at 100 μL / well, incubated at 37°C for 1 hour, washed three times with PBST, and then 50 μL of TMB (SURMOPICS) was added for reaction. The reaction was terminated with 1 M H2SO4, and the OD value was measured on a microplate reader at 450 nm-570 nm. As shown in Figure 5A, the GC90 monoclonal antibody and its mutants of the present invention all have very good binding affinity to the GPC3 protein.

[0250] 5.2 ELISA detection of affinity-matured monoclonal antibody molecules

[0251] The relative binding activity of each antibody to human GPC3 protein was determined by ELISA. The specific operation was as follows: recombinant human GPC3 protein (1 μg / mL) was coated in a 96-well plate at 100 μL / well and incubated at 4°C overnight. The plates were then blocked with 1% BSA in PBST (containing 0.05% Tween-20) for 2 hours at 37°C (200 μL / well), washed three times with PBST, and serially diluted with 1% BSA in PBST before being added to a 96-well plate (100 μL / well). The working concentrations of humanized mAbs GC90-4mu, GC90LH6, GC90LH7, and GC90LH8 were 10,000 ng / ml, 3,333.33 ng / ml, and 1,111.11 ng / ml, followed by 3-fold dilutions for a total of 10 points. The working concentrations of GC90LH8 and GC90LH10 were 10,000 ng / ml, 2,500 ng / ml, and 625 ng / ml, followed by 4-fold dilutions for a total of 8 points. The plates were incubated at 37°C for 1 hour and washed three times with PBST. Then, 100 μL / well of Anti-Human IgG-FC-HRP (Sigma, 1 / 30,000 dilution) was added, incubated at 37°C for 1 hour, washed three times with PBST, and then 50 μL of TMB (SURMOPICS) was added for reaction. The reaction was terminated with 1 M H₂SO₄, and the OD value was measured on a microplate reader at 450 nm-570 nm. As shown in Figure 5B, the GC90 monoclonal antibody mutants of the present invention all exhibited excellent binding affinity to the GPC3 protein.

[0252] Example 6

[0253] Flow cytometry detection of humanized monoclonal antibodies and bispecific antibodies binding to tumor cells

[0254] Flow cytometry was used to determine the affinity of each anti-GPC3 antibody to HepG2, Hep3B, or Huh-7 cells. The specific operation was as follows: each humanized monoclonal antibody or bispecific antibody was serially diluted with FACS Buffer (PBS + 5% FBS) and added to a 96-well U-shaped plate in sequence. The specific operation was as follows: the working concentration of each monoclonal antibody and bispecific antibodies GB5 and GB6 was: 100nM, 25nM, 6.25nM and then diluted 5 or 6 times in multiples of 4; bispecific antibodies GB9 to GB14 were diluted to 80nM with FACS Buffer and mixed with cells at a 1:1 ratio with an initial concentration of 40nM, and then diluted 40nM, 10nM, 2.5nM and then diluted 4 times in multiples of 4 for a total of 8 points. HepG2, Hep3B, and Huh-7 cells were digested with trypsin, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in FACS Buffer at a concentration of 2×10 6Cells were centrifuged at 4°C for 3 minutes at 3500 rpm, and the supernatant was discarded. The cells were then resuspended in 250 μL of pre-cooled FACS Buffer and centrifuged twice. 100 μL of diluted PE anti-human IgG FC fluorescent secondary antibody (BioLegend, 0.5 μL / 1×10 5 The cells were prepared using a 400 μL flow cytometer (prepared with 100 cells) and incubated on ice in the dark for 30 min. The supernatant was discarded and washed twice, and the cells were finally resuspended in 200 μL FACS Buffer. The MFI values ​​were measured using an Attune NxT flow cytometer (Thermo), and the data were processed using GraphPad Prism software. The results are shown in Figures 2A to 2C and Figures 6A to 6F. Based on these results, it can be seen that the monoclonal antibodies or bispecific antibodies of the present invention have good affinity for HepG2 cells, Hep3B cells, and Huh-7 cells.

[0255] Example 7

[0256] 7.1 Flow cytometry to detect the internalization efficiency of humanized monoclonal antibodies / bispecific antibodies

[0257] In HepG2 and Hep3B cells, flow cytometry was used to determine the internalization efficiency of each anti-GPC3 antibody. The specific operation was as follows: each humanized monoclonal antibody or bispecific antibody was diluted to 50nM with FACS Buffer, mixed with cells at an initial concentration of 25nM in a 1:1 ratio, and then diluted in multiples of 3 for a total of 8 points; or each monoclonal antibody or bispecific antibody was diluted to 80nM with FACS Buffer, mixed with cells at a 1:1 ratio to an initial concentration of 40nM, and then diluted in multiples of 4 for a total of 8 points; 50μL / well was added to a 96-well U-shaped plate. The binding of antibodies and cells was carried out as in Example 6. Each antibody and cell was incubated for different times: the sample was incubated at 37°C for 4h and immediately transferred to ice after incubation. 200 μL FACS buffer was added to each well and mixed, centrifuged at 3500 rpm for 3 min, the supernatant was discarded, and 100 μL pre-cooled PE anti-human IgG FC fluorescent secondary antibody (BioLegend, 1 μL / 2×10 5The cells were incubated on ice in the dark for 60 minutes. The supernatant was discarded and washed twice. Finally, the cells were resuspended in 200 μL FACS buffer, and the MFI values ​​were measured on a FACS analyzer. The data were processed using GraphPad Prism software. The cellular internalization efficiency of the antibody was calculated as follows: (1-antibody MFI value at 37°C / antibody MFI value at 4°C) × 100%. Specific results are shown in Figures 3A to 3C. These results show that the monoclonal antibodies or bispecific antibodies of the present invention have good internalization efficiency in both HepG2 and Hep3B cells.

[0258] 7.2 Antibody internalization assay

[0259] HepG2 cells were digested, harvested, and resuspended in assay buffer (or corresponding complete medium). The cell density was adjusted and the cell suspension was transferred to a 96-well assay plate (10,000 cells / well). The assay plates were incubated overnight in a cell culture incubator (37°C / 5% CO2). Test / control working solutions and labeling reagent working solution (4×) were prepared in assay buffer. The test sample GC90, h1B12-G34R, GB-11, and hIgG1 working solutions were mixed with the labeling reagent working solution at a 1:3 molar ratio and a 1:1 volume ratio. The mixture was incubated in a cell culture incubator (37°C / 5% CO2) for 15 minutes to allow for complete coupling. The coupling mixture was transferred to the corresponding wells of the 96-well assay plate containing the cell suspension. The assay plates were incubated in a cell culture incubator (37°C / 5% CO2) for 48 hours and images were taken at designated time points using an Incucyte instrument with a wavelength compatible with the detection band. The parameter used to characterize internalization is Total Red Object Integrated Intensity (RCU×μm2 / Image). The specific results are shown in Figure 3D. It can be seen that the bispecific antibody of the present invention has a significantly improved internalization effect compared to the monoclonal antibody.

[0260] Example 8

[0261] Preparation of Antibody-Drug Conjugates

[0262] 8.1 Site-directed coupling

[0263] The 239th residue of the constant region of the antibody heavy chain was mutated to cysteine ​​(S239C, EU numbering, corresponding to position 243 of the GC90-4mu heavy chain), thereby site-specifically coupling the drug through the linker through the cysteine. Specifically, the monoclonal antibody or bispecific antibody stock solution was replaced with 20mM PBS buffer (pH = 7.2) and the concentration was adjusted to about 5mg / mL, and 250mM EDTA solution was added at a volume ratio of 50:1 (antibody: EDTA) and mixed thoroughly. Then, according to different monoclonal antibodies / bispecific antibodies and different linker-payload (LP) combinations, tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added at a 1-12 times excess molar ratio (relative to the antibody), mixed thoroughly, and placed at room temperature (25°C) for reduction reaction for 3 hours. Add an appropriate amount of DMSO to the reaction solution, followed by a 6-12-fold molar excess (relative to the antibody) of the LP drug (pre-dissolved in DMSO at 5mM / 10mM), ensuring that the DMSO volume 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 allow to stand at room temperature for 10 minutes to terminate the reaction.

[0264] Ultrafiltration was used for desalting. The reaction solution was transferred to a 10KD ultrafiltration tube (Millipore), supplemented with PBS buffer (pH 6.0), and concentrated to the desired volume by centrifugation at 3500 g. PBS was added and the centrifugation was repeated five times. The product was filtered through a 0.22 μm filter membrane (Millipore) and stored at -80°C.

[0265] Table 10 ADC product detection analysis

[0266] 8.2 Random Coupling

[0267] After the coupling reaction, various ADC products were obtained. ADC product purity 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). The monoclonal antibody or dual-antigen solution was replaced with 20mM NaAc-HAc buffer (pH 5.5) and adjusted to a concentration of 5±1 mg / mL. Tris(2-carboxyethyl)phosphine (TCEP) was added to the reaction system at a molar ratio of 20:1, mixed thoroughly, and then reduced at 37°C for 3 hours. After the reduction reaction, the solution was replaced with 20mM NaAc / Tris, 1mM EDTA, pH 7.0 buffer (3500×g, four times) using a 10kD ultrafiltration centrifuge tube and adjusted to a concentration of 5±1 mg / mL. An appropriate amount of DMSO (~10%) was added to the reaction mixture. The desired amount of LP solution (5-10 mM, pre-dissolved in DMSO) was added to the reaction system at a molar ratio of LP drug to antibody of 10:1, mixed thoroughly, and allowed to react at 21-25°C for 1 hour. N-acetylcysteine ​​(NAC) solution was then added at a molar ratio of NAC to antibody of 40:1, mixed thoroughly, and the reaction was terminated at 21-25°C for 1 hour. Following termination, the buffer was exchanged with 20 mM His / His-HCl, pH 6.0 buffer (3500 × g, 4 times) using a 10 kD ultrafiltration centrifuge tube and adjusted to the target concentration. The buffer was then filtered through a 0.22 μm sterilizing filter and stored at ≤-20°C.

[0268] After the coupling reaction, various ADC products were obtained. The ADC product purity was analyzed by size exclusion chromatography (SEC), and the drug-antibody coupling ratio (DAR) and naked antibody ratio were analyzed by hydrophobic interaction chromatography (HIC). The DAR values ​​of GB-11-DXD and IgG1-DXD were 9.42 and 9.2, respectively.

[0269] Example 9

[0270] 9.1 Binding of Antibody-Drug Conjugates to Tumor Cells

[0271] Flow cytometry was used to determine the affinity of antibody-drug conjugates to HepG2, Hep3B or Huh-7 cells. The specific operation was as follows: each antibody-drug conjugate was serially diluted with FACS Buffer (PBS + 5% FBS) and added to a 96-well U-shaped plate in sequence. The specific operation was as follows: the working concentrations of h1B12-G34R-PBD and GB-11-PBD were: 100nM, 25nM, 6.25nM and then diluted 8 points in a multiple of 4; the initial concentration of IgG1-PBD was 100nM or 25nM and then diluted 8 points in a multiple of 4. HepG2, Hep3B and Huh-7 cells were digested with trypsin, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended in FACS Buffer at a concentration of 2×10 6 Cells were centrifuged at 4°C for 3 minutes at 3500 rpm, and the supernatant was discarded. The cells were then resuspended in 250 μL of pre-cooled FACS Buffer and centrifuged twice. 100 μL of diluted PE anti-human IgG FC fluorescent secondary antibody (BioLegend, 0.5 μL / 1×10 5 Cells were prepared using a 400 μL flow cytometer (prepared with 100 μL of cells) and incubated on ice in the dark for 30 min. The supernatant was discarded, and the cells were washed twice, finally resuspended in 200 μL FACS buffer. MFI values ​​were measured using an Attune NxT flow cytometer (Thermo), and the data were processed using GraphPad Prism software. The results are shown in Figure 7. These results show that the antibody-drug conjugates of the present invention have good affinity for HepG2 cells, Hep3B cells, and Huh-7 cells.

[0272] 9.2 Antibody-drug conjugates for tumor cell killing

[0273] The cell cytotoxicity of each antibody-drug conjugate against HepG2, Hep3B, and Huh-7 liver cancer cells, as well as HEK293T and A375 cells (low GPC3 expression) was determined using the Cell Counting Kit-8 (Dojindo). Specifically, HepG2, Hep3B, Huh-7, HEK293T, and A375 cells were cultured in 10% FBS (Gibco) + DMEM medium (Corning). When the cell confluence reached 75% or higher, the cells were digested with trypsin (0.25% Trypsin-EDTA) and counted. The cells were counted at 1.5 × 10 4Cells / mL, 160 μL / well (2400 cells / well) were plated into 96-well plates and cultured overnight at 37°C and 5% CO2. Then, 10% FBS+DMEM medium was used to dilute each antibody-drug conjugate to 333.5 nM, and 40 μL / well was added to a 96-well plate containing 160 ul / well for 5-fold dilution. The initial concentration was 66.7 nM. 6 or 8 concentration points were serially diluted, and duplicate wells were made. After that, the cells were cultured in a 37°C, 5% CO2 constant temperature incubator. On the 4th day, the viability of tumor cells was detected using Cell Counting Kit-8. The specific killing results are shown in Figures 4A to 4F (IC 50 Unit: nM). As the administration concentration increases, the antibody-drug conjugate of the present invention shows a good cell killing effect on HepG2 cells, Hep3B cells and Huh-7 cells, while basically no non-specific cell killing occurs (HEK293T, A375 cells).

[0274] Example 10

[0275] Anti-tumor trials of test substances

[0276] 10.1 Subcutaneous xenograft tumor-bearing nude mouse model of human hepatocellular carcinoma Hep3B cell line

[0277] Hep3B cells in the exponential growth phase were collected and resuspended in 1:1 PBS and Matrigel to adjust the cell density to 5×10 7 / mL, the experimental mice were subcutaneously inoculated with 5×10 6 Hep3B cells (0.1 mL / cell) were added, and tumor growth was observed regularly. When the tumor grew to an average volume of 80-150 mm 3 The mice were randomly divided into groups according to tumor size and body weight for drug administration, with 6 mice in each group. The day of grouping was defined as Day 0. On Day 0, mice were given a single tail vein injection of IgG1-PBD (5 mg / kg), GB-11-PBD (2.5 mg / kg), FH4-PBD (2.5 mg / kg), GB-11-DUBA (5 mg / kg), and IgG1-DUBA (10 mg / kg). The body weight and tumor size of the mice were measured twice a week. The volume was calculated as follows: Tumor volume (mm 3 )=1 / 2×(a×b 2 )(wherein a represents the major diameter and b represents the minor diameter), the in vivo tumor inhibition effect of the antibody-coupled PBD is shown in FIG8A , and the in vivo inhibition effect of the antibody-coupled DUBA is shown in FIG8B .

[0278] 10.2 Subcutaneous xenograft tumor-bearing nude mouse model of human hepatocellular carcinoma Hep3B cell line

[0279] Hep3B cells in the exponential growth phase were collected and resuspended in 1:1 PBS and Matrigel to adjust the cell density to 5×10 7 / mL, the experimental mice were subcutaneously inoculated with 5×10 6 Hep3B cells (0.1 mL / cell) were added, and tumor growth was observed regularly. When the tumor grew to an average volume of 80-150 mm 3 The mice were randomly divided into groups according to tumor size and weight, with 6 mice in each group. The second day of grouping was defined as day 0. On day 0, GB-11-DXD (10 mg / kg), GC90-4mu-DXD (10 mg / kg), and PBS were injected into the tail vein. The body weight and tumor size of the mice were measured twice a week. The volume was calculated as follows: tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter), the in vivo tumor inhibition effect is shown in FIG9 .

[0280] 10.3 Human Hepatocellular Carcinoma HuH7 Cell Line Subcutaneous Xenograft Tumor-Bearing Nude Mouse Model

[0281] HuH7 cells in the exponential growth phase were collected and resuspended in 1:1 PBS and Matrigel to adjust the cell density to 4 × 10 7 / mL, inoculated subcutaneously in the right flank of experimental mice, 50μl / mouse, i.e. 2×10 6 The tumor growth was observed regularly. When the tumor grew to an average volume of 50-80 mm 3 Around 24 hours after the onset of treatment, the tumor-bearing mice were divided into groups according to the tumor volume, with 6 mice in each group. The day of grouping was defined as Day 0. On Day 0, GB-11-DXD (22.5 nmol / kg) and IgG1-DXD (22.5 nmol / kg) were injected into the tail vein of the mice. The body weight and tumor size of the mice were measured twice a week. The volume was calculated as follows: Tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter), the in vivo tumor inhibition effect is shown in FIG10 .

[0282] Example 11

[0283] Functional characterization of GC90 antibody

[0284] 11.1 Species Cross-Reactivity

[0285] GC90 binding to GPC3 proteins from different species was determined using an ELISA method. Specifically, 100 μL / well of each GPC3 protein (6 μg / mL) was coated onto a 96-well plate and incubated overnight at 4°C. The plates were then blocked with 1% BSA in PBST (containing 0.05% Tween-20) for 2 hours at 37°C (200 μL / well), washed three times with PBST, and serially diluted GC90 in PBST containing 1% BSA. The plates were then added to the 96-well plate (100 μL / well) at a working concentration of 30 nM, 15 nM, and 15 nM, followed by eight dilutions in multiples of four. The plates were incubated at 37°C for 1 hour, and washed three times with PBST. Then, 100 μL / well of Anti-Human IgG-FC-HRP (Sigma, 1 / 30000 dilution) was added, incubated at 37°C for 1 h, washed three times with PBST, and then 50 μL of TMB (SURMOPICS) was added to react. The reaction was terminated with 1 M H2SO4, and the OD value was measured at 450 nm by a microplate reader. The results are shown in Table 8.

[0286] Table 11 Binding of GC90 to GPC3 proteins from different species

[0287] Note: √ represents affinity recognition, and × represents no recognition.

[0288] 11.2 Fine Mapping of Recognition Epitopes

[0289] Previous studies have shown that GC90 only recognizes the GPC3-B peptide, but not the GPC3-A peptide. To further precisely identify the epitope recognized by GC90, the amino acid sequence of the GPC3-B peptide was divided into multiple overlapping peptides, the amino acid sequences of which are shown in Table 9. Overlapping peptides were synthesized and the binding properties of each peptide to GC90 were measured using ELISA. Specifically, 100 μL / well of each overlapping peptide (6 μg / mL) was coated onto a 96-well plate and incubated overnight at 4°C. The plates were then blocked with 1% BSA in PBST (containing 0.05% Tween-20) for 2 hours at 37°C (200 μL / well), washed three times with PBST, and serially diluted GC90 in PBST containing 1% BSA (negative control: 1% BSA) and added to the 96-well plate (100 μL / well). The working concentrations were 30 nM, 15 nM, and 15 nM, followed by 8 dilutions in 4 steps. Incubate at 37°C for 1 hour and wash three times with PBST. Then, add 100 μL / well of Anti-Human IgG-FC-HRP (Sigma, 1 / 30000 dilution), incubate at 37°C for 1 hour, wash three times with PBST, and then add 50 μL of TMB (SURMOPICS) to react. The reaction is terminated with 1M H2SO4 and the OD value is measured at 450 nm using a microplate reader. The results are shown in Figure 11. The OD value of the antigen-antibody complex is measured. 450 The values ​​show the binding strength between GC90 and each peptide. It can be seen that the antibody has strong binding activity only with GPC3-B3 or long fragments containing GPC3-B3. It can be determined that the GPC3 antigen epitope region is located in GPC3-B3, namely, residues 485-496 of the human GPC3 protein (DKNLDEEGFESG).

[0290] Table 12 Amino acid sequences of overlapping peptides

[0291] Further, amino acid sequence analysis of the homology between human and mouse GPC3 proteins ( FIG12 ) revealed two amino acid residue differences within the GPC3-B3 region (monkey GPC3 protein reference Genbank ID: XP_005594665.1, also including GPC3-B3), namely, amino acid residue 487 (N) and amino acid residue 493 (F) in the human GPC3 protein (reference NP_004475.1). Combined with the results of cross-species detection, it was determined that GC90 was unable to bind to the mouse GPC3 protein due to mutations in the mouse GPC3 protein at positions corresponding to asparagine 487 and phenylalanine 493 in the human GPC3 protein, indicating that positions 487 and / or 493 of the human GPC3 protein are one of the core binding sites in the antigen epitope region.

[0292] FIG13 summarizes the antigen recognition regions of various anti-GPC3 antibodies on the GPC3 protein. It can be seen that the antigen epitope bound by GC90 is far away from the antigen binding regions of other reported anti-GPC3 antibodies, and it recognizes a completely new GPC3 antigen epitope.

[0293] Although the present invention has been described by one or more embodiments, it should be understood that the present invention is not limited to these embodiments, and the present description is intended to cover all substitutions, modifications and variations that fall within the spirit and broad scope of the appended claims. All references cited in the present invention are incorporated into the present invention by reference in their entirety.

Claims

1. A bispecific antibody or antigen-binding fragment thereof targeting GPC3, characterized in that: The bispecific antibody or antigen-binding fragment thereof targeting GPC3 contains a first GPC3 antigen-binding domain and a second GPC3 antigen-binding domain. The first GPC3 antigen binding domain comprises a first heavy chain variable region (AVH) and a first light chain variable region (AVL); the second GPC3 antigen binding domain comprises a second heavy chain variable region (BVH) and a second light chain variable region (BVL); The first heavy chain variable region (AVH) comprises a CDR region, wherein the CDR region comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR region of any one of the amino acid sequences SEQ ID Nos: 1-6; The first light chain variable region (AVL) comprises a CDR region comprising a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR region of the amino acid sequence SEQ ID No: 7; The second heavy chain variable region (BVH) comprises a CDR region comprising a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR region of the amino acid sequence of SEQ ID Nos: 8 or 9; The second light chain variable region (BVL) comprises a CDR region, wherein the CDR region comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CDR region of any one of the amino acid sequences SEQ ID Nos: 10-19; The CDR region comprises CDR1, CDR2 and CDR3 regions; the CDR1, CDR2 and CDR3 regions are defined according to IMGT, Kabat, Chothia, AbM or Contact.

2. The bispecific antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The AVH comprises a CDR region, and the CDR region comprises a sequence identical to the CDR region of any one of the amino acid sequences SEQ ID Nos: 1-6, 38-43; the AVL comprises a CDR region, and the CDR region comprises a sequence identical to the CDR region of the amino acid sequence SEQ ID No: 7; The BVH comprises a CDR region, and the CDR region comprises a sequence identical to the CDR region of the amino acid sequence SEQ ID Nos: 8 or 9; the BVL comprises a CDR region, and the CDR region comprises a sequence identical to the CDR region of any one of the amino acid sequences SEQ ID Nos: 10-19; Preferably, the CDR regions of AVH and AVL contain the same sequences as the CDR regions of SEQ ID NOs: 1 and 7, respectively, and the CDR regions of BVH and BVL contain the same sequences as the CDR regions of SEQ ID NOs: 8 and 11, respectively; or, the CDR regions of AVH and AVL contain the same sequences as the CDR regions of SEQ ID NOs: 6 and 7, respectively, and the CDR regions of BVH and BVL contain the same sequences as the CDR regions of SEQ ID NOs: 8 and 11, respectively.

3. The bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The AVH comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID Nos: 1-6, 38-43; The AVL comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No: 7; The BVH comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID Nos: 8 or 9; The BVL comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID Nos: 10-19.

4. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The AVH comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID Nos: 1 or 6; The AVL comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No: 7; The BVH comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No: 8; The BVL comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No:

11.

5. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The AVH and AVL contain SEQ ID NOs: 1 and 7, respectively; the AVH and AVL contain SEQ ID NOs: 6 and 7, respectively; the AVH and AVL contain SEQ ID NOs: 38 and 7, respectively; the AVH and AVL contain SEQ ID NOs: 39 and 7, respectively; the AVH and AVL contain SEQ ID NOs: 40 and 7, respectively; the AVH and AVL contain SEQ ID NOs: 41 and 7, respectively; the AVH and AVL contain SEQ ID NOs: 42 and 7, respectively; or the AVH and AVL contain SEQ ID NOs: 43 and 7, respectively; The BVH and BVL contain SEQ ID NOs: 9 and 19, respectively; the BVH and BVL contain SEQ ID NOs: 8 and 10, respectively; the BVH and BVL contain SEQ ID NOs: 8 and 11, respectively; the BVH and BVL contain SEQ ID NOs: 8 and 14, respectively; the BVH and BVL contain SEQ ID NOs: 8 and 15, respectively; the BVH and BVL contain SEQ ID NOs: 8 and 16, respectively; the BVH and BVL contain SEQ ID NOs: 8 and 17, respectively; the BVH and BVL contain SEQ ID NOs: 8 and 18, respectively; Preferably, the AVH and AVL comprise SEQ ID NOs: 1 and 7, respectively, and the BVH and BVL comprise SEQ ID NOs: 8 and 11, respectively; or the AVH and AVL comprise SEQ ID NOs: 6 and 7, respectively, and the BVH and BVL comprise SEQ ID NOs: 8 and 11, respectively.

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

7. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that: The bispecific antibody comprises a first polypeptide chain and a second polypeptide chain; Wherein, the structure of the first polypeptide chain can be selected from any one of the following (b1)-(b10): (b1)[BVH]-[L1]-[BVL]-[L2]-[AVH]-[CH]; (b2)[BVL]-[L1]-[BVH]-[L2]-[AVH]-[CH]; (b3)[AVH]-[L1]-[AVL]-[L2]-[BVH]-[CH]; (b4)[AVL]-[L1]-[AVH]-[L2]-[BVH]-[CH]; (b5)[AVH]-[CH]-[L2]-[BVH]-[L1]-[BVL]; (b6)[AVH]-[CH]-[L2]-[BVL]-[L1]-[BVH]; (b7) [AVH]-[CH]; (b8)[BVH]-[CH]-[L2]-[AVH]-[L1]-[AVL]; (b9)[BVH]-[CH]-[L2]-[AVL]-[L1]-[AVH]; (b10)[BVH]-[CH]; wherein, [L1] and [L2] independently represent linkers, and the linkers are selected from (GGGGS)n, wherein n is an integer of 1, 2, 3, 4, 5 or 6; the [CH] represents a heavy chain constant region, comprising CH1, CH2 and CH3; the AVH represents a first heavy chain variable region; the AVL represents a first light chain variable region; the BVH represents a second heavy chain variable region; and the BVL represents a second light chain variable region; And / or, the structure of the second polypeptide chain may be selected from any one of the following (c1)-(c10): (c1)[AVL]-[CL]; (c2) [BVL]-[CL]; (c3)[BVH]-[L1]-[BVL]-[L2]-[AVL]-[CL]; (c4)[BVL]-[L1]-[BVH]-[L2]-[AVL]-[CL]; (c5)[AVL]-[CL]-[L2]-[BVL]-[L1]-[BVH]; (c6)[AVL]-[CL]-[L2]-[BVH]-[L1]-[BVL]; (c7)[AVH]-[L1]-[AVL]-[L2]-[BVL]-[CL]; (c8)[AVL]-[L1]-[AVH]-[L2]-[BVL]-[CL]; (c9)[BVL]-[CL]-[L2]-[AVL]-[L1]-[AVH]; (c10)[BVL]-[CL]-[L2]-[AVH]-[L1]-[AVL]; Wherein, [CL] is the antibody light chain constant region; Preferably, the structures of the first and second polypeptide chains are selected from (b1) and (c1); (b7) and (c3); (b3) and (c2); or, (b10) and (c7).

8. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, characterized in that: The first polypeptide chain of the bispecific antibody or antigen-binding fragment thereof comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID Nos: 20-24, 44-47.

9. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, characterized in that: The second polypeptide chain of the bispecific antibody or antigen-binding fragment thereof comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID Nos: 25-29, 48-51.

10. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that: The first and second polypeptide chains of the bispecific antibody or antigen-binding fragment thereof are selected from one of the following combinations: 1) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 20, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 25; 2) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 21, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 26; 3) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 21, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 27; 4) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 22, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 25; 5) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 23, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 25; 6) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 21, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 28; 7) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 21, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 29; 8) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 24, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 25; 9) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 44, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 48; 10) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 45, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 49; 11) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 45, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 50; 12) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 45, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 51; 13) the first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 46, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No: 25; or 14) The first polypeptide chain is selected from the amino acid sequence of SEQ ID No: 47, and the second polypeptide chain is selected from the amino acid sequence of SEQ ID No:

25.

11. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, characterized in that: The bispecific antibody or antigen-binding fragment thereof targeting GPC3 is a humanized bispecific antibody or antigen-binding fragment thereof.

12. A bispecific antibody or antigen-binding fragment thereof targeting GPC3, characterized in that: The bispecific antibody or antigen-binding fragment thereof targeting GPC3 contains a first GPC3 antigen-binding domain and a second GPC3 antigen-binding domain, wherein the first GPC3 antigen-binding domain and the second GPC3 antigen-binding domain specifically bind to different epitopes of GPC3; The first GPC3 antigen binding domain recognizes the same GPC3 epitope as antibody A, wherein antibody A comprises a heavy chain variable region as shown in SEQ ID NO: 1 or 6 and a light chain variable region as shown in SEQ ID NO: 7; The second GPC3 antigen binding domain recognizes the same GPC3 epitope as antibody B, and the antibody B comprises a heavy chain variable region as shown in SEQ ID NO: 8 and a light chain variable region as shown in SEQ ID NO: 10 or 11.

13. The bispecific antibody or antigen-binding fragment thereof according to claim 12, characterized in that: The epitope recognized by the first GPC3 antigen binding domain comprises at least the 487th amino acid residue (N) and / or the 493rd amino acid residue (F) of the human GPC3 protein; preferably, the bispecific antibody or antigen-binding fragment thereof is selected from the bispecific antibody or antigen-binding fragment thereof targeting GPC3 according to any one of claims 1 to 11.

14. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

15. A polynucleotide, characterized in that The polynucleotide encodes the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

16. An expression vector, characterized in that: The expression vector comprises the polynucleotide of claim 15.

17. A host cell, characterized in that The host cell comprises the polynucleotide of claim 15 or the expression vector of claim 16.

18. An antibody-drug conjugate, characterized in that: It comprises the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 and a drug or toxin.

19. The antibody-drug conjugate according to claim 18, characterized in that: The drug or toxin is selected from: one or more of SN-38, MMAE, PBD dimer, DX-8951 (DXd) or DUBA; Preferably, the antibody-drug conjugate is selected from the group consisting of: (1) The bispecific antibody comprises a heavy chain as shown in SEQ ID NO: 23 and a light chain as shown in SEQ ID NO: 25, which is coupled to PBD via a maleimide-dPEG8-VA-PABA linker; (2) The bispecific antibody comprises a heavy chain as shown in SEQ ID NO: 21 and a light chain as shown in SEQ ID NO: 28, which is coupled to PBD via a maleimide-dPEG8-VA-PABA linker; (3) The bispecific antibody comprises a heavy chain as shown in SEQ ID NO: 23 and a light chain as shown in SEQ ID NO: 25, which is coupled to DUBA via a Vc-seco linker; (4) The bispecific antibody comprises a heavy chain as shown in SEQ ID NO: 21 and a light chain as shown in SEQ ID NO: 28, which is coupled to DUBA via a Vc-seco linker; (5) The bispecific antibody comprises a heavy chain as shown in SEQ ID NO: 45 and a light chain as shown in SEQ ID NO: 50, which is coupled to DXd via a maleimide-GGFG linker; (6) the bispecific antibody comprises the heavy chain shown in SEQ ID NO: 45 and the light chain shown in SEQ ID NO: 50, which is coupled to DUBA via a Vc-seco linker; or (7) The bispecific antibody comprises a heavy chain as shown in SEQ ID NO: 45 and a light chain as shown in SEQ ID NO: 50, which is coupled to PBD via a maleimide-dPEG8-VA-PABA linker.

20. A pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or the antibody-drug conjugate according to any one of claims 18 to 19 and one or more pharmaceutically acceptable carriers.

21. A kit comprising the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or the antibody-drug conjugate according to any one of claims 18 to 19 22. Use of the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, the antibody-drug conjugate according to any one of claims 18 to 19, the pharmaceutical composition according to claim 20, or the kit according to claim 21 in the preparation of a medicament for treating or preventing cancer, wherein the cancer is preferably liver cancer.

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