Anti-GPC3 antibody

Abstract

Provided are an anti-GPC3 antibody or an antigen-binding fragment thereof, and a multispecific antibody or antibody conjugate comprising same. Also provided is the use of the antibody or the antigen-binding fragment thereof, the multispecific antibody or the antibody conjugate.

Description

Antibodies against gpc3 TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine. Specifically, the present application relates to an antibody or antigen-binding fragment thereof against GPC3, and a multispecific antibody or antibody conjugate comprising the same. The present application also relates to the use of the antibody or antigen-binding fragment thereof, multispecific antibody or antibody conjugate.

[0002] BACKGROUND

[0003] Liver cancer is the sixth most common and the fourth most deadly malignancy worldwide. Hepatocellular carcinoma (HCC) is the major histopathological type of liver cancer, accounting for about 75-85%. According to the statistics in 2020, there are 410,000 new cases of liver cancer in China each year, ranking fifth among malignant tumors, and 390,000 deaths each year, ranking second among malignant tumors. The risk factors for HCC include chronic hepatitis B and C, alcohol addiction, metabolic liver disease (especially non-alcoholic fatty liver disease), and exposure to dietary toxins such as aflatoxin and aristolochic acid. All of these risk factors are preventable, and early detection and diagnosis of liver cancer are key factors in determining the prognosis of liver cancer patients. The 5-year survival rate of early liver cancer patients is more than 50%, while the 5-year survival rate of advanced liver cancer patients is less than 20%. Early liver cancer can be treated radically by local ablation, surgical resection or liver transplantation. The choice of treatment regimen depends on tumor characteristics, the severity of underlying liver dysfunction, age and other medical comorbidities. Transcatheter arterial chemoembolization (TACE) is currently the classic standard treatment for advanced liver cancer, providing more possibilities for the treatment of unresectable liver cancer. Tyrosine kinase inhibitors (TKIs), immune checkpoint inhibitors (ICIs) and anti-vascular endothelial growth factor (VEGF) monoclonal antibodies have made good progress in the treatment of HCC, and various combination regimens have been proven to be effective for advanced liver cancer. Although the treatment options for liver cancer are becoming more diverse and combined, the overall treatment options are still very limited, especially for patients with advanced liver cancer, who have only a few second-line and subsequent treatment options. Therefore, there is still a huge unmet clinical need for patients with advanced HCC. Therefore, it is of great clinical significance and application value to find specific surface molecules of HCC for targeted therapy of liver cancer.

[0004] GPC3 (Glypican-3) is a cell surface glycoprotein mainly involved in cell proliferation, differentiation, migration and apoptosis. GPC3 is expressed in the liver and kidney of fetuses and is almost not expressed in healthy human tissues, but GPC3 is highly expressed in 70-80% of HCC tissues and is also expressed in small amounts in other tumors such as ovarian clear cell carcinoma, melanoma, lung squamous cell carcinoma, etc. Although the function of GPC3 is not fully understood, a large number of studies have shown that GPC3 is closely related to the occurrence, metastasis and prognosis of HCC. Therefore, GPC3 has become a new target for the diagnosis and immunotherapy of HCC. Immunotherapy targeting GPC3 protein mainly includes monoclonal antibodies, antibody-drug conjugates, tumor vaccines, chimeric antigen receptor T cells (CAR-T) and bispecific antibodies, etc. Currently, there is no GPC3 targeted drug on the market worldwide. Among them, the bispecific antibody targeting GPC3 and CD3 can form an immunological synapse between T cells and tumor cells, summon T cells to the tumor site, activate T cell immune mechanisms, and mediate T cell killing of GPC3 overexpressing tumor cells. As a powerful expansion of monoclonal antibody therapy, a number of bispecific antibody projects are undergoing preclinical and clinical research, showing good development prospects.

[0005] SUMMARY

[0006] The present application provides at least the following embodiments:

[0007] Embodiment 1. An antibody (such as a monoclonal antibody) or an antigen binding fragment thereof against GPC3, wherein the antibody comprises a heavy chain variable region and a light chain variable region,

[0008] the heavy chain variable region comprises:

[0009] a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having substitution of D at position 1 with A relative to SEQ ID NO: 3,

[0010] a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having substitution of G at position 7 with N, substitution of D at position 8 with K, and / or substitution of Q at position 13 with N relative to SEQ ID NO: 4, and

[0011] a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having 1 or 2 or 3 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 5;

[0012] and

[0013] the light chain variable region comprises:

[0014] a VL CDR1 comprising an amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence having 1 or 2 or 3 amino acid residue substitutions, deletions, or additions to SEQ ID NO: 6, wherein the G at position 11 is substituted with K, the N at position 12 is substituted with D, and / or the L at position 15 is substituted with V,

[0015] a VL CDR2 comprising an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having 1 or 2 or 3 amino acid residue substitutions, deletions, or additions to SEQ ID NO: 7, and

[0016] a VL CDR3 comprising an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having Y at position 5 substituted with M, K, or G to SEQ ID NO: 8,

[0017] the heavy chain variable region does not comprise the amino acid sequence set forth in SEQ ID NO: 1 and / or the light chain variable region does not comprise the amino acid sequence set forth in SEQ ID NO: 2.

[0018] Embodiment 2. The antibody or antigen binding fragment thereof against GPC3 of embodiment 1, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and

[0019] the VH CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NO: 39;

[0020] the VH CDR2 has an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 40;

[0021] the VH CDR3 has an amino acid sequence selected from the group consisting of SEQ ID NO: 5;

[0022] the VL CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 42, and SEQ ID NO: 44;

[0023] the VL CDR2 has an amino acid sequence selected from the group consisting of SEQ ID NO: 7;

[0024] the VL CDR3 has an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 41, and SEQ ID NO: 43.

[0025] Embodiment 3. The antibody or antigen binding fragment thereof against GPC3 of embodiment 1 or 2, wherein

[0026] i) the heavy chain variable region has a VH CDR1 as set forth in SEQ ID NO: 39, a VH CDR2 as set forth in SEQ ID NO: 40, a VH CDR3 as set forth in SEQ ID NO: 5; and / or the light chain variable region has a VL CDR1 as set forth in SEQ ID NO: 6, a VL CDR2 as set forth in SEQ ID NO: 7, a VL CDR3 as set forth in SEQ ID NO: 41 ;

[0027] ii) the heavy chain variable region has a VH CDR1 as set forth in SEQ ID NO: 39, a VH CDR2 as set forth in SEQ ID NO: 40, a VH CDR3 as set forth in SEQ ID NO: 5; and / or the light chain variable region has a VL CDR1 as set forth in SEQ ID NO: 42, a VL CDR2 as set forth in SEQ ID NO: 7, a VL CDR3 as set forth in SEQ ID NO: 8;

[0028] iii) the heavy chain variable region has a VH CDR1 as set forth in SEQ ID NO: 39, a VH CDR2 as set forth in SEQ ID NO: 40, a VH CDR3 as set forth in SEQ ID NO: 5; and / or the light chain variable region has a VL CDR1 as set forth in SEQ ID NO: 42, a VL CDR2 as set forth in SEQ ID NO: 7, a VL CDR3 as set forth in SEQ ID NO: 43;

[0029] iv) the heavy chain variable region has a VH CDR1 as set forth in SEQ ID NO: 39, a VH CDR2 as set forth in SEQ ID NO: 40, a VH CDR3 as set forth in SEQ ID NO: 5; and / or the light chain variable region has a VL CDR1 as set forth in SEQ ID NO: 44, a VL CDR2 as set forth in SEQ ID NO: 7, a VL CDR3 as set forth in SEQ ID NO: 8; or

[0030] v) the heavy chain variable region has a VH CDR1 as set forth in SEQ ID NO: 39, a VH CDR2 as set forth in SEQ ID NO: 4, a VH CDR3 as set forth in SEQ ID NO: 5; and / or the light chain variable region has a VL CDR1 as set forth in SEQ ID NO: 42, a VL CDR2 as set forth in SEQ ID NO: 7, a VL CDR3 as set forth in SEQ ID NO: 41.

[0031] Embodiment 4. The antibody or antigen binding fragment thereof against GPC3 of any one of embodiments 1-3, the heavy chain variable region has T at position 28 substituted with Q, V, or M relative to SEQ ID NO: 1.

[0032] Embodiment 5. The antibody or antigen-binding fragment thereof against GPC3 of any one of embodiments 1-4, wherein the heavy chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or more sequence identity to SEQ ID NO: 1.

[0033] Embodiment 6. The antibody or antigen-binding fragment thereof against GPC3 of embodiment 4, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 13, 16, 19, and 22.

[0034] Embodiment 7. The antibody or antigen-binding fragment thereof against GPC3 of any one of embodiments 1-6, wherein the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or more sequence identity to SEQ ID NO: 2.

[0035] Embodiment 8. The antibody or antigen-binding fragment thereof against GPC3 of embodiment 7, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, 14, 17, 20, and 23.

[0036] Embodiment 9. The antibody or antigen-binding fragment thereof against GPC3 of any one of embodiments 1-8, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein

[0037] i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11;

[0038] ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 13 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14;

[0039] iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 16 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 17;

[0040] iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 19 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 20; or

[0041] v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 22 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 23.

[0042] Embodiment 10. The antibody or antigen binding fragment thereof against GPC3 according to any one of embodiments 1 to 9, wherein the antibody or antigen binding fragment thereof against GPC3 is a scFv against GPC3, wherein the heavy chain variable region and the light chain variable region are connected by a linker, such as a peptide linker set forth in SEQ ID NO: 25.

[0043] Embodiment 11. The antibody or antigen binding fragment thereof against GPC3 according to embodiment 10, wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 15, 18, 21 and 24.

[0044] Embodiment 12. The antibody or antigen binding fragment thereof against GPC3 according to any one of embodiments 1 to 11, further comprising an immunoglobulin Fc region, preferably the Fc region is a Fc region of human IgGl, such as comprising the amino acid sequence of SEQ ID NO: 45.

[0045] Embodiment 13. The antibody or antigen binding fragment thereof against GPC3 according to any one of embodiments 1 to 12, which is selected from the group consisting of a monoclonal antibody, a multispecific antibody, a human antibody, a non-human antibody, a humanized antibody, a chimeric antibody, an intrabody, and an antibody fragment, such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a disulfide linked Fv (dsFv), a Fd fragment, a Fd' fragment, a single chain Fv (scFv), a single chain Fab (scFab).

[0046] Embodiment 14. The antibody or antigen binding fragment thereof against GPC3 according to any one of embodiments 1 to 13, wherein the antibody or antigen binding fragment thereof against GPC3 binds to GPC3 with a KD value of less than 1 x 10 -7 M, preferably less than 1 x 10 -8 M, more preferably less than 1 x 10 -9 M, more preferably less than 1 x 10 -10 M, even more preferably less than 1 x 10 -11 M.

[0047] Embodiment 15. A multispecific antibody comprising at least one antibody or antigen binding fragment thereof against GPC3 according to any one of embodiments 1 to 14.

[0048] Embodiment 16. The multispecific antibody according to embodiment 15, which is a bispecific antibody comprising at least one antibody or antigen binding fragment thereof against GPC3 according to any one of embodiments 1 to 14, and at least one antibody or antigen binding fragment thereof against another antigen, such as at least one antibody or antigen binding fragment thereof against CD3.

[0049] Embodiment 17. The multispecific antibody of embodiment 16, wherein the antibody against CD3 comprises a heavy chain variable region set forth in SEQ ID NO: 27, and a light chain variable region set forth in SEQ ID NO: 28.

[0050] Embodiment 18. The multispecific antibody of any one of embodiments 15-17, wherein the antibody or antigen binding fragment thereof is a scFv.

[0051] Embodiment 19. The multispecific antibody of embodiment 18, wherein the multispecific antibody comprises a scFv against GPC3 of embodiment 10 or 11.

[0052] Embodiment 20. The multispecific antibody of embodiment 19, wherein the multispecific antibody comprises a scFv against CD3, for example, the scFv against CD3 comprises an amino acid sequence set forth in SEQ ID NO: 29.

[0053] Embodiment 21. The multispecific antibody of embodiment 20, wherein the scFv against GPC3 and the scFv against CD3 are connected by a linker, for example, a peptide linker set forth in SEQ ID NO: 26.

[0054] Embodiment 22. The multispecific antibody of embodiment 21, wherein the multispecific antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-36.

[0055] Embodiment 23. A nucleic acid molecule encoding the antibody or antigen binding fragment thereof against GPC3 of any one of embodiments 1-14 or the multispecific antibody of any one of embodiments 15-22.

[0056] Embodiment 24. An expression vector comprising the nucleic acid molecule of embodiment 23.

[0057] Embodiment 25. A host cell comprising the expression vector of embodiment 23 or the nucleic acid molecule of embodiment 24 integrated in the genome.

[0058] Embodiment 26. An antibody conjugate comprising the antibody or antigen binding fragment thereof against GPC3 of any one of embodiments 1-14 and a functional molecule conjugated to the antibody or antigen binding fragment thereof.

[0059] Embodiment 27. The antibody conjugate of embodiment 26, wherein the functional molecule is selected from the group consisting of a molecule targeting a tumor surface marker such as an antibody, a molecule inhibiting a tumor such as a cytotoxin or a small molecule drug, a molecule targeting a surface marker of an immune cell such as an antibody, a detectable label, a radioisotope.

[0060] Embodiment 28. The antibody conjugate of embodiment 26 or 27, wherein the functional molecule is conjugated (fused) to the antibody or antigen-binding fragment thereof against GPC3 directly or through a peptide linker; or the functional molecule is conjugated to the antibody or antigen-binding fragment thereof against GPC3 through a chemical linker.

[0061] Embodiment 29. Use of the antibody conjugate of any one of embodiments 26-28 for the manufacture of an anti-tumor medicament, for the manufacture of a reagent for detecting a cell or tissue expressing GPC3, or for the manufacture of a chimeric antigen receptor modified immune cell.

[0062] Embodiment 30. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof against GPC3 of any one of embodiments 1-14, the multispecific antibody of any one of embodiments 15-22, the nucleic acid molecule of embodiment 23, the expression vector of embodiment 24, the antibody conjugate of any one of embodiments 26-28, and a pharmaceutically acceptable carrier.

[0063] Embodiment 31. Use of the antibody or antigen-binding fragment thereof against GPC3 of any one of embodiments 1-14, the multispecific antibody of any one of embodiments 15-22, the nucleic acid molecule of embodiment 23, the expression vector of embodiment 24, the antibody conjugate of any one of embodiments 26-28 for the manufacture of a medicament for treating and / or preventing a GPC3 related disease, such as a malignant tumor, in a subject.

[0064] Embodiment 32. A method of treating and / or preventing a GPC3 related disease, such as a malignant tumor, in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof against GPC3 of any one of embodiments 1-14, the multispecific antibody of any one of embodiments 15-22, the nucleic acid molecule of embodiment 23, the expression vector of embodiment 24, the antibody conjugate of any one of embodiments 26-28, or the pharmaceutical composition of embodiment 30.

[0065] Embodiment 33. The use of embodiment 31 or the method of embodiment 32, wherein the GPC3 related disease is a tumor that highly expresses GPC3, such as a liver cancer, e.g. hepatocellular carcinoma (HCC), a lung cancer, e.g. lung squamous cell carcinoma (SqCC), a gastric cancer, an ovarian cancer, e.g. ovarian clear cell, a melanoma or a pediatric embryonal tumor.

[0066] BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1, SDS-PAGE results of purified antibodies.

[0068] Figure 2, Fc binding flow results plot.

[0069] DETAILED DESCRIPTION

[0070] I. DEFINITIONS

[0071] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the terms and techniques employed herein relating to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and immunological procedures and laboratory procedures steps are those well known and commonly used in the corresponding art. In addition, the following definitions are provided for certain terms used herein.

[0072] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether naturally occurring or produced in whole or in part synthetically (e.g., recombinantly), including any fragment thereof that retains the ability of a full-length immunoglobulin to bind with specificity to an immunoglobulin antigen, including at least a portion of the variable region of the immunoglobulin molecule. Thus, antibodies include any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding domain (antibody binding site). Antibodies include antibody fragments, such as anti-tumor cell antibody fragments. As used herein, the term antibody therefore includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above. Antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass (e.g., IgG2a and IgG2b) of antibodies.

[0073] As used herein, an "antibody fragment" or "antigen binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length, but which at least contains a portion of the variable region (e.g., one or more CDRs and / or one or more antibody binding sites) of the antibody that binds antigen, and thus retains the binding specificity and at least a portion of the specific binding ability of the full-length antibody. Thus, an antigen binding fragment refers to an antibody fragment that contains an antigen binding portion that binds the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, e.g., recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single chain Fv (scFv), Fv, dsFv, bispecific antibodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p3-25, Kipriyanov). The fragments can include multiple chains linked together, e.g., by disulfide bonds and / or by peptide linkers. Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by substitution of the corresponding region), acquires the ability to immunospecifically bind (i.e., exhibit at least or at least about 10 7 -10 8 M -1 fold increase in Ka) an antigen.

[0074] As used herein, a "monoclonal antibody" refers to a population of identical antibodies, indicating that each individual antibody molecule in the population of monoclonal antibodies is identical to other antibody molecules. This property is in contrast to polyclonal populations of antibodies, which contain antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by a number of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalizing B cells, e.g., by fusion with myeloma cells to produce hybridoma cell lines or by infection of B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies from clonal populations of host cells by transforming host cells with plasmids carrying artificial sequences encoding the nucleotides of the antibodies.

[0075] As used herein, a "conventional antibody" refers to an antibody comprising two heavy chains (which can be designated H and H') and two light chains (which can be designated L and L') and two antigen binding sites, wherein each heavy chain can be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen binding ability (e.g., a heavy chain includes but is not limited to V H heavy chain, V H -C H 1 heavy chain, and V H -C H 1-C H 2-C H 3 heavy chain), and each light chain can be a full-length light chain or any functional region thereof (e.g., a light chain includes but is not limited to V L heavy chain, and V L -C L heavy chain). Each heavy chain (H and H') pairs with one light chain (L and L', respectively).

[0076] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., V H -C H 1-C H 2-C H 3 or V H -C H 1-C H 2-C H 3-C H 4) and two full-length light chains (V L -C L ) and a hinge region, such as an antibody naturally produced by an antibody secreting B cell and an antibody synthetically produced with the same domains.

[0077] As used herein, a dsFv refers to an Fv with an engineered intermolecular disulfide bond stabilizing the V H -V L pair.

[0078] As used herein, a Fab fragment is an antibody fragment obtained by digestion of a full-length immunoglobulin with papain, or a fragment with the same structure synthetically produced, e.g., by recombinant methods. A Fab fragment comprises a light chain (comprising V L and C L ) and another chain comprising a variable domain of a heavy chain (V H ) and one constant region domain of a heavy chain (C H 1).

[0079] As used herein, an F(ab')2 fragment is an antibody fragment resulting from pepsin digestion of an immunoglobulin at pH 4.0-4.5, or a fragment of the same structure produced synthetically, for example, by recombinant means. An F(ab')2 fragment essentially comprises two Fab fragments, wherein each heavy chain portion comprises an additional few amino acids, including a cysteine which forms a disulfide bridge linking the two fragments.

[0080] As used herein, a Fab' fragment is a fragment comprising one half (one heavy chain and one light chain) of an F(ab')2 fragment.

[0081] As used herein, an scFv fragment refers to an antibody fragment comprising a variable light chain (VL) and a variable heavy chain (VH) covalently linked in any order by a polypeptide linker. The linker is of a length such that the two variable domains are essentially unimpeded in their bridging. An exemplary linker is (Gly-Ser)4, which is interspersed with a few Glu or Lys residues to increase solubility. L H As used herein, an scFv fragment refers to an antibody fragment comprising a variable light chain (VL) and a variable heavy chain (VH) covalently linked in any order by a polypeptide linker. The linker is of a length such that the two variable domains are essentially unimpeded in their bridging. An exemplary linker is (Gly-Ser)4, which is interspersed with a few Glu or Lys residues to increase solubility. n

[0082] The term "multispecific antibody" includes bispecific antibodies, trispecific antibodies, and tetraspecific antibodies, and the like, which are capable of recognizing multiple (e.g., two, three, four, or more) different antigenic determinants. Multispecific antibodies can include bivalent or multivalent antibodies. The antigenic determinants can be different antigenic determinants on the same antigen or different antigens.

[0083] The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0084] In addition, it is possible to mutate amino acid residues within the CDR1, CDR2, and / or CDR3 regions of the VH and / or VL, thereby improving one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, by PCR-mediated mutagenesis, the effects of which on the binding or other functional properties of the antibody can be evaluated using the in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Further, typically no more than three mutations are made within a CDR. Accordingly, the humanized antibodies of the application also encompass antibodies comprising no more than 3 amino acid mutations within a CDR, for example, comprising 1 or two amino acid mutations.

[0085] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which a paratope of an antibody binds. Epitope determinants typically comprise chemically active surface groupings of molecules such as amino acids or sugar side chains and typically have specific three-dimensional structural characteristics, as well as specific charge characteristics.​​

[0086] As used herein, a variable domain or variable region is a particular Ig domain of an antibody heavy or light chain that comprises an amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has one variable region domain, V L and V H The variable domain provides antigen specificity and is thus responsible for antigen recognition. Each variable region comprises CDRs and framework regions (FRs), the CDRs being part of the antigen binding site domain.

[0087] As used herein, "antigen binding domain" and "antigen-binding site" are used synonymously to refer to the domain within an antibody that recognizes and physically interacts with a cognate antigen. A native, conventional full-length antibody molecule has two conventional antigen-binding sites, each comprising a heavy chain variable region portion and a light chain variable region portion. A conventional antigen-binding site comprises loops connecting antiparallel beta strands within the variable region domain. An antigen-binding site can comprise other portions of the variable region domain. Each conventional antigen-binding site comprises 3 hypervariable regions from the heavy chain and 3 hypervariable regions from the light chain. Hypervariable regions are also known as complementarity determining regions (CDRs).

[0088] As used herein, "hypervariable region," "HV," "complementarity determining region," and "CDR," and "antibody CDR," are used interchangeably to refer to one of a number of portions within each variable region that together form an antigen-binding site of an antibody. Each variable region domain comprises 3 CDRs, designated CDR1, CDR2, and CDR3. For example, a light chain variable region domain comprises 3 CDRs, designated VL CDR1, VL CDR2, and VL CDR3; a heavy chain variable region domain comprises 3 CDRs, designated VH CDR1, VH CDR2, and VH CDR3. The 3 CDRs in a variable region are non-contiguous along the linear amino acid sequence, but are proximate in the folded polypeptide. The CDRs are located within loops connecting the parallel beta strands of the variable domain's beta sheet. As described herein, one of skill in the art knows and can identify CDRs based on Kabat or Chothia numbering (see, e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).

[0089] As used in this article, the frame region (FR) is a domain located within the antibody variable region domain within the β-sheet; in terms of amino acid sequence, the FR region is relatively more conserved than the hypervariable region.

[0090] As used in this article, the "constant region" domain is a domain in the antibody heavy or light chain that contains a more conserved amino acid sequence than the variable region domain. In a typical full-length antibody molecule, each light chain has a single light chain constant region (C0). L ) structural domains, and each heavy chain contains one or more heavy chain constant regions (C H ) structural domain, including C H 1. C H 2. C H 3 and C H 4. Full-length IgA, IgD, and IgG isotypes include C. H 1. C H 2. C H 3 and hinge region, while IgE and IgM contain C H 1. C H 2. C H 3 and C H 4. C H 1 and C L The structural domain extends the Fab arm of the antibody molecule, thus facilitating interaction with antigens and the rotation of the antibody arm. The antibody constant region can serve effector functions, such as, but not limited to, clearing antigens, pathogens, and toxins specifically bound to the antibody, for example, through interactions with various cells, biomolecules, and tissues.

[0091] As used in this article, the functional region of an antibody is a region containing at least V of the antibody. H V L C H (e.g., C) H 1. C H 2 or C H 3) C L Or the hinge region structural domain or at least the antibody portion of its functional region.

[0092] As used in this article, V H The functional area of ​​a structural domain is to preserve the complete V H At least part of the binding specificity of the domain (e.g., by preserving the complete V) H The complete V of one or more CDRs of the structural domain H At least a portion of the structural domain, thus the V H The functional regions of the structural domain, either alone or in combination with another antibody structural domain (e.g., V), L The antigen is bound to a domain or a combination of its regions. Example V H The functional area of ​​a structural domain is the area containing V HThe regions of CDR1, CDR2 and / or CDR3 of the structural domain.

[0093] As used in this article, V L The functional area of ​​a structural domain is to preserve the complete V L At least part of the binding specificity of the domain (e.g., by preserving the complete V) L The complete V of one or more CDRs of the structural domain L At least a portion of the structural domain, thus the V L The functional regions of the structural domain, either alone or in combination with another antibody structural domain (e.g., V), H The antigen is bound to a domain or a combination of its regions. Example V L The functional area of ​​a structural domain is the area containing V L The regions of CDR1, CDR2 and / or CDR3 of the structural domain.

[0094] As used herein, an antibody or antigen-binding fragment thereof targeting a specific antigen refers to the antibody or antigen-binding fragment thereof specifically binding to the specific antigen.

[0095] As used herein, the terms “specific binding” and “immune-specific binding” for antibodies or their antigen-binding fragments are used interchangeably and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with the same antigen through a non-covalent interaction between the antibody and the antigen’s antibody-binding site. The antigen may be an isolated antigen or present in tumor cells. Typically, antibodies that immune-specifically bind (or specifically bind) antigens are present in quantities of approximately 1 × 10⁻⁶. 7 M -1 Or 1x 10 8 M -1 Or a larger affinity constant Ka (or 1 x 10⁻⁶) -7 M or 1×10 -8 M or a lower dissociation constant (K) dThe affinity constant can be determined by standard kinetic methods of antibody reactions, such as immunoassay, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); also see U.S. Patent No. 7,229,619, which describes exemplary SPR and ITC methods for calculating the binding affinity of an antibody). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0096] As used herein, a polypeptide refers to two or more amino acids covalently linked together. The terms "polypeptide" and "protein" are used interchangeably herein.

[0097] "Isolated protein," "isolated polypeptide," or "isolated antibody" means that the protein, polypeptide, or antibody (1) is not associated with its natural associated component in its native state, (2) does not contain other proteins from the same species, (3) is expressed by cells from a different species, or (4) does not occur naturally. Therefore, a chemically synthesized polypeptide or a polypeptide synthesized in a cellular system of a different natural source cell will be "isolated" from its natural associated component. Isolation can also render a protein substantially free of its natural associated component, i.e., using protein purification techniques well known in the art.

[0098] In peptides or proteins, suitable conserved amino acid substitutions are known to those skilled in the art and can generally be performed without altering the biological activity of the resulting molecule. Typically, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0099] As used herein, the terms “polynucleotide” and “nucleic acid molecule” refer to oligomers or polymers containing at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which are typically linked together by phosphodiester bonds.

[0100] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in natural sources of nucleic acid molecules. “Isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding provided antibody or antigen-binding fragments.

[0101] Sequence “identity” has a generally accepted meaning in the art, and the percentage of sequence similarity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of that molecule. (See, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are many methods for measuring the similarity between two polynucleotides or polypeptides, the term "similarity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0102] As used herein, “operably linked” in relation to a nucleic acid sequence, region, element, or domain indicates that the nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide, thereby regulating or mediating the transcription of that nucleic acid.

[0103] As used herein, “expression” refers to the process by which a polypeptide is produced through the transcription and translation of polynucleotides. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from host cells. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assays, and Bradford protein assays.

[0104] As used herein, a “host cell” is a cell used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When a host cell divides, the nucleic acids contained in the vector replicate, thereby amplifying the nucleic acids. Host cells can be eukaryotic or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0105] “Codon optimization” refers to methods of modifying nucleic acid sequences to enhance expression in host cells of interest by replacing at least one codon of the natural sequence with codons that are used more frequently or most frequently in the gene in the host cell (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons while maintaining the natural amino acid sequence). Different species exhibit specific preferences for certain codons of specific amino acids. Codon preference (differences in codon use between organisms) is often associated with the translation efficiency of messenger RNA (mRNA), which is thought to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell generally reflects the codons most frequently used for peptide synthesis. Therefore, genes can be customized to achieve optimal gene expression in a given organism based on codon optimization. Codon utilization tables are readily available, for example, in the Codon Usage Database available at www.kazusa.orjp / codon / , and these tables can be adapted in various ways. See Nakamura. Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0106] As used herein, a "vector" is a reproducible nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can typically be introduced via restriction enzyme digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplification of nucleic acids or for expression / display of the polypeptide encoded by the nucleic acid. Vectors are typically kept free but can be designed to integrate genes or portions thereof into the chromosome of the genome. Vectors for artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes, are also considered. The selection and use of such vectors are well known to those skilled in the art.

[0107] As used in this article, vectors also include “viral vectors” or “vectors of viruses.” Viral vectors are engineered viruses that are operatively linked to a foreign gene to transfer (as a medium or shuttle) the foreign gene into cells.

[0108] As used herein, "expression vector" includes a vector capable of expressing DNA operatively linked to regulatory sequences, such as promoter regions, that influence the expression of such DNA fragments. These additional fragments may include promoter and terminator sequences and optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into a suitable host cell, results in the expression of clonal DNA. Suitable expression vectors are well known to those skilled in the art and include reproducible expression vectors in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain free or are integrated into the host cell genome.

[0109] As used herein, “treatment” for an individual suffering from a disease or disease condition means that the individual’s symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing underlying disease and / or preventing the worsening of symptoms or the development of disease. Treatment also includes any antibodies or antigen-binding fragments thereof provided, and any pharmaceutical use of the compositions provided herein.

[0110] As used in this article, “therapeutic effect” refers to the effect resulting from treatment of an individual, which alters, usually improves or enhances the symptoms of a disease or condition, or cures a disease or condition.

[0111] As used herein, "therapeutic effective amount" or "therapeutic effective dose" refers to an amount of substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition.

[0112] As used herein, "preventive effective dose" or "preventive effective amount" refers to the amount of a substance, compound, material, or composition containing a compound that, when applied to a subject, would have the intended preventive effect, such as preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. A fully preventive effective dose does not necessarily occur through the administration of a single dose and can occur only after a series of doses have been administered. Therefore, a preventive effective dose can be administered in one or more applications.

[0113] As used in this article, the term "object" refers to mammals, such as humans.

[0114] II. Antibodies against GPC3

[0115] In one aspect, the present invention provides an antibody (such as a monoclonal antibody) or an antigen-binding fragment thereof targeting GPC3, wherein the antibody comprises a heavy chain variable region and a light chain variable region.

[0116] The heavy chain variable region includes:

[0117] VH CDR1, comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having one, two, or three amino acid residues substituted, deleted, or added relative to SEQ ID NO:3,

[0118] VH CDR2, comprising the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having one, two, or three amino acid residues substituted, deleted, or added relative to SEQ ID NO:4, and

[0119] VH CDR3, which contains the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having one, two, or three amino acid residues substituted, deleted, or added relative to SEQ ID NO:5;

[0120] The light chain variable region includes:

[0121] VL CDR1, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having one, two, or three amino acid residues substituted, deleted, or added relative to SEQ ID NO:6,

[0122] VL CDR2, comprising the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having one, two, or three amino acid residues substituted, deleted, or added relative to SEQ ID NO:7, and

[0123] VL CDR3, comprising the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having one, two, or three amino acid residues substituted, deleted, or added relative to SEQ ID NO:8.

[0124] In some embodiments, the heavy chain variable region does not contain the amino acid sequence shown in SEQ ID NO:1 and / or the light chain variable region does not contain the amino acid sequence shown in SEQ ID NO:2.

[0125] In some embodiments, the GPC3 is human GPC3, for example, which contains the amino acid sequence shown in SEQ ID NO:31.

[0126] In some embodiments, the VH CDR1 comprises an amino acid sequence in which D is replaced by A at position 1 relative to SEQ ID NO:3;

[0127] In some embodiments, the VH CDR2 comprises an amino acid sequence in which G at position 7 is replaced by N, D at position 8 is replaced by K, and / or Q at position 13 is replaced by N, relative to SEQ ID NO:4.

[0128] In some embodiments, the VL CDR1 comprises an amino acid sequence in which G at position 11 is replaced by K, N at position 12 is replaced by D, and / or L at position 15 is replaced by V, relative to SEQ ID NO:6.

[0129] In some embodiments, the VL CDR3 comprises an amino acid sequence in which the Y at position 5 of SEQ ID NO:8 is replaced by M, K, or G.

[0130] In some implementations, the heavy chain variable region comprises:

[0131] VH CDR1, which contains the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence in which D is replaced by A at position 1 relative to SEQ ID NO:3.

[0132] VH CDR2, comprising the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence in which G at position 7 is replaced by N, D at position 8 is replaced by K, and / or Q at position 13 is replaced by N, and

[0133] VH CDR3, which contains the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having one, two, or three amino acid residues substituted, deleted, or added relative to SEQ ID NO:5;

[0134] and

[0135] The light chain variable region includes:

[0136] VL CDR1, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence relative to SEQ ID NO:6 in which G at position 11 is replaced by K, N at position 12 is replaced by D, and / or L at position 15 is replaced by V.

[0137] VL CDR2, comprising the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having one, two, or three amino acid residues substituted, deleted, or added relative to SEQ ID NO:7, and

[0138] VL CDR3, which contains the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence in which Y at position 5 of SEQ ID NO:8 is replaced by M, K or G.

[0139] In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region.

[0140] The heavy chain variable region includes:

[0141] VH CDR1 contains an amino acid sequence in which D is replaced by A at position 1 relative to SEQ ID NO:3.

[0142] VH CDR2, comprising the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence in which G at position 7 is replaced by N, D at position 8 is replaced by K, and / or Q at position 13 is replaced by N, and

[0143] VH CDR3 contains the amino acid sequence shown in SEQ ID NO:5;

[0144] The light chain variable region includes:

[0145] VL CDR1, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence relative to SEQ ID NO:6 in which G at position 11 is replaced by K, N at position 12 is replaced by D, and / or L at position 15 is replaced by V.

[0146] VL CDR2, which contains the amino acid sequence shown in SEQ ID NO:7, and

[0147] VL CDR3, which contains the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence in which Y at position 5 of SEQ ID NO:8 is replaced by M, K or G.

[0148] In some implementation schemes, where

[0149] VH CDR1 has an amino acid sequence selected from the group consisting of: SEQ ID NO:39;

[0150] VH CDR2 has an amino acid sequence selected from the following groups: SEQ ID NO:4 and SEQ ID NO:40;

[0151] VH CDR3 has an amino acid sequence selected from the group consisting of: SEQ ID NO:5;

[0152] VL CDR1 has an amino acid sequence selected from the group consisting of: SEQ ID NO:6, SEQ ID NO:42 and SEQ ID NO:44;

[0153] VL CDR2 has an amino acid sequence selected from the group consisting of: SEQ ID NO:7;

[0154] VL CDR3 has an amino acid sequence selected from the group consisting of: SEQ ID NO:8, SEQ ID NO:41 and SEQ ID NO:43.

[0155] In some implementation schemes, where

[0156] i) The heavy chain variable region has VH CDR1 shown in SEQ ID NO:39, VH CDR2 shown in SEQ ID NO:40, and VH CDR3 shown in SEQ ID NO:5; and / or the light chain variable region has VL CDR1 shown in SEQ ID NO:6, VL CDR2 shown in SEQ ID NO:7, and VL CDR3 shown in SEQ ID NO:41;

[0157] ii) The heavy chain variable region has VH CDR1 shown in SEQ ID NO:39, VH CDR2 shown in SEQ ID NO:40, and VH CDR3 shown in SEQ ID NO:5; and / or the light chain variable region has VL CDR1 shown in SEQ ID NO:42, VL CDR2 shown in SEQ ID NO:7, and VL CDR3 shown in SEQ ID NO:8;

[0158] iii) The heavy chain variable region has VH CDR1 shown in SEQ ID NO:39, VH CDR2 shown in SEQ ID NO:40, and VH CDR3 shown in SEQ ID NO:5; and / or the light chain variable region has VL CDR1 shown in SEQ ID NO:42, VL CDR2 shown in SEQ ID NO:7, and VL CDR3 shown in SEQ ID NO:43;

[0159] iv) The heavy chain variable region has VH CDR1 shown in SEQ ID NO:39, VH CDR2 shown in SEQ ID NO:40, and VH CDR3 shown in SEQ ID NO:5; and / or the light chain variable region has VL CDR1 shown in SEQ ID NO:44, VL CDR2 shown in SEQ ID NO:7, and VL CDR3 shown in SEQ ID NO:8; or

[0160] v) The heavy chain variable region has VH CDR1 shown in SEQ ID NO:39, VH CDR2 shown in SEQ ID NO:4, and VH CDR3 shown in SEQ ID NO:5; and / or the light chain variable region has VL CDR1 shown in SEQ ID NO:42, VL CDR2 shown in SEQ ID NO:7, and VL CDR3 shown in SEQ ID NO:41.

[0161] In some embodiments, the heavy chain variable region at position 28 of SEQ ID NO:1 is replaced by Q, V, or M.

[0162] In one aspect, the present invention provides an antibody (such as a monoclonal antibody) or an antigen-binding fragment thereof targeting GPC3, wherein the antibody comprises a heavy chain variable region and a light chain variable region.

[0163] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence in which T at position 28 is substituted with Q, V, or M, D at position 31 is substituted with A, G at position 56 is substituted with N, D at position 57 is substituted with K, and / or Q at position 62 is substituted with N; and / or

[0164] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence in which G at position 34 is replaced by K, N at position 35 is replaced by D, L at position 38 is replaced by V, and / or Y at position 98 is replaced by M, K or G relative to SEQ ID NO:2.

[0165] In some embodiments, the heavy chain variable region comprises an amino acid sequence in which T at position 28 is replaced by Q, V or M, D at position 31 is replaced by A, G at position 56 is replaced by N, D at position 57 is replaced by K, and / or Q at position 62 is replaced by N; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2.

[0166] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1; the light chain variable region comprises an amino acid sequence in which G at position 34 is replaced by K, N at position 35 is replaced by D, L at position 38 is replaced by V, and / or Y at position 98 is replaced by M, K or G, relative to SEQ ID NO:2.

[0167] In some embodiments, the heavy chain variable region comprises an amino acid sequence in which T at position 28 of SEQ ID NO:1 is replaced by Q, V, or M, D at position 31 is replaced by A, G at position 56 is replaced by N, D at position 57 is replaced by K, and / or Q at position 62 is replaced by N; the light chain variable region comprises an amino acid sequence in which G at position 34 of SEQ ID NO:2 is replaced by K, N at position 35 is replaced by D, L at position 38 is replaced by V, and / or Y at position 98 is replaced by M, K, or G.

[0168] In some embodiments of various aspects of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:1.

[0169] In some embodiments of various aspects of the present invention, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO: 10, 13, 16, 19 and 22.

[0170] In some embodiments of various aspects of the present invention, the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:2.

[0171] In some embodiments of various aspects of the present invention, the light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 11, 14, 17, 20 and 23.

[0172] In some embodiments of various aspects of the present invention, the antibody comprises a heavy chain variable region and a light chain variable region, wherein

[0173] i) The heavy chain variable region contains the amino acid sequence of SEQ ID NO:10, and the light chain variable region contains the amino acid sequence of SEQ ID NO:11;

[0174] ii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO:13, and the light chain variable region contains the amino acid sequence of SEQ ID NO:14;

[0175] iii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO:16, and the light chain variable region contains the amino acid sequence of SEQ ID NO:17;

[0176] iv) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:19, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:20; or

[0177] v) The heavy chain variable region contains the amino acid sequence of SEQ ID NO:22, and the light chain variable region contains the amino acid sequence of SEQ ID NO:23.

[0178] In some embodiments of various aspects of the present invention, the antibody (such as a monoclonal antibody) or its antigen-binding fragment against GPC3 is an isolated antibody or its antigen-binding fragment.

[0179] In some embodiments of various aspects of the present invention, the antibody (e.g., a monoclonal antibody) or its antigen-binding fragment against GPC3 is a scFv targeting GPC3. In some embodiments, in the scFv, the heavy chain variable region and the light chain variable region are linked by a linker, for example, the peptide linker shown in SEQ ID NO:25.

[0180] In some embodiments of various aspects of the present invention, the scFv comprises an amino acid sequence selected from SEQ ID NO: 12, 15, 18, 21 and 24.

[0181] In some embodiments of various aspects of the present invention, the antibody against GPC3 (such as a monoclonal antibody) or its antigen-binding fragment further comprises an immunoglobulin Fc region. The Fc region used in the present invention can be derived from different subtypes of immunoglobulins, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM. Preferably, the Fc region is the Fc region of human IgG1, for example, containing the amino acid sequence of SEQ ID NO:45.

[0182] In some embodiments of the present invention, the KD value of the antibody (e.g., monoclonal antibody) or its antigen-binding fragment targeting GPC3 may be less than 1 × 10⁻⁶. -7 M, preferably less than 1×10 -8 M, more preferably less than 1×10 -9 M, more preferably less than 1×10 -10 M, especially preferred to be less than 1×10 -11 M.

[0183] In another aspect, the present invention provides a multispecific antibody comprising at least one (e.g., 1, 2, 3 or more) of the antibody against GPC3 of the present invention or an antigen-binding fragment thereof. In some embodiments, the multispecific antibody further comprises at least one (e.g., 1, 2, 3 or more) of an antibody against other antigens or an antigen-binding fragment thereof.

[0184] In some embodiments, the multispecific antibody is a bispecific antibody comprising at least one (e.g., 1, 2, 3 or more) of the present invention’s antibody against GPC3 or an antigen-binding fragment thereof, and at least one (e.g., 1, 2, 3 or more) of an antibody against another antigen or an antigen-binding fragment thereof.

[0185] In some embodiments, the antibody against another antigen or its antigen-binding fragment is an antibody against CD3 or its antigen-binding fragment.

[0186] In some embodiments, the antibody or its antigen-binding fragment in the bispecific antibody is scFv.

[0187] In some embodiments, the antibody against CD3 includes the heavy chain variable region shown in SEQ ID NO:27 and the light chain variable region shown in SEQ ID NO:28.

[0188] In some embodiments, the bispecific antibody comprises the scFv of the present invention targeting GPC3 and the scFv targeting CD3.

[0189] In some embodiments, the scFv for GPC3 and the scFv for CD3 of the present invention are connected by a connector, such as the peptide connector shown in SEQ ID NO:26.

[0190] In some embodiments, the CD3-targeted scFv contains the amino acid sequence shown in SEQ ID NO:29.

[0191] In some embodiments, the bispecific antibody comprises an amino acid sequence selected from SEQ ID NO:32-36.

[0192] III. Methods for producing nucleic acids, vectors, and antibodies

[0193] On the other hand, the present invention provides isolated nucleic acid molecules encoding the aforementioned antibody against GPC3 of the present invention or its antigen-binding fragment, or the multispecific antibody of the present invention. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized for the host cell used for expression. In some embodiments, the nucleic acid molecule of the present invention is operatively linked to an expression regulatory sequence.

[0194] The present invention also provides an expression vector comprising at least one of the aforementioned nucleic acid molecules of the present invention.

[0195] The present invention also provides a host cell comprising at least one of the aforementioned nucleic acid molecules or expression vectors of the present invention. In some embodiments, the host cell has a nucleic acid molecule of the present invention integrated into its genome.

[0196] In another aspect, the present invention provides a method for producing the antibody against GPC3 of the present invention or its antigen-binding fragment, or the multispecific antibody of the present invention, comprising:

[0197] (i) The host cells of the present invention are cultured under conditions suitable for expression of the nucleic acid molecules or expression vectors, and

[0198] (ii) Isolate and purify the antibody against GPC3 of the present invention or its antigen-binding fragment or the multispecific antibody of the present invention expressed by the host cells.

[0199] The present invention also relates to antibodies against GPC3 or antigen-binding fragments thereof or multispecific antibodies obtained by the methods of the present invention described above, which are capable of specifically binding to GPC3.

[0200] IV. Antibody Conjugates

[0201] This invention also provides an antibody conjugate comprising the antibody against GPC3 of this invention or its antigen-binding fragment thereof, and a functional molecule conjugated to the antibody or its antigen-binding fragment thereof. In some embodiments, the functional molecule may be a molecule targeting tumor surface markers such as antibodies, a tumor-inhibiting molecule such as cytotoxins or small molecule drugs, a molecule targeting immune cell surface markers such as antibodies, a detectable marker, a radioisotope, etc. In some embodiments, the functional molecule may be conjugated (fused) to the antibody against GPC3 of this invention or its antigen-binding fragment thereof directly or via a peptide linker. In some embodiments, the functional molecule may be conjugated to the antibody against GPC3 of this invention or its antigen-binding fragment thereof via a chemical linker.

[0202] The present invention also provides the use of the antibody conjugate for the preparation of antitumor drugs, for the preparation of reagents for the detection of cells or tissues expressing GPC3, or for the preparation of chimeric antigen receptor modified immune cells.

[0203] V. Pharmaceutical compositions, disease treatment and / or prevention

[0204] The present invention also provides a pharmaceutical composition comprising an antibody against GPC3 of the present invention or an antigen-binding fragment thereof, a multispecific antibody of the present invention, a nucleic acid molecule of the present invention, an expression vector of the present invention or an antibody conjugate of the present invention, and a pharmaceutically acceptable carrier. The pharmaceutical composition is intended for the prevention and / or treatment of GPC3-related diseases such as malignancies in subjects.

[0205] The present invention also provides the use of the antibody against GPC3 of the present invention or its antigen-binding fragment, the multispecific antibody of the present invention, the nucleic acid molecule of the present invention, the expression vector of the present invention, or the antibody conjugate of the present invention in the preparation of a medicament for use in and / or in the prevention of GPC3-related diseases such as malignant tumors.

[0206] The term "pharmaceutically acceptable carrier" as used herein includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0207] The actual dose level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response to a specific subject, composition, and route of administration, without toxicity to the subject. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the specific composition of the present invention applied, the route of administration, the time of administration, the excretion rate of the specific compound applied, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition applied, the age, sex, weight, condition, general health status, and medical history of the subject receiving treatment, and similar factors known in the medical field.

[0208] An "effective amount" of the antibody of the present invention or its antigen-binding fragment or the multispecific antibody of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of damage or disability caused by disease suffering. For example, in the treatment of tumors, an "effective amount" of the antibody of the present invention or its antigen-binding fragment or the multispecific antibody of the present invention preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system for predicting the efficacy of treatment against human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro by assays known to those skilled in the art. An effective amount of the antibody of the present invention or its antigen-binding fragment or the multispecific antibody can reduce tumor size or otherwise alleviate symptoms in the subject, such as prevention and / or treatment of metastasis or recurrence. Those skilled in the art can determine this amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration chosen.

[0209] The antibodies or antigen-binding fragments thereof of the present invention, the multispecific antibodies of the present invention, or the pharmaceutical compositions of the present invention can be administered via one or more routes of administration using one or more methods known in the art. Those skilled in the art will understand that the route and / or manner of administration varies depending on the desired outcome. Preferred routes of administration for the antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as injection or infusion. The phrase “parenteral administration” as used herein refers to a mode of administration other than enteral and local administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intra-capsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions.

[0210] The present invention also provides a method for treating and / or preventing GPC3-related diseases, such as malignant tumors, in a subject, the method comprising administering to the subject an effective amount of an antibody against GPC3 of the present invention or an antigen-binding fragment thereof, or a multispecific antibody of the present invention, a nucleic acid molecule of the present invention, an expression vector of the present invention, an antibody conjugate of the present invention, or a pharmaceutical composition of the present invention.

[0211] GPC3-related diseases, such as tumors that highly express (as opposed to healthy tissue) GPC3, including but not limited to liver cancer such as hepatocellular carcinoma (HCC), lung cancer such as squamous cell carcinoma (SqCC), gastric cancer, ovarian cancer such as clear cell ovarian cancer, melanoma, or pediatric embryonal tumors, can be treated and / or prevented by the antibodies against GPC3 or their antigen-binding fragments, the multispecific antibodies of the present invention, the nucleic acid molecules of the present invention, the expression vectors of the present invention, the antibody conjugates of the present invention, the pharmaceutical compositions of the present invention, or the methods of the present invention.

[0212] In some embodiments, the antibody against GPC3 of the present invention or its antigen-binding fragment, the multispecific antibody of the present invention, the nucleic acid molecule of the present invention, the expression vector of the present invention, the antibody conjugate of the present invention, the pharmaceutical composition of the present invention, or the method of the present invention can be used in combination with chemotherapeutic agents, immune checkpoint inhibitors, antibodies targeting other tumor-specific antigens, or radiotherapy.

[0213] There are no particular limitations on the antibodies or antigen-binding fragments of the present invention targeting GPC3, the multispecific antibodies of the present invention, the nucleic acid molecules of the present invention, the expression vectors of the present invention, the antibody conjugates of the present invention, the chemotherapeutic agents used in combination with the pharmaceutical compositions of the present invention, immune checkpoint inhibitors, or antibodies targeting other tumor antigens. Examples of the chemotherapeutic agents, immune checkpoint inhibitors, and antibodies targeting other tumor antigens include, but are not limited to: ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, melphalan, enoxabin, capecitabine, carmoflurane, cladribine, gemcitabine, cytarabine, tegafur, tegafur-uracil, TS-1, deoxyfluorouridine, nerabine, hydroxyurea, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine, methotrexate, irinotecan, etoposide, eribulin, sobuzosen, docetaxel, paclitaxel, vinorelbine, vincristine, vindesine, actinomycin D, arubicin, amrubicin, idarubicin, epirubicin, doxorubicin, daunorubicin, doxorubicin. Pirarubicin, Bleomycin, Pyromycin, Mitomycin C, Mitoxantrone, Oxaliplatin, Carboplatin, Cisplatin, Nedaplatin, Anastrozole, Exemestane, Ethinylestradiol, Chlormadinone, Goserelin, Tamoxifen, Dexamethasone, Bicalutamide, Toremifene, Flutamide, Prednisolone, S-estradiol, Mitotan, Methyltestosterone, Leuprorelin, Letrozole, Methylprogesterone acetate, Ti... Imoxicillin, imatinib, everolimus, erlotinib, gefitinib, sunitinib, cetuximab, sorafenib, dasatinib, tamibarbitine, trastuzumab, retinoic acid, perumumab, bevacizumab, bortezomib, lapatinib, atezolizumab, pembrolizumab, tislelizumab, camrelizumab, sugemalimab, and nivolumab, etc.

[0214] The antibodies against GPC3 or their antigen-binding fragments, the multispecific antibodies of the present invention, the nucleic acid molecules of the present invention, the expression vectors of the present invention, the antibody conjugates of the present invention, the pharmaceutical compositions of the present invention, and the chemotherapeutic agents, immune checkpoint inhibitors, or antibodies targeting other tumor antigens of the present invention can all be administered at once or separately. When administered separately (using different administration regimens), they can be administered continuously without interruption or at predetermined intervals.

[0215] The present invention does not impose any particular limitation on the combined dosage of the antibody against GPC3 or its antigen-binding fragment, the multispecific antibody of the present invention, the nucleic acid molecule of the present invention, the expression vector of the present invention, the antibody conjugate of the present invention, the pharmaceutical composition of the present invention, and the chemotherapeutic agent, immune checkpoint inhibitor, or antibody targeting other tumor antigens in the pharmaceutical composition of the present invention. As mentioned above, the dosage of the antibody of the present invention can be determined by referring to the dosage when the antibody is used alone. The chemotherapeutic agent, immune checkpoint inhibitor, and antibody targeting other tumor antigens can be used according to the dosage specified in their respective pharmaceutical descriptions or can be reduced (taking into account the combination effect with the antibody of the present invention).

[0216] The antibodies against GPC3 or their antigen-binding fragments, the multispecific antibodies of the present invention, the nucleic acid molecules of the present invention, the expression vectors of the present invention, the antibody conjugates of the present invention, and the pharmaceutical compositions of the present invention can also be combined with radiotherapy, for example, including the administration of ionizing radiation to a subject earlier, during, and / or later than the administration of the antibodies or pharmaceutical compositions of the present invention. Example

[0217] Example 1: Preparation and identification of parental BiTE-FASEBA samples

[0218] The parental antibodies are GPC3 ScFv and CD3 ScFv linked together to form a BiTE. The GPC3 ScFv sequence (SEQ ID NO:9) is derived from the humanized Y035 sequence of patent CN111018986 B, and the CD3 ScFv sequence (SEQ ID NO:29) is derived from the CD3 antibody sequence of Amgen's Tarlatamab. The two are linked together by a linker (SEQ ID NO:26).

[0219] The affinity of parental antibodies was enhanced using the FASEBA (Fast Screening of Expression Biophysical-properties Affinity) high-throughput screening platform (GenScript), and BiTE molecules with higher affinity for human GPC3 were screened out.

[0220] A DNA sequence encoding the parental BiTE antibody was synthesized and inserted into the FASEBA vector to construct the parental expression plasmid. The FASEBA plasmid was then transformed into E. coli TG1 competent cells. After selecting positive clones for culture, parental BiTE-FASEBA expression was induced by IPTG.

[0221] The affinity of parental BiTE-FASEBA supernatant for human GPC3 antigen protein (SEQ ID NO:31) was determined using a Biacore 8K (Cytiva) sensor, with GPC3 scfv-FASEBA supernatant serving as a control. FASEBA supernatant was captured on the sensor chip, using the antigen as the analyte, with association time defined, and a buffer flow maintained for dissociation. Dissociation (kd) and association (ka) rate constants were obtained using the Biacore 8K evaluation software; specific parameters are shown in Table 1.

[0222] Table 1. Affinity Detection Parameters

[0223] Experiment Example 2: Construction and Screening of PML (Precise Mutagenesis Library) Mutation Libraries

[0224] The amino acid residues in the parental BiTE CDR region were mutated using E. coli optimal codons to obtain 18 other required amino acids (excluding parental amino acids and cysteine). DNA oligonucleotide libraries were synthesized on a programmable microarray and cloned into the parental BiTE-FASEBA vector as daughter libraries. Each PML library was generated based on the FASEBA platform, and NGS sequencing was used to ensure library quality and guarantee a minimum coverage of 90%.

[0225] Forty-seven clones were randomly selected from each single-point mutation in the PML library, resulting in a total of 2256 clones, and their expression was induced in 96-well plates. The secreted proteins in the culture medium were detected by ELISA. BSA and human GPC3 antigen proteins were selected to assess the expression levels and binding affinity of the clones in the PML library, respectively. The positive control was parental BiTE-FASEBA supernatant, and the negative controls were irrelevant FASEBA supernatant (NC) and 2-YT medium (Blank).

[0226] The selected mutants were sorted according to their dissociation rate constant (dissociation rate, kd). Thirty-six clones with enhanced affinity for GPC3 were selected for sequencing and ranked by affinity to obtain the "beneficial mutants".

[0227] Experiment Example 3: Construction and Screening of Combinatorial Libraries

[0228] Based on the identified "beneficial mutants," a total of 13 residues led to a significant increase in affinity for GPC3 (Table 2). Combinatorial libraries with random mutant combinations of 13 residues were constructed by PCR. The library design is shown in Table 2. The first amino acid of either the heavy chain variable region or the light chain variable region was named 1, and then the positions of the beneficial mutations were numbered.

[0229] Table 2. Beneficial mutations screened from PML libraries

[0230] 552 clones were randomly selected from the combinatorial library and screened for initial affinity with human GPC3 antigen protein by ELISA. Then, clones with the highest initial affinity were selected, and their affinity with human GPC3 antigen protein was determined by SPR (Table 3). Combination antibodies of the "beneficial mutants" with the highest affinity enhancement were selected (Table 4). M311, M312, M317, M319, and M329 were selected for protein expression and purification.

[0231] Table 3. Affinity assay results of FASEBA supernatant from affinity-enhanced clones with human GPC3 antigen protein.

[0232] Table 4. Beneficial mutation combinations screened from combinatorial libraries

[0233] Experiment 4: Construction, eukaryotic expression, and purification of BiTE

[0234] The variable regions encoding the heavy and light chains of the affinity maturation antibody were synthesized and inserted into the pCDNA3.4 vector to construct a full-length BiTE expression vector. The BiTE plasmid was transfected into CHO-S cells for expression, encoding a recombinant protein with the format "guide sequence (SEQ ID NO:37)-GPC3 / CD3 BiTE-HHHHHH". The recombinant antibody secreted into the culture medium was purified using His-labeled protein affinity chromatography. The SDS-PAGE results are shown in Figure 1.

[0235] The affinity of purified BiTE antibody for human GPC3 antigen protein was determined using a Biacore 8K sensor. The antigen was immobilized on a sensor chip, and the antibody was used as the analyte. The dissociation (kd) and binding (ka) rate constants were obtained using Biacore evaluation software. The equilibrium dissociation constant (KD) was calculated based on the ratio of kd to ka. Specific parameters are shown in Table 5, and the measurement results are shown in Table 6.

[0236] Table 5. Affinity Detection Parameters

[0237] Table 6. Results of Affinity Measurement

[0238] Experiment 5: Construction, eukaryotic expression, and purification of anti-GPC3 scfv-Fc

[0239] Anti-GPC3 scfv DNA sequences of WT, M311, M312, M317, M319, and M329 were synthesized and inserted into a commercially available PTT5 vector to construct an scfv-hIgG1 Fc expression plasmid encoding a recombinant protein in the form of "guide sequence (SEQ ID NO:38)-anti-GPC3 scfv-huIgG1". The plasmid was transfected into CHO-S cells for expression. The recombinant antibody secreted into the culture medium was purified using Protein A beads to obtain the scFv fused with hIgG1 Fc (SEQ ID NO:45).

[0240] Experimental Example 6: Binding assay of anti-GPC3 scfv-hIgG1 Fc

[0241] Binding assays were performed using the HepG2 cell line. HepG2 cells were cultured until approximately 80% confluence in T75 flasks. They were first washed with PBS (Gibco), then digested with 3 mL of trypsin-EDTA (0.25%) (Gibco) at 37°C for 3 min. Digestion was stopped with DMEM medium (Gibco) containing 10% FBS. The cells were centrifuged at 400g for 5 min, the supernatant was discarded, and the cells were washed once more with PBS. Cells were counted and diluted with PBS to a concentration of 2.5e5 cells / 25 μL. 25 μL of single-cell suspension was added to seven tubes, along with 2 μg of anti-GPC3 scfv-Fc protein (WT, M311, M312, M317, M319, M329) and an equal volume of PBS (as the NC control group). The tubes were incubated at room temperature for 30 min, washed twice with PBS, centrifuged at 400g for 5 min, and the supernatant was discarded. Next, add the secondary antibody Rabbit anti-Human IgG Fc Secondary Antibody (FITC, Invitrogen) diluted 1:50 with cell staining buffer (Biolegend) as the solvent to all tubes. Incubate at room temperature in the dark for 30 min, wash twice with cell staining buffer, centrifuge at 400g for 5 min, and discard the supernatant. Finally, add 200 μL of cell staining buffer for instrumentation.

[0242] The experimental results showed that M311, M312, M317, M319, and M329 bound to HepG2 cells more strongly than WT (Table 7 and Figure 2).

[0243] Table 7. Statistics on average fluorescence intensity and positive rate

[0244] Sequence information:

[0245] SEQ ID NO:1 WT GPC3 antibody heavy chain variable region

[0246] SEQ ID NO:2 Light chain variable region of WT GPC3 antibody

[0247] SEQ ID NO:3 VH CDR1

[0248] SEQ ID NO:4 VH CDR2:

[0249] SEQ ID NO:5 VH CDR3:

[0250] SEQ ID NO:6 VL CDR1:

[0251] SEQ ID NO:7 VL CDR2:

[0252] SEQ ID NO:8 VL CDR3:

[0253] SEQ ID NO:9 WT-ScFv(GPC3):

[0254] SEQ ID NO:10 M311-VH(GPC3)

[0255] SEQ ID NO:11 M311-VL(GPC3)

[0256] SEQ ID NO:12 M311-ScFv(GPC3)

[0257] SEQ ID NO:13 M312-VH(GPC3)

[0258] SEQ ID NO:14 M312-VL(GPC3)

[0259] SEQ ID NO:15 M312-ScFv(GPC3)

[0260] SEQ ID NO:16 M317-VH(GPC3)

[0261] SEQ ID NO:17 M317-VL(GPC3)

[0262] SEQ ID NO:18 M317-ScFv(GPC3)

[0263] SEQ ID NO:19 M319-VH(GPC3)

[0264] SEQ ID NO:20 M319-VL(GPC3)

[0265] SEQ ID NO:21 M319-ScFv(GPC3)

[0266] SEQ ID NO:22 M329-VH(GPC3)

[0267] SEQ ID NO:23 M329-VL(GPC3)

[0268] SEQ ID NO:24 M329-ScFv(GPC3)

[0269] SEQ ID NO:25 linker

[0270] SEQ ID NO:26 Linker

[0271] SEQ ID NO:27 CD3 antibody VH

[0272] SEQ ID NO:28 CD3 antibody VL

[0273] SEQ ID NO:29 ScFv(CD3)

[0274] SEQ ID NO:30 WT BiTE-ScFv(GPC3)-ScFv(CD3):

[0275] SEQ ID NO:31 Amino acid sequence of human GPC3 protein

[0276] SEQ ID NO:32 BiTE-M311 ScFv(GPC3)-ScFv(CD3):

[0277] SEQ ID NO:33 BiTE-M312 ScFv(GPC3)-ScFv(CD3):

[0278] SEQ ID NO:34 BiTE-M317 ScFv(GPC3)-ScFv(CD3):

[0279] SEQ ID NO:35 BiTE-M319 ScFv(GPC3)-ScFv(CD3):

[0280] SEQ ID NO:36 BiTE-M329 ScFv(GPC3)-ScFv(CD3):

[0281] SEQ ID NO:37 Leader sequence

[0282] SEQ ID NO:38 Leader sequence

[0283] SEQ ID NO:45 hIgG1 Fc amino acid sequence

Claims

1. An antibody (such as a monoclonal antibody) targeting GPC3 or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes: VH CDR1, which contains the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence in which D is replaced by A at position 1 relative to SEQ ID NO:

3. VH CDR2, comprising the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence in which G at position 7 is replaced by N, D at position 8 is replaced by K, and / or Q at position 13 is replaced by N, and VH CDR3, which contains the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having one, two, or three amino acid residues substituted, deleted, or added relative to SEQ ID NO:5; and The light chain variable region includes: VL CDR1, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence relative to SEQ ID NO:6 in which G at position 11 is replaced by K, N at position 12 is replaced by D, and / or L at position 15 is replaced by V. VL CDR2, comprising the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having one, two, or three amino acid residues substituted, deleted, or added relative to SEQ ID NO:7, and VL CDR3, which contains the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence in which the Y at position 5 of SEQ ID NO:8 is replaced by M, K or G. The heavy chain variable region does not contain the amino acid sequence shown in SEQ ID NO:1 and / or the light chain variable region does not contain the amino acid sequence shown in SEQ ID NO:

2.

2. The antibody against GPC3 of claim 1, or its antigen-binding fragment, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and VH CDR1 has an amino acid sequence selected from the group consisting of: SEQ ID NO:39; VH CDR2 has an amino acid sequence selected from the following groups: SEQ ID NO:4 and SEQ ID NO:40; VH CDR3 has an amino acid sequence selected from the group consisting of: SEQ ID NO:5; VL CDR1 has an amino acid sequence selected from the group consisting of: SEQ ID NO:6, SEQ ID NO:42 and SEQ ID NO:44; VL CDR2 has an amino acid sequence selected from the group consisting of: SEQ ID NO:7; VL CDR3 has an amino acid sequence selected from the group consisting of: SEQ ID NO:8, SEQ ID NO:41 and SEQ ID NO:

43.

3. The antibody against GPC3 of claim 1 or 2, or the antigen-binding fragment thereof, wherein... i) The heavy chain variable region has VH CDR1 shown in SEQ ID NO:39, VH CDR2 shown in SEQ ID NO:40, and VH CDR3 shown in SEQ ID NO:5; and / or the light chain variable region has VL CDR1 shown in SEQ ID NO:6, VL CDR2 shown in SEQ ID NO:7, and VL CDR3 shown in SEQ ID NO:41; ii) The heavy chain variable region has VH CDR1 shown in SEQ ID NO:39, VH CDR2 shown in SEQ ID NO:40, and VH CDR3 shown in SEQ ID NO:5; and / or the light chain variable region has VL CDR1 shown in SEQ ID NO:42, VL CDR2 shown in SEQ ID NO:7, and VL CDR3 shown in SEQ ID NO:8; iii) The heavy chain variable region has VH CDR1 shown in SEQ ID NO:39, VH CDR2 shown in SEQ ID NO:40, and VH CDR3 shown in SEQ ID NO:5; and / or the light chain variable region has VL CDR1 shown in SEQ ID NO:42, VL CDR2 shown in SEQ ID NO:7, and VL CDR3 shown in SEQ ID NO:43; iv) The heavy chain variable region has VH CDR1 shown in SEQ ID NO:39, VH CDR2 shown in SEQ ID NO:40, and VH CDR3 shown in SEQ ID NO:5; and / or the light chain variable region has VL CDR1 shown in SEQ ID NO:44, VL CDR2 shown in SEQ ID NO:7, and VL CDR3 shown in SEQ ID NO:8; or v) The heavy chain variable region has VH CDR1 shown in SEQ ID NO:39, VH CDR2 shown in SEQ ID NO:4, and VH CDR3 shown in SEQ ID NO:5; and / or the light chain variable region has VL CDR1 shown in SEQ ID NO:42, VL CDR2 shown in SEQ ID NO:7, and VL CDR3 shown in SEQ ID NO:

41.

4. An antibody against GPC3 or an antigen-binding fragment thereof according to any one of claims 1-3, wherein the heavy chain variable region at position 28 of SEQ ID NO:1 is replaced by Q, V or M.

5. An antibody against GPC3 or an antigen-binding fragment thereof according to any one of claims 1-4, wherein the heavy chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:

1.

6. The antibody against GPC3 of claim 4 or its antigen-binding fragment, wherein the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO: 10, 13, 16, 19 and 22.

7. An antibody against GPC3 or an antigen-binding fragment thereof according to any one of claims 1-6, wherein the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:

2.

8. The antibody against GPC3 of claim 7 or an antigen-binding fragment thereof, wherein the light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 11, 14, 17, 20 and 23.

9. An antibody against GPC3 according to any one of claims 1-8, or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein... i) The heavy chain variable region contains the amino acid sequence of SEQ ID NO:10, and the light chain variable region contains the amino acid sequence of SEQ ID NO:11; ii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO:13, and the light chain variable region contains the amino acid sequence of SEQ ID NO:14; iii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO:16, and the light chain variable region contains the amino acid sequence of SEQ ID NO:17; iv) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:19, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:20; or v) The heavy chain variable region contains the amino acid sequence of SEQ ID NO:22, and the light chain variable region contains the amino acid sequence of SEQ ID NO:

23.

10. An antibody against GPC3 or an antigen-binding fragment thereof according to any one of claims 1-9, wherein the antibody against GPC3 or the antigen-binding fragment thereof is an scFv against GPC3, wherein the heavy chain variable region and the light chain variable region are connected by a linker, such as the peptide linker shown in SEQ ID NO:

25.

11. The antibody against GPC3 of claim 10 or an antigen-binding fragment thereof, wherein the scFv comprises an amino acid sequence selected from SEQ ID NO: 12, 15, 18, 21 and 24.

12. An antibody against GPC3 or an antigen-binding fragment thereof according to any one of claims 1-11, further comprising an immunoglobulin Fc region, preferably, said Fc region being the Fc region of human IgG1, for example comprising the amino acid sequence of SEQ ID NO:

45.

13. The antibody against GPC3 or the antigen-binding fragment thereof of any one of claims 1-12, wherein the antibody is selected from monoclonal antibodies, multispecific antibodies, human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), and single-chain Fab (scFab).

14. An antibody against GPC3 or an antigen-binding fragment thereof according to any one of claims 1-13, wherein the KD value of the antibody against GPC3 or the antigen-binding fragment thereof is less than 1 × 10⁻⁶. -7 M, preferably less than 1×10 -8 M, more preferably less than 1×10 -9 M, more preferably less than 1×10 -10 M, especially preferred to be less than 1×10 -11 M.

15. A multispecific antibody comprising at least one antibody against GPC3 according to any one of claims 1-14 or an antigen-binding fragment thereof.

16. The multispecific antibody of claim 15, which is a bispecific antibody, comprising at least one antibody against GPC3 of any one of claims 1-14 or an antigen-binding fragment thereof, and at least one antibody against another antigen or an antigen-binding fragment thereof, for example, at least one antibody against CD3 or an antigen-binding fragment thereof.

17. The multispecific antibody of claim 16, wherein the antibody against CD3 comprises the heavy chain variable region shown in SEQ ID NO:27 and the light chain variable region shown in SEQ ID NO:

28.

18. The multispecific antibody of any one of claims 15-17, wherein the antibody or its antigen-binding fragment is scFv.

19. The multispecific antibody of claim 18, wherein the multispecific antibody comprises the scFv for GPC3 of claim 10 or 11.

20. The multispecific antibody of claim 19, wherein the multispecific antibody comprises a CD3-targeting scFv, for example, the CD3-targeting scFv comprising the amino acid sequence shown in SEQ ID NO:

29.

21. The multispecific antibody of claim 20, wherein the scFv targeting GPC3 and the scFv targeting CD3 are linked by a linker, for example, the peptide linker shown in SEQ ID NO:

26.

22. The multispecific antibody of claim 21, wherein the multispecific antibody comprises an amino acid sequence selected from SEQ ID NO:32-36.

23. A nucleic acid molecule encoding an antibody against GPC3 of any one of claims 1-14 or an antigen-binding fragment thereof, or a multispecific antibody of any one of claims 15-22.

24. An expression vector comprising the nucleic acid molecule of claim 23.

25. A host cell comprising the expression vector of claim 23 or the genome in which the nucleic acid molecule of claim 24 is integrated.

26. An antibody conjugate comprising an antibody against GPC3 of any one of claims 1-14 or an antigen-binding fragment thereof, and a functional molecule conjugated to said antibody or antigen-binding fragment thereof.

27. The antibody conjugate of claim 26, wherein the functional molecule is selected from molecules that target tumor surface markers such as antibodies, molecules that inhibit tumors such as cytotoxins or small molecule drugs, molecules that target immune cell surface markers such as antibodies, detectable markers, and radioisotopes.

28. The antibody conjugate of claim 26 or 27, wherein the functional molecule is conjugated (fused) to the antibody against GPC3 or its antigen-binding fragment directly or via a peptide linker; or the functional molecule is conjugated to the antibody against GPC3 or its antigen-binding fragment via a chemical linker.

29. Use of the antibody conjugate of any one of claims 26-28 for the preparation of antitumor drugs, for the preparation of reagents for detecting cells or tissues expressing GPC3, or for the preparation of chimeric antigen receptor-modified immune cells.

30. A pharmaceutical composition comprising an antibody against GPC3 of any one of claims 1-14 or an antigen-binding fragment thereof, a multispecific antibody of any one of claims 15-22, a nucleic acid molecule of claim 23, an expression vector of claim 24, an antibody conjugate of any one of claims 26-28, and a pharmaceutically acceptable carrier.

31. Use of an antibody against GPC3 of any one of claims 1-14 or an antigen-binding fragment thereof, a multispecific antibody of any one of claims 15-22, a nucleic acid molecule of claim 23, an expression vector of claim 24, or an antibody conjugate of any one of claims 26-28 in the preparation of a medicament for use in the treatment and / or prevention of GPC3-related diseases such as malignant tumors in subjects.

32. A method for treating and / or preventing GPC3-related diseases, such as malignant tumors, in a subject, the method comprising administering to the subject an effective amount of an antibody against GPC3 of any one of claims 1-14 or an antigen-binding fragment thereof, a multispecific antibody of any one of claims 15-22, a nucleic acid molecule of claim 23, an expression vector of claim 24, an antibody conjugate of any one of claims 26-28, or a pharmaceutical composition of claim 30.

33. The use of claim 31 or the method of claim 32, wherein the GPC3-related disease is a tumor that highly expresses GPC3, such as liver cancer like hepatocellular carcinoma (HCC), lung cancer like squamous cell carcinoma of the lung (SqCC), gastric cancer, ovarian cancer like clear cell ovarian cancer, melanoma, or pediatric embryonal tumor.

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