Anti-Glypican 3 Antibody

Abstract

Anti-Glypican 3 Antibodies. The present disclosure provides anti-GPC3 antibodies, including murine antibodies, humanized antibodies, and antibodies with further optimized CDR sequences. The newly disclosed antibodies exhibit higher cell binding efficiency than the leading anti-GPC3 candidate antibodies in clinical development.

Description

[0001] This application is a divisional application of 202280083725.4. The original application is a PCT application entering the Chinese national phase, with an application date of October 17, 2022, an application number of 202280083725.4, and the name of the invention is: Anti-Glypican 3 Antibody. The original application claims the following priority: PCT / CN2021 / 124182, application date: 2021-10-15. Background Art

[0002] Glypican 3 (GPC3) is a member of the Glypican-related integral membrane heparan sulfate proteoglycan (GRIPS) family present on the cell surface. The protein core of GPC3 consists of two subunits, of which the N-terminal subunit is approximately 40 kDa in size and the C-terminal subunit is approximately 30 kDa in size. Six GPCs (GPC1-6) have been identified in mammals. GPC3 plays an important role in regulating cell proliferation, differentiation, adhesion, and migration. GPC3 interacts with Wnt and Frizzled receptors (FZDs) to form a complex and trigger downstream signaling. The core protein of GPC3 can act as a co-receptor or receptor for Wnt.

[0003] Glypican 3 immunostaining can be used to differentiate hepatocellular carcinoma (HCC) from dysplastic development in cirrhotic liver. GPC3 protein is expressed in HCC but not in normal liver and cholangiocarcinoma. GPC3 is also expressed to a lesser extent in melanoma, ovarian clear cell carcinoma, yolk sac tumor, neuroblastoma, hepatoblastoma, Wilms tumor cells, and other tumors.

[0004] GPC3 is a promising therapeutic target for the treatment of cancers such as liver cancer. A variety of therapeutic anti-GPC3 antibodies have been developed, including GC33 and YP7. Some of these antibodies inhibit Wnt signaling in liver cancer cells. In addition, chimeric antigen receptor (CAR) T cell immunotherapy for the treatment of cancer is in different stages of development. In mice with xenografts or primary liver tumors, CAR-T cells can eliminate GPC3-positive cancer cells by inducing perforin- and granzyme-mediated cell death and reducing Wnt signaling in tumor cells. Summary of the invention

[0005] In various embodiments of the present disclosure, antibodies and antigen-binding fragments specific for human GPC3 protein are provided. Experimental testing has shown that these newly identified antibodies outperform the benchmark GC33 antibody.

[0006] Therefore, according to one embodiment of the present disclosure, an anti-GPC3 antibody or a fragment thereof is provided, which is specific to human GPC3 protein and comprises a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 71-76.

[0007] In some embodiments, the VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 25, 59 and 63; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 19, 23, 26, 29, 30, 60 and 65; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 20, 24 and 33; the VL CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 21, 27 and 31; the VL CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 61, 64 and 66; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 22, 28, 32, 34 and 62.

[0008] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the following amino acid sequence:

[0009] SEQ ID NO: 13, 19, 15, 21, 17 and 18;

[0010] SEQ ID NOs: 13, 19, 20, 21, 17 and 22;

[0011] SEQ ID NO: 13-18;

[0012] SEQ ID NO: 13, 23, 24, 21, 17 and 18;

[0013] SEQ ID NOs: 25, 26, 20, 27, 17 and 28;

[0014] SEQ ID NOs: 25, 29, 20, 27, 17 and 28;

[0015] SEQ ID NOs: 25, 30, 15, 31, 17 and 32;

[0016] SEQ ID NO: 59, 60, 15, 21, 61 and 62;

[0017] SEQ ID NO: 63, 19, 15, 21, 64 and 62; or

[0018] SEQ ID NO: 13, 65, 15, 21, 66 and 62.

[0019] In some embodiments, the VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 59 and 63; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 60 and 65; the VH CDR3 comprises the amino acid sequence shown in SEQ ID NO: 15; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO: 21; the VL CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 61, 64 and 66; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 62.

[0020] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 13, 19, 15, 21, 17 and 18. In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-39; and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-44.

[0021] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 36, and the VL comprises the amino acid sequence shown in SEQ ID NO: 41. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 37, and the VL comprises the amino acid sequence shown in SEQ ID NO: 43.

[0022] In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:36, and the VL comprises the amino acid sequence as shown in SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:36, and the VL comprises the amino acid sequence as shown in SEQ ID NO:43. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:36, and the VL comprises the amino acid sequence as shown in SEQ ID NO:44. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:37, and the VL comprises the amino acid sequence as shown in SEQ ID NO:41. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:37, and the VL comprises the amino acid sequence as shown in SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:37, and the VL comprises the amino acid sequence as shown in SEQ ID NO:44. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:38, and the VL comprises the amino acid sequence as shown in SEQ ID NO:41. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:38, and the VL comprises the amino acid sequence as shown in SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:38, and the VL comprises the amino acid sequence as shown in SEQ ID NO:43. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:38, and the VL comprises the amino acid sequence as shown in SEQ ID NO:44. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:39, and the VL comprises the amino acid sequence as shown in SEQ ID NO:41. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:39, and the VL comprises the amino acid sequence as shown in SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:39, and the VL comprises the amino acid sequence as shown in SEQ ID NO:43. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:39, and the VL comprises the amino acid sequence as shown in SEQ ID NO:44.

[0023] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 59, 60, 15, 21, 61 and 62, respectively. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 53, and the VL comprises the amino acid sequence shown in SEQ ID NO: 54.

[0024] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 63, 19, 15, 21, 64 and 62, respectively. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 55, and the VL comprises the amino acid sequence shown in SEQ ID NO: 56.

[0025] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 13, 65, 15, 21, 66 and 62, respectively. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 57, and the VL comprises the amino acid sequence shown in SEQ ID NO: 58.

[0026] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 13, 19, 20, 21, 17 and 22, respectively. In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 45-50; and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 51 and 52. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 48, and the VL comprises the amino acid sequence shown in SEQ ID NO: 51. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 48, and the VL comprises the amino acid sequence shown in SEQ ID NO: 52.

[0027] In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:45, and the VL comprises the amino acid sequence as shown in SEQ ID NO:51. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:45, and the VL comprises the amino acid sequence as shown in SEQ ID NO:52. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:46, and the VL comprises the amino acid sequence as shown in SEQ ID NO:51. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:46, and the VL comprises the amino acid sequence as shown in SEQ ID NO:52. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:47, and the VL comprises the amino acid sequence as shown in SEQ ID NO:51. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:47, and the VL comprises the amino acid sequence as shown in SEQ ID NO:52. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:49, and the VL comprises the amino acid sequence as shown in SEQ ID NO:51. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 49, and the VL comprises the amino acid sequence shown in SEQ ID NO: 52. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 50, and the VL comprises the amino acid sequence shown in SEQ ID NO: 51. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 50, and the VL comprises the amino acid sequence shown in SEQ ID NO: 52.

[0028] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 13-18. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 1, and the VL comprises the amino acid sequence shown in SEQ ID NO: 2.

[0029] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 13, 23, 24, 21, 17 and 18. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 5, and the VL comprises the amino acid sequence shown in SEQ ID NO: 6.

[0030] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 25, 26, 20, 27, 17 and 28, respectively. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises the amino acid sequence shown in SEQ ID NO: 8.

[0031] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 25, 29, 20, 27, 17 and 28, respectively. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 9, and the VL comprises the amino acid sequence shown in SEQ ID NO: 10.

[0032] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 25, 30, 15, 31, 17 and 32. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 11, and the VL comprises the amino acid sequence shown in SEQ ID NO: 12.

[0033] In some embodiments, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:13; the VHCDR2 comprises the amino acid sequence shown in SEQ ID NO:19; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:15, 20, 24 and 33; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:21; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:17; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:18, 22, 28, 32 and 34.

[0034] In some embodiments, the antibodies or fragments thereof are humanized.

[0035] In one embodiment, an antibody-drug conjugate is also provided, characterized in that the antibody-drug conjugate comprises: (a) an antibody or a fragment thereof disclosed herein; and (b) a coupling portion coupled to the antibody or fragment, wherein the coupling portion is selected from: a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0036] In one embodiment, a multispecific antibody is also provided, which comprises the antibody of the present disclosure and one or more second antibodies or antigen-binding fragments having binding specificity to a target antigen other than GPC3.

[0037] Also provided is a chimeric antigen receptor (CAR) comprising the antibody or fragment thereof of the present disclosure, a transmembrane domain, a co-stimulatory domain and a CD3 intracellular domain.

[0038] In another embodiment, polynucleotides encoding the antibodies or fragments thereof of the present disclosure, cells comprising the polynucleotides, and compositions comprising the antibodies or fragments thereof and a pharmaceutically acceptable carrier are provided. Methods and uses of using the antibodies or fragments thereof to treat cancer are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Shown is the binding affinity of the 52H5D3B8 antibody to the GPC3 protein (human GPC3-his tag) measured using Biacore.

[0040] Figure 2A-2B It was shown that the 52H5D3B8 antibody bound to GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells, and Huh-7 cells with higher affinity than GC33.

[0041] Figure 3ELISA binding activity of 52H5D3B8 to human, mouse and cynomolgus monkey GPC3 is shown.

[0042] Figure 4 It was shown that the affinity of 52H5D3B8-6# chimeric monoclonal antibody (mAb) binding to GPC3-CHOK1 cells was higher than that of other optimized 52H5D3B8 chimeric monoclonal antibodies.

[0043] Figure 5 The results showed that the affinity of 52H5D3B8-6# chimeric monoclonal antibody binding to GPC3-CHOK1 cells, Hep3B cells and Huh-7 cells was the highest among 52H5D3B8 chimeric monoclonal antibody and GC33 antibody.

[0044] Figure 6 The binding affinity of 52H5D3B8-6# antibody to GPC3 protein (human GPC3-his tag) measured using Biacore 8K for chimeric antibodies is shown.

[0045] Figure 7 The EC of each humanized antibody against GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells, and Huh-7 cells is shown. 50 .

[0046] Figure 8 The EC of each humanized antibody against GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells, and Huh-7 cells is shown. 50 .

[0047] Fig. 9 The binding of 52H5D3B8-6#-VH+VL, 52H5D3B8-6#-VH1+VL1, 52H5D3B8-6#-VH1+VL3, 52H5D3B8-6#-VH1+VL4, 52H5D3B8-6#-VH2+VL4, and 52H5D3B8-6#-VH3+VL1 to recombinant GPC3 protein (human GPC3-his tag) tested using Biacore with capture method is shown.

[0048] Fig.10 Shown are the results of antibody binding to CHOK1 cells and HEPG2 cells overexpressing human GPC3.

[0049] Fig.11 Shown are the binding results of affinity matured antibodies to CHOK1 cells overexpressing human GPC3.

[0050] Fig.12 The results showing that the antibodies induce GPC3 antibody-mediated ADCC signaling.

[0051] Fig.13 It was shown that the antibody activated the function of NK cells.

[0052] Fig.14 Shows the degranulation of NK cells upon antibody stimulation.

[0053] Fig.15 Cytokine production in stimulated NK cells is shown.

[0054] Fig.16 It was shown that the binding efficiency of all candidate humanized antibodies was comparable to that of the chimeric antibody on GPC3-CHOK1 cells and superior to that of the chimeric antibody on Huh-7 cells.

[0055] Fig.17 The results of antibody binding to CHOK1 cells, Huh-7 cells, and HEPG2 cells overexpressing human GPC3 are shown.

[0056] Fig.18 Results of antibody internalization in HEPG2 cells are shown.

[0057] Detailed description

[0058] definition

[0059] It should be noted that the term "a" or "an" entity refers to one or more of the entity; for example, "an antibody" is understood to represent one or more antibodies. Therefore, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0060] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. Therefore, the term "antibody" includes any protein- or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule that has biological activity of binding to an antigen. Such examples include, but are not limited to, a complementarity determining region (CDR) of a heavy or light chain or its ligand-binding portion, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, or at least one portion of a binding protein.

[0061] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, such as F(ab') 2 、F(ab) 2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, antibody fragments can bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror images and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0062] The term "antibody" encompasses various major classes of polypeptides that can be distinguished by biochemical methods. It will be appreciated by those skilled in the art that heavy chains can be classified as γ, μ, α, δ, or ε, with some subclasses (e.g., γ1-γ4). The nature of the chain determines the "class" of the antibody, which is IgG, IgM, IgA, IgG, or IgE, respectively.

[0063] Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and known to have functional specificity. In view of the present disclosure, modified versions of each of these classes and isotypes are easily discernible to those skilled in the art and are therefore within the scope of the present invention. All immunoglobulin classes are obviously within the scope of the present disclosure, and the following discussion is generally directed to IgG class immunoglobulin molecules. For IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000 Daltons. These four chains are typically connected by disulfide bonds into a "Y" shaped structure, in which the light chain starts from the mouth of the "Y" shaped structure and extends all the way to the variable region, surrounding the heavy chain.

[0064] The antibodies, antigen-binding polypeptides, variants or derivatives thereof of the present disclosure include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies or chimeric antibodies, single-chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab') 2 , Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising VK or VH domains, Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to antibodies disclosed herein). The immunoglobulin or antibody molecules of the present disclosure can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0065] As used herein, the term "chimeric antibody" refers to any antibody in which the immunologically active region or site is obtained or derived from a first species, while the constant region (which may be complete, partial or modified, depending on the disclosure) is obtained from a second species. In certain embodiments, the target binding region or site will be from a non-human source (such as a mouse or primate), while the constant region is human.

[0066] The antibodies disclosed herein can be from any animal origin, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies. In some embodiments, the variable region can be from a condricthoid (e.g., from a shark).

[0067] As used herein, the term "recombinant" in reference to a polypeptide or polynucleotide means a form of the polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which can be produced by combining polynucleotides that do not normally occur together.

[0068] Hybridoma technology can be performed under conditions of varying "stringency". Generally, low stringency hybridization reactions are performed at a temperature of about 40°C in about 10×SSC or a solution of equivalent ionic strength / temperature. Moderate stringency hybridizations are typically performed in about 6×SSC at about 50°C, and high stringency hybridization reactions are typically performed in about 1×SSC at about 60°C. Hybridization reactions can also be performed under "physiological conditions" well known to those skilled in the art. Non-limiting examples of physiological conditions are the temperature, ionic strength, pH, and Mg that are normally present in cells. 2+ concentration.

[0069] GPC-3 Antibody

[0070] As demonstrated in the accompanying experimental examples, the inventors were able to generate potent murine anti-GPC3 antibodies 18C1D9, 52H5D3B8, 163E7D12, 172B4E9, 152E9F7 and 171F9C5 (Table 1). For example, as shown in Example 3, the 52H5D3B8 antibody exhibited a higher cell binding efficiency than the leading anti-GPC3 antibody GC33 in clinical development. In addition, experimental data showed that these new antibodies were more active in inducing cellular internalization. This property makes these new antibodies particularly suitable for use in the field of antibody-drug conjugates (ADCs).

[0071] In addition, the sequence of the 52H5D3B8 antibody was further optimized by amino acid substitutions in the VH and VL CDR3s. For example, one of such optimized antibodies, 52H5D3B8-6#, bound to GPC3-CHOK1 cells, Hep3B cells, and Huh-7 cells with higher affinity than the parental 52H5D3B8 chimeric mAb and the reference GC33 antibody.

[0072] A humanized version of the 52H5D3B8-6# antibody was generated and compared to GC33 and another benchmark antibody, Y035 (as disclosed in US patent application US20190046659). Fig.10 As shown in Table 14, humanized antibodies 52H5D3B8-6#-VH1+VL1 and 52H5D3B8-6#-VH2+VL3 retained the high affinity of the parental antibodies and were significantly superior to GC33 and Y035.

[0073] The humanized antibodies were further affinity matured to further improve the binding efficiency. Fig.11 As shown in Table 16, it is encouraging that all affinity-matured antibodies showed enhanced binding efficiency compared to the parental antibody (52H5D3B8-6#-VH2+VL3) and the benchmark antibody GC33. It is also surprising that when targeting cells with moderate GPC3 expression levels, the humanized antibodies showed significantly higher ADCC signaling efficacy than GC33 ( Fig.12 and Table 17).

[0074] These humanized and affinity-matured antibodies were further tested for their ability to activate NK cells in the presence of cells expressing GPC3. Fig.13 As shown in Table 18, all of these new antibodies have higher activity than GC33. Compared with GC33, they induced higher IFN-γ and TNF-α production in NK cells ( Fig.15 ).

[0075] The parent antibody 52H5D3B8 was also humanized, and the binding and internalization activities of two humanized antibodies Hu52H5D3B8-7 and Hu 52H5D3B8-8 were tested with GC33 and Y035 as references. Figure 17-18 As shown in Tables 24-25, these humanized antibodies outperformed the two reference antibodies. Interestingly, when the CDRs of Hu 52H5D3B8-7 / 8 were inserted into the framework of Y035 or the framework regions of Hu52H5D3B8-7 / 8 were used to support the CDRs of Y035, the resulting grafted antibodies were superior to Y035 (Table 24), emphasizing the excellence of the CDR and framework sequences of these new antibodies.

[0076] It was observed that these antibodies and their CDR sequences were highly homologous to each other, indicating that the CDR sequences were interchangeable (see Table A).

[0077] Table A. CDR sequences

[0078]

[0079]

[0080] Therefore, in one embodiment, the present disclosure provides an anti-glypican 3 antibody comprising a VH (heavy chain variable region) and a VL (light chain variable region). The VH and VL regions include VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, such as those shown in Tables A, 1A-1F, 5A-50, 8A and 8B.

[0081] In some embodiments, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:71; the VHCDR2 comprises the amino acid sequence shown in SEQ ID NO:72; the VH CDR3 comprises the amino acid sequence shown in SEQ ID NO:73; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:74; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:75; and the VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:76.

[0082] SEQ ID NO:71 is X 1 YE X 2 H, where X 1 Can be D, R or G, X 2 It can be I or M. SEQ ID NO: 72 is AI X 1 P X 2 X 3 X 4 X 5 TAY X 6 X 7 X 8 FKG, where X 1 Can be D, G or H, X 2 Can be A, E, X 3 Can be S or T, X 4 Can be D or G, X 5 Can be D, G, N or S, X 6 Can be N, S or T, X 7 Can be Q or S, and X 8 It can be R, SEQ ID NO: 73 is X 1 YS X 2 AY, where X 1 Can be F or Y, and X 2 It can be F or Y. SEQ ID NO: 74 is RS X 1 QS X 2 VH X 3 NG X 4 TYL X 5 , where X 1 Can be R or S, X 2Can be L or P, X 3 Can be S, R or T, X 4 Can be H or N, and X 5 It can be H or Q. SEQ ID NO: 75 is KVSNRX 1 X 2 , where X 1 Can be F or Y, and X 2 It can be S, A or P. SEQ ID NO: 76 is X 1 X 2 X 3 X 4 HVPYT, where X 1 Can be F, S or V, X 2 Can be Q or E, X 3 Can be S or T, and X 4 Can be I or T.

[0083] In some embodiments, the VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 25; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 19, 23, 26, 29 and 30; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 20, 24 and 33; the VL CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 21, 27 and 31; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO: 17; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 22, 28, 32 and 34.

[0084] In some embodiments, the VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 25, 59 and 63; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 19, 23, 26, 29, 30, 60 and 65; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 20, 24 and 33; the VL CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 21, 27 and 31; the VL CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 61, 64 and 66; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 22, 28, 32, 34 and 62.

[0085] In some embodiments, anti-GPC3 antibodies and antigen-binding fragments that compete with any of the antibodies disclosed herein for binding to human GPC3 are also provided. In some embodiments, anti-GPC3 antibodies and antigen-binding fragments that bind to the same epitope as any of the antibodies disclosed herein are also provided. In some embodiments, anti-GPC3 antibodies and antigen-binding fragments are also provided, which comprise CDR1, CDR2, and CDR3 of the VH and VL of the antibodies disclosed herein.

[0086] In some embodiments, antibodies or antigen-binding fragments comprising the CDRs of antibody 52H5D3B8-6# or a humanized / affinity matured version thereof are provided. In some embodiments, the VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 59, and 63; the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 60, and 65; the VH CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 15; the VLCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 21; the VL CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 61, 64, and 66; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 62. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 13, 19, 15, 21, 17 and 18. In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-39; the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-44.

[0087] Exemplary humanized antibodies or antigen-binding fragments derived from 52H5D3B8-6# include those shown in Table 8A. Examples include a VH of SEQ ID NO:36 and a VL of SEQ ID NO:41, a VH of SEQ ID NO:36 and a VL of SEQ ID NO:42, a VH of SEQ ID NO:36 and a VL of SEQ ID NO:43, a VH of SEQ ID NO:36 and a VL of SEQ ID NO:44, a VH of SEQ ID NO:37 and a VL of SEQ ID NO:41, a VH of SEQ ID NO:37 and a VL of SEQ ID NO:42, a VH of SEQ ID NO:37 and a VL of SEQ ID NO:43, a VH of SEQ ID NO:37 and a VL of SEQ ID NO:44, a VH of SEQ ID NO:38 and a VL of SEQ ID NO:41, a VH of SEQ ID NO:38 and a VL of SEQ ID NO:42, a VH of SEQ ID NO:38 and a VL of SEQ ID NO:43, or a VH of SEQ ID NO:38 and a VL of SEQ ID NO:44.

[0088] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 36, and the VL comprises the amino acid sequence shown in SEQ ID NO: 41. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 37, and the VL comprises the amino acid sequence shown in SEQ ID NO: 43.

[0089] In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:36, and the VL comprises the amino acid sequence as shown in SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:36, and the VL comprises the amino acid sequence as shown in SEQ ID NO:43. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:36, and the VL comprises the amino acid sequence as shown in SEQ ID NO:44. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:37, and the VL comprises the amino acid sequence as shown in SEQ ID NO:41. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:37, and the VL comprises the amino acid sequence as shown in SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:37, and the VL comprises the amino acid sequence as shown in SEQ ID NO:44. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:38, and the VL comprises the amino acid sequence as shown in SEQ ID NO:41. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:38, and the VL comprises the amino acid sequence as shown in SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:38, and the VL comprises the amino acid sequence as shown in SEQ ID NO:43. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:38, and the VL comprises the amino acid sequence as shown in SEQ ID NO:44. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:39, and the VL comprises the amino acid sequence as shown in SEQ ID NO:41. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:39, and the VL comprises the amino acid sequence as shown in SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:39, and the VL comprises the amino acid sequence as shown in SEQ ID NO:43. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:39, and the VL comprises the amino acid sequence as shown in SEQ ID NO:44.

[0090] Affinity matured versions of these antibodies are also provided, such as 52H5D3B8-18#-VH2+VL3, 52H5D3B8-19#-VH2+VL3, and 52H5D3B8-20#-VH2+VL3 (Table 15). In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 59, 60, 15, 21, 61, and 62, respectively. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 53, and the VL comprises the amino acid sequence shown in SEQ ID NO: 54.

[0091] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 63, 19, 15, 21, 64 and 62. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 55, and the VL comprises the amino acid sequence shown in SEQ ID NO: 56.

[0092] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 13, 65, 15, 21, 66 and 62. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 57, and the VL comprises the amino acid sequence shown in SEQ ID NO: 58.

[0093] In some embodiments, antibodies or antigen-binding fragments comprising the CDRs of antibody 52H5D3B8 are provided. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 13, 19, 20, 21, 17, and 22, respectively. In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 45-50; the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 51, and 52.

[0094] Exemplary humanized antibodies or antigen-binding fragments derived from 52H5D3B8 include those shown in Table 19. Examples include a VH of SEQ ID NO:45 and a VL of SEQ ID NO:51, a VH of SEQ ID NO:46 and a VL of SEQ ID NO:51, a VH of SEQ ID NO:47 and a VL of SEQ ID NO:51, a VH of SEQ ID NO:48 and a VL of SEQ ID NO:51, a VH of SEQ ID NO:49 and a VL of SEQ ID NO:51, a VH of SEQ ID NO:50 and a VL of SEQ ID NO:51, a VH of SEQ ID NO:45 and a VL of SEQ ID NO:52, a VH of SEQ ID NO:46 and a VL of SEQ ID NO:52, a VH of SEQ ID NO:47 and a VL of SEQ ID NO:52, a VH of SEQ ID NO:48 and a VL of SEQ ID NO:52, a VH of SEQ ID NO:49 and a VL of SEQ ID NO:52, or a VH of SEQ ID NO:50 and a VL of SEQ ID NO:52.

[0095] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 48, and the VL comprises the amino acid sequence shown in SEQ ID NO: 51. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 48, and the VL comprises the amino acid sequence shown in SEQ ID NO: 52.

[0096] In some embodiments, antibodies or antigen-binding fragments derived from antibody 52H5D3B8 are provided, such as those having alternative VH CDR3 and VL CDR3 as shown in Tables 5A-5O. In some embodiments, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO: 13; the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO: 19; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 20, 24 and 33; the VLCDR1 comprises the amino acid sequence shown in SEQ ID NO: 21; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO: 17; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 22, 28, 32 and 34.

[0097] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:35, and the VL comprises the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:67, and the VL comprises the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:3, and the VL comprises the amino acid sequence shown in SEQ ID NO:40. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:3, and the VL comprises the amino acid sequence shown in SEQ ID NO:68. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:69, and the VL comprises the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:35, and the VL comprises the amino acid sequence shown in SEQ ID NO:68. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:67, and the VL comprises the amino acid sequence shown in SEQ ID NO:40. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:67, and the VL comprises the amino acid sequence as shown in SEQ ID NO:68. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:3, and the VL comprises the amino acid sequence as shown in SEQ ID NO:70. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:67, and the VL comprises the amino acid sequence as shown in SEQ ID NO:70. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:35, and the VL comprises the amino acid sequence as shown in SEQ ID NO:70. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:69, and the VL comprises the amino acid sequence as shown in SEQ ID NO:68. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:69, and the VL comprises the amino acid sequence as shown in SEQ ID NO:40. In some embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:69, and the VL comprises the amino acid sequence as shown in SEQ ID NO:70.

[0098] In some embodiments, an antibody or antigen-binding fragment comprising the CDRs of antibody 18C1D9 is provided. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 13-18, respectively. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 1, and the VL comprises the amino acid sequence shown in SEQ ID NO: 2.

[0099] In some embodiments, an antibody or antigen-binding fragment comprising the CDRs of antibody 163E7D12 is provided. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 13, 23, 24, 21, 17 and 18, respectively. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 5, and the VL comprises the amino acid sequence shown in SEQ ID NO: 6.

[0100] In some embodiments, an antibody or antigen-binding fragment comprising the CDRs of antibody 172B4E9 is provided. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 25, 26, 20, 27, 17 and 28, respectively. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises the amino acid sequence shown in SEQ ID NO: 8.

[0101] In some embodiments, antibodies or antigen-binding fragments comprising the CDRs of antibody 152E9F7 are provided. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 25, 29, 20, 27, 17, and 28, respectively. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 9, and the VL comprises the amino acid sequence shown in SEQ ID NO: 10.

[0102] In some embodiments, an antibody or antigen-binding fragment comprising the CDRs of antibody 171F9C5 is provided. In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 25, 30, 15, 31, 17 and 32, respectively. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 11, and the VL comprises the amino acid sequence shown in SEQ ID NO: 12.

[0103] Antibody-drug conjugates

[0104] Preliminary data suggest that these new antibodies have higher activity in inducing cellular internalization, a property that makes them particularly suitable for use in areas such as antibody-drug conjugates (ADCs).

[0105] In some embodiments, the antibody or fragment may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent or PEG.

[0106] In one embodiment, the antibody or fragment of the present disclosure is covalently linked to a drug moiety. The drug moiety can be or be modified to include a group that reacts with a coupling point on the antibody. For example, the drug molecule can be connected by alkylation (e.g., at the N-terminus of ε-aminolysine or the antibody), reductive amination of oxidized carbohydrates, esterification between hydroxyl and carboxyl groups, amidation of amino or carboxyl groups, and coupling with thiol.

[0107] In some embodiments, the average number of drug moieties conjugated per antibody molecule (p) ranges from 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, the average value of p ranges from 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is an average of 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average value of p ranges from about 1 to about 20, about 1 to about 10, about 2 to about 10, about 2 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2. In some embodiments, p ranges from about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, or about 2 to about 3.

[0108] For example, when chemical activation of the protein results in the formation of free thiol groups, the protein can be coupled with a sulfhydryl reactive agent. In one aspect, the reagent is a reagent that is substantially specific for free thiol groups. Such reagents include, for example, maleimide, haloacetamide (e.g., iodine, bromine or chlorine), halogenated esters (e.g., iodine, bromine or chlorine), halomethyl ketones (e.g., iodine, bromine or chlorine), benzyl halides (e.g., iodide, bromide or chloride), vinyl sulfones and pyridylthio.

[0109] The drug can be connected to the antibody or fragment by a joint. Suitable joints include, for example, cleavable and non-cleavable joints. Cleavable joints are usually easy to cut under intracellular conditions. Suitable cleavable joints include, for example, peptide joints that can be cut by intracellular proteases such as lysosomal proteases or endosomal proteases. In an exemplary embodiment, the joint can be a dipeptide joint, such as valine-citrulline (val-cit), phenylalanine-lysine (phe-lys) joints or maleimidocaproic acid-valine-citrulline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PABA) joints. Another joint is sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate (smcc). Sulfo-smcc coupling occurs through a maleimide group, which reacts with a sulfhydryl group (thiol, -SH), and its Sulfo-NHS ester is reactive to primary amines (such as those found in lysine and protein or peptide N-terminals). The other linker is maleimide Acyl (mc). Other suitable linkers include linkers that are hydrolyzable at a specific pH or pH range, such as hydrazone linkers. The cleaved linker includes a disulfide bond linker. The linker can be covalently bound to the antibody so that the antibody must be degraded in the cell to release the drug, such as the mc linker.

[0110] The linker may include a group for connecting an antibody. For example, the linker may include an amino, hydroxyl, carboxyl or sulfhydryl reactive group (e.g., maleimide, haloacetamide (e.g., iodine, bromine or chloride), halogenated ester (e.g., iodine, bromine or chloride), halomethyl ketone (e.g., iodine, bromine or chloride), benzyl halide (e.g., iodide, bromide or chloride), vinyl sulfone and pyridinethio).

[0111] In some embodiments, the drug moiety is a cytotoxic or cytostatic agent, an immunosuppressant, a radioisotope, a toxin, or the like. The conjugate can be used to inhibit the proliferation of tumor cells or cancer cells, induce apoptosis of tumor or cancer cells, or be used to treat cancer in patients. The conjugate can be used in a variety of settings for treating animal cancer accordingly. The conjugate can be used to deliver drugs to tumor cells or cancer cells. Without being bound by theory, in some embodiments, the conjugate binds or associates with cancer cells expressing GPC3, and the conjugate and / or the drug can be taken up by tumor cells or cancer cells through receptor-mediated endocytosis.

[0112] Once inside the cell, one or more specific peptide sequences in the conjugate (e.g., in the linker) are hydrolyzed and cleaved by one or more tumor cell or cancer cell-associated proteases, resulting in drug release. The released drug can migrate freely within the cell and induce cytotoxicity, cell inhibition or other activities. In some embodiments, the drug is cleaved from the antibody outside the tumor cell or cancer cell, and then the drug penetrates the cell or acts on the cell surface.

[0113] Examples of drug moieties or payloads are selected from DM1 (maytansine, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)- or N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), mc-MMAD (6-maleimido-monomethyllaurin-D or N-methyl-L-valyl-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[ (1S)-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidinyl]-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valinamide), mc-MMAF (maleimido-monomethyl auristatin F or N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-N-methyl-L-valinyl-L-valinyl-(3R,4S,5S)-3-methyl Oxy-5-methyl-4-(methylamino)heptanoyl-(αR,βR,2S)-β-methoxy-α-methyl-2-pyrrolidinepropionyl-L-phenylalanine) and mc-Val-Cit-PABA-MMAE (6-maleimido-ValcCit-(p-aminobenzyloxycarbonyl)-monomethyl auristatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-valyl-N5- [(aminocarbonyl)-L-ornithine]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide). DM1 is a derivative of the microtubule inhibitor maytansine, while MMAD, MMAE and MMAF are derivatives of auristatin. In some embodiments, the drug moiety is selected from the group consisting of mc-MMAF and mc-Val-Cit-PABA-MMAE. In some embodiments, the drug moiety is a maytansine compound or auristatin.

[0114] The antibody or fragment may be coupled or fused to a therapeutic agent which may include a detectable label, such as a radioactive label, an immunomodulatory agent, a hormone, an enzyme, an oligonucleotide, a photosensitive therapeutic or diagnostic agent, a cytotoxic agent which may be a drug or toxin, an ultrasound enhancing agent, a non-radioactive label, combinations thereof, and other such agents known in the art.

[0115] The antibody can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent labeled antigen-binding polypeptide can then be determined by detecting the luminescence produced during the chemical reaction. Examples of particularly effective chemiluminescent labeling compounds are luminol, isoluminol, thermal acridinium esters, imidazoles, acridinium salts, and oxalate esters.

[0116] The antibodies can also be detectably labeled with fluorescent emitting metals (e.g., 152Eu or other lanthanides). These metals can be attached to the antibodies via metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). The techniques for coupling various moieties to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In CancerTherapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin etal. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The PreparationAnd Cytotoxic Properties Of Antibody-Toxin Conjugates",Immunol.Rev.(52:119-58(1982)).

[0117] Multifunctional molecules

[0118] As a molecule preferentially expressed on tumor cells, an antibody or antigen-binding fragment specific for GPC3 can be combined with a second or more fragments specific for immune cells to generate a bispecific antibody or a multispecific antibody.

[0119] In some embodiments, the immune cell is selected from the group consisting of T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells.

[0120] In some embodiments, the second specificity is for CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, CMET, 4-1BB, OX40, SIRPA CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.

[0121] Different forms of bispecific antibodies are also provided. In some embodiments, the anti-GPC3 fragment and the second fragment are each independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment.

[0122] Also provided are bifunctional molecules that include not only antibodies or antigen binding fragments. As tumor antigen targeting molecules, GPC3-specific antibodies or antigen binding fragments, such as those described herein, can be optionally combined with immune cytokines or ligands through peptide linkers. Relevant immune cytokines or ligands include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT, and GITRL. This bifunctional molecule can combine immune checkpoint blocking effects with local immune regulation at the tumor site.

[0123] Chimeric antigen receptors, polynucleotides, and methods for preparing antibodies

[0124] In one embodiment, a chimeric antigen receptor (CAR) is also provided, which includes an antibody or fragment thereof disclosed herein as a targeting unit. In some embodiments, CAR comprises an antibody or fragment thereof disclosed herein, a transmembrane domain, a costimulatory domain, and a CD3 ξ intracellular domain.

[0125] The transmembrane domain can be designed to be fused to an extracellular domain including an antibody or fragment, optionally through a hinge domain. Similarly, it can also be fused to an intracellular domain (e.g., a costimulatory domain). In some embodiments, the transmembrane domain may include a natural transmembrane domain of a costimulatory domain (e.g., a TM region of CD28T or 4-IBB used as a costimulatory domain) or a natural transmembrane domain of a hinge region (e.g., a TM region of CD8α or CD28T used as a hinge domain).

[0126] In some embodiments, a transmembrane domain may include a sequence that spans the cell membrane but extends into the cytoplasm and / or extracellular space of the cell. For example, a transmembrane domain may include a transmembrane sequence, which itself may further include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids that extend into the cytoplasm and / or extracellular space of the cell. Thus, a transmembrane domain includes a transmembrane region and may further include amino acids that extend beyond the inner or outer surface of the membrane itself; such a sequence may still be considered a "transmembrane domain."

[0127] In some embodiments, the transmembrane domain is fused to the cytoplasmic domain via a short linker. Optionally, a short peptide or polypeptide connector preferably between 2 and 10 amino acids in length can form a connection between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. Glycine-serine doublets (GS), glycine-serine-glycine triplet (GSG) or alanine-alanine-alanine triplet (AAA) provide suitable linkers.

[0128] In some embodiments, CAR further comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain is located between the transmembrane domain and the activation domain. Exemplary co-stimulatory domains include, but are not limited to, CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (T FRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79a (B cell antigen receptor complex-associated alpha chain), CD79b (B cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (HECT), CD100 (SEMA4D), CD 103(ITGAE), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD158A(KIR2DL1), CD158B1( KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KTR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD 229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(T FSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (KG2D), CD319 (SLAMF7), CD335 (K-p46), CD336 (K-p44), CD337 (K-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF 18), inducible T cell co-stimulator (ICOS), LFA-1 (CD 1la / CD18), KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activated NK cell receptor, Toll ligand receptor and fragments or combinations thereof.

[0129] In some embodiments, the cytoplasmic portion of CAR also includes a signal transduction / activation domain. In one embodiment, the signal transduction / activation domain is a CD3 ξ domain, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity with a CD3 ξ domain.

[0130] The present disclosure also provides polynucleotides or nucleic acid molecules encoding antibodies, variants or derivatives thereof, or separations of CARs of the present disclosure. The polynucleotides of the present disclosure can encode the entire heavy chain variable region and light chain variable region of antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on different polynucleotide molecules. In addition, the polynucleotides of the present disclosure can encode partial heavy chain variable regions and light chain variable regions of antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on different polynucleotide molecules.

[0131] Methods for preparing antibodies are well known in the art and described herein. In certain embodiments, the variable and constant regions of the antigen-binding polypeptides disclosed herein are fully human. Fully human antibodies can be made using techniques described in the art and methods described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigenic attack, but whose endogenous loci have been disabled. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Patents (6,150,584; 6,458,592; 6,420,140), the entire contents of which are incorporated herein by reference.

[0132] Treatment and uses

[0133] As described herein, the antibodies, variants, derivatives or antibody-drug conjugates of the present disclosure may be used in certain therapeutic and diagnostic methods.

[0134] The present disclosure also relates to antibody-based treatment methods, which involve administering the antibodies, fragments or antibody-drug conjugates of the present disclosure to patients, such as animals, mammals and humans, to treat one or more diseases or conditions described herein. The therapeutic compounds of the present disclosure include, but are not limited to, antibodies of the present disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0135] The antibodies disclosed herein can also be used to treat or inhibit cancer. As described above, GPC3 can be overexpressed in tumor cells, particularly liver cancer, gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer and lung cancer. Inhibition of GPC3 has been shown to be useful for treating tumors.

[0136] Thus, in some embodiments, methods for treating cancer in a patient in need thereof are provided. In one embodiment, the method entails administering to the patient an effective amount of an antibody, fragment, or antibody-drug conjugate of the present disclosure. In some embodiments, at least one cancer cell (e.g., stromal cell) in the patient overexpresses GPC3.

[0137] The present disclosure also provides cell therapy, such as chimeric antigen receptor (CAR) T cell therapy. Suitable cells can be used, the cells are transduced with a vector encoding CAR, or contacted with CAR, the CAR comprising the anti-GPC3 antibody disclosed herein (or engineered to express the anti-GPC3 antibody disclosed herein). After such contact or engineering, the cells can be introduced into the body of a cancer patient in need of treatment. The cancer patient may suffer from any type of cancer disclosed herein. The cell (e.g., T cell) can be, for example, a tumor infiltrating T lymphocyte, a CD4+T cell, a CD8+T cell, or a combination thereof, but is not limited thereto.

[0138] In some embodiments, the cells are isolated from the cancer patient himself. In some embodiments, the cells are provided by a donor or come from a cell bank. When the cells are isolated from the cancer patient himself, unnecessary immune responses can be minimized.

[0139] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer and thyroid cancer. In some embodiments, the cancer is one or more of gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer and lung cancer.

[0140] Other diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed and / or predicted using the antibodies or variants or derivatives thereof of the present disclosure include, but are not limited to, progression and / or metastasis of malignancies and related diseases, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia) (including myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroblastic leukemia)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including, but not limited to, sarcomas and carcinomas, such as fibrosarcomas, myxosarcoma, liposarcoma , chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0141] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, variant or derivative thereof used, the patient's age, weight, general health, sex and diet, as well as the time of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The judgment of these factors by the medical caregiver is within the ordinary skill of the art. The dosage also depends on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage can be determined according to pharmacological and pharmacokinetic principles well known in the art.

[0142] The method of administration of antibodies, fragments or antibody-drug conjugates includes but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. Antigen-binding polypeptides or compositions can be administered by any convenient route, such as by infusion or push injection, by absorption of epithelial or mucocutaneous linings (e.g., oral mucosa, rectum and intestinal mucosa, etc.), and can be used together with other bioactive agents. Therefore, pharmaceutical compositions containing antigen-binding polypeptides of the present disclosure can be administered orally, rectally, parenterally, intracisternal, intravaginal, intraperitoneally, or locally (such as by powders, ointments, drops or transdermal patches), orally or as oral or nasal sprays.

[0143] As used herein, the term "parenteral" refers to routes of administration including intravenous, intramuscular, intraperitoneal, intraperitoneal, subcutaneous and intraarticular injection and infusion.

[0144] Administration can be systemic or local. In addition, the antibodies of the present disclosure can be introduced into the central nervous system by any suitable route, including intravenous injection and intrathecal injection; intravenous injection can be achieved by intravenous catheter, for example, connected to a reservoir (such as an Ommaya reservoir). Pulmonary administration can also be used, for example, by using an inhaler or nebulizer, and formulations with an atomizer.

[0145] It may be desirable to administer the antigen-binding polypeptides or compositions of the present disclosure locally to the area in need of treatment; this may be accomplished, for example but not limited to, by local infusion during surgery, local administration (e.g., in conjunction with a wound dressing after surgery), by injection, by catheter, by suppository, or by implant, the implant being a porous, non-porous, or gel-like material, including membranes, such as silicone rubber membranes or fibers. Preferably, when administering proteins of the present disclosure, including antibodies, care must be taken to use materials that are not absorbed by the protein.

[0146] The effective dosage of the disclosed antibodies, fragments or antibody-drug conjugates in the treatment, inhibition and prevention of inflammatory, immune or malignant diseases, disorders or conditions can be determined by standard clinical techniques. In addition, in vitro tests can be optionally used to help determine the optimal dosage range. The precise dosage used in the formulation also depends on the route of administration and the severity of the disease, disorder or condition, and should be determined according to the judgment of the physician and the circumstances of each patient. The effective dose can be inferred from the dose-response curve of an in vitro or animal model test system.

[0147] In general, the dosage of the antibody, fragment or antibody-drug conjugate of the present disclosure to the patient is usually 0.001 mg / kg to 100 mg / kg (based on the patient's body weight), or 0.01 mg / kg to 20 mg / kg (based on the patient's body weight), or 0.5 mg / kg to 10 mg / kg (based on the patient's body weight). In general, due to the immune response to foreign polypeptides, human antibodies have a longer half-life in the human body than antibodies of other species. Therefore, lower doses of human antibodies and lower dosing frequencies are generally possible. In addition, the uptake and tissue penetration of antibodies (e.g., into the brain) can be enhanced by modification (e.g., lipidation), thereby reducing the dosage and frequency of administration of the antibodies of the present disclosure.

[0148] In another embodiment, the compositions of the present disclosure are administered in combination with cytokines. Cytokines that can be administered with the compositions of the present disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0149] In additional embodiments, the compositions of the present disclosure are administered in conjunction with other therapeutic or preventive regimens, such as radiation therapy.

[0150] Composition

[0151] The present disclosure also provides pharmaceutical compositions. These compositions include an effective amount of an antibody, fragment or antibody-drug conjugate, and an acceptable carrier. In some embodiments, the composition also includes a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0152] In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, more particularly in humans. In addition, a "pharmaceutically acceptable carrier" is generally a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or any type of formulation auxiliary.

[0153] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle used together with the therapeutic agent. Such a pharmaceutical carrier can be a sterile liquid, such as water and oil, including oils from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum powder, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol, etc. If desired, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffer, such as acetate, citrate or phosphate. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting tension such as sodium chloride or glucose are also contemplated. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition can be formulated into suppositories together with conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of an antigen-binding polypeptide, preferably in a purified form, and an appropriate amount of a carrier to provide a suitable form of administration to the patient. The formulation should be suitable for the mode of administration. The parent formulation can be contained in an ampoule, disposable syringe, or multiple-dose vial made of glass or plastic.

[0154] In one embodiment, the composition is formulated into a pharmaceutical composition suitable for human intravenous administration according to conventional procedures. Usually, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally speaking, these components are provided separately or mixed together in a unit dose form, such as a dry lyophilized powder or anhydrous concentrate, and are contained in a sealed container (such as an ampoule or a pouch indicating the amount of the active agent). If the composition is administered by infusion, an infusion bottle equipped with sterile pharmaceutical grade water or saline may be used. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that each component may be mixed before administration.

[0155] The compounds of the present disclosure may be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and those formed with cations, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. DETAILED DESCRIPTION

[0156] Example 1: Generation of mouse antibodies against human GPC3

[0157] This example describes the generation of mouse monoclonal antibodies against human-GPC3 using hybridoma technology.

[0158] Antigen: Human GPC3-His protein.

[0159] Immunization: To generate mouse monoclonal antibodies targeting human GPC3, Balb / c mice and C57BL / 6 mice were first immunized with GPC3-His protein. Immunized mice were then boosted with GPC3-His protein. To select mice that produced antibodies that bound to GPC3 protein, the sera of immunized mice were evaluated for antibody titers by ELISA. Briefly, microtiter plates were filled with ELISA coating buffer containing 0.5 μg / mL human GPC3 protein was used for coating, 100 μL / well, and incubated at 4°C. overnight, and then 150 μL / well of 1% BSA blocked. Diluted serum from immunized mice was added to each well and incubated at 37°C for 1-2h. The plates were washed with PBS / Tween and then incubated with anti-mouse IgG antibody coupled to horseradish peroxidase (HRP) at 37°C for 1h. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450nm. After several rounds of immunization, the immune response against the GPC3-CHOK1 cell line was also detected by serum FACS, and the CHOK1 parental cell line was used as a negative control. The resulting mice were used for fusion. Hybridoma supernatants were screened by ELISA method.

[0160] Cell fusion: Fusion was performed by electrofusion. Each fusion was performed by seeding 50 96-well plates.

[0161] Screening: The supernatants were screened by ELISA against recombinant human (rh) GPC3-His protein and counter-screening antigen. Positive supernatants were then confirmed by binding to cells of the GPC3-CHOK1 cell line.

[0162] Subcloning and screening: Positive primary clones from each fusion were subcloned by limiting dilution to ensure that the subclones were derived from a single parental cell. Subclones were screened in the same manner as primary clones, and culture supernatants from positive clones were additionally screened by affinity sorting.

[0163] Hybridoma clones 18C1D9, 52H5D3B8, 163E7D12, 172B4E9, 152E9F7 and 171F9C5 were selected for further analysis. The amino acid sequences of the variable regions of 18C1D9, 52H5D3B8, 163E7D12, 172B4E9, 152E9F7 and 171F9C5 are listed in Table 1 below.

[0164] Table 1. Sequences of the variable regions of 18C1D9, 52H5D3B8, 163E7D12, 172B4E9, 152E9F7 and 171F9C5

[0165]

[0166] Table 1A. CDR sequences of 18C1D9

[0167] 18C1D9 sequence SEQ ID NO: CDRH1 DYEM 13 CDRH2 AIDPETGGTAYNQKFKG 14 CDRH3 YYSYAY 15 CDRL1 RSRQSLVHSNGNTYLQ 16 CDRL2 KVSNRFS 17 CDRL3 FQSIHVPY 18

[0168] Table 1B. CDR sequences of 52H5D3B8

[0169] 52H5D3B8 sequence SEQ ID NO: CDRH1 DYEM 13 CDRH2 AIHPGSGGTAYNQKFKG 19 CDRH3 FYSYAY 20 CDRL1 RSSQSLVHSNGNTYLQ 21 CDRL2 KVSNRFS 17 CDRL3 SQSIHVPYT 22

[0170] Table 1 C. CDR sequences of 163E7D12

[0171] 163E7D12 sequence SEQ ID NO: CDRH1 DYEM 13 CDRH2 AIGPETGSTAYNQRFKG 23 CDRH3 YYSFAY 24 CDRL1 RSSQSLVHSNGNTYLQ 21 CDRL2 KVSNRFS 17 CDRL3 FQSIHVPY 18

[0172] Table 1D. CDR sequences of 172B4E9

[0173] 172B4E9 sequence SEQ ID NO: CDRH1 DYEIH 25 CDRH2 AIDPATGDTAYTQKFKG 26 CDRH3 FYSYAY 20 CDRL1 RSSQSLVHTNGHTYLQ 27 CDRL2 KVSNRFS 17 CDRL3 SQSTHVPYT 28

[0174] Table 1 CDR sequences of E.152E9F7

[0175] 152E9F7 sequence SEQ ID NO: CDRH1 DYEIH 25 CDRH2 AIDPETGDTAYTQKFKG 29 CDRH3 FYSYAY 20 CDRL1 RSSQSLVHTNGHTYLQ 27 CDRL2 KVSNRFS 17 CDRL3 SQSTHVPYT 28

[0176] Table 1F. CDR sequences of 171F9C5

[0177] 171F9C5 sequence SEQ ID NO: CDRH1 DYEIH 25 CDRH2 AIDPETDNTAYSQKFKG 30 CDRH3 YYSYAY 15 CDRL1 RSSQSPVHRNGNTYLH 31 CDRL2 KVSNRFS 17 CDRL3 SQTTHVPYT 32

[0178] Example 2: Binding affinity of mouse and chimeric monoclonal antibodies

[0179] Affinity ranking of mouse monoclonal antibodies

[0180] Mouse monoclonal antibodies were tested for binding to human GPC3-his using a capture method using Biacore. Antibodies were captured using a CM5 sensor chip. A single dose of human GPC3-his tagged protein was injected onto the captured antibody at a flow rate of 30 μL / min for 2 min. Antigen was allowed to dissociate for 360 s. All experiments were performed on a Biacore 8K. Data analysis was performed using the Biacore 8K evaluation software.

[0181] As shown in the results of Table 2A, all mouse monoclonal antibodies showed high binding affinity to the recombinant GPC3 protein.

[0182] Table 2A. Affinity ranking using Biacore assay

[0183]

[0184]

[0185] The variable regions of mouse antibodies 18C1D9, 52H5D3B8, 163E7D12, 172B4E9, 152E9F7, and 171F9C5 were then fused to human IgG1 to generate chimeric monoclonal antibodies.

[0186] Full kinetic affinity of 52H5D3B8 determined using Biacore

[0187] The binding of chimeric 52H5D3B8 monoclonal antibody to human GPC3-his was tested using the capture method using Biacore. The 52H5D3B8 monoclonal antibody was captured using a CM5 sensor chip. Serial dilutions of human GPC3-his tag protein were injected onto the captured antibody at a flow rate of 30 μL / min for 2 min. The antigen was allowed to dissociate for 360 s. All experiments were performed on a Biacore 8K. Data analysis was performed using the Biacore 8K evaluation software.

[0188] like Figure 1 As shown in Table 2B below, the 52H5D3B8 antibody exhibited high binding affinity to the recombinant GPC3 protein.

[0189] Table 2B. Affinity determined using Biacore

[0190]

[0191] Example 3: Binding activity to GPC3 antigen

[0192] FACS detection of chimeric antibodies

[0193] Cell-based binding: The binding activity of chimeric monoclonal antibodies (18C1D9, 52H5D3B8, 163E7D12, 172B4E9, 152E9F7, and 171F9C5) and the known GPC3 antibody GC33 (Chugai Pharmaceutical) to CHOK1 cells overexpressing human GPC3 was evaluated using FACS.

[0194] Briefly, GPC3-CHOK1 cells were washed with FACS buffer, and then 3-fold serial dilutions of chimeric mAb or GC33 mAb were added to each well, starting at 5 μg / mL, and incubated at 4°C for 40 min. 647AffiniPure goat anti-human IgG (H+L), incubated at 4°C for 30 min. Samples were washed twice with FACS buffer. The mean fluorescence intensity (MFI) of 647. Table 3A below lists the EC values ​​of each monoclonal antibody against GPC3-CHOK1 cells. 50 The 52H5D3B8 chimeric antibody showed higher cell binding efficiency than GC33 ( Figure 2A Table 3A. Binding activity to GPC3-CHOK1 cells

[0195]

[0196] FACS detection of 52H5D3B8 chimeric antibody

[0197] Cell-based binding: FACS was used to evaluate the binding activity of 52H5D3B8 chimeric monoclonal antibody and known GPC3 antibody GC33 (Chugai Pharmaceutical) on CHOK1 cells, HEPG2 cells, Hep3B cells, and Huh-7 cells overexpressing human GPC3.

[0198] Briefly, GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells, or Huh-7 cells were first incubated with Fc blocking agent at 4°C for 15 min. GPC3-CHOK1 cells were washed with FACS buffer, and then 3-fold serial dilutions of 52H5D3B8 chimeric monoclonal antibody or GC33 monoclonal antibody were added to each well, starting at 30 μg / mL, and placed at 4°C for 60 min. Cells were washed with FACS buffer and fixed with 2% PFA for 15 min at room temperature. After washing with FACS buffer, Alexa Fluor was added to each well. 647AffiniPure goat anti-human IgG and incubated at room temperature for 30 min. Samples were washed twice with FACS buffer. The mean fluorescence intensity (MFI) of 647 was measured by MACSQuant Analyzer 16. Figure 2B As shown in Figure 3, the binding efficiency of 52H5D3B8 to GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells or Huh-7 cells is comparable and higher than that of GC33. 50 The results are listed in Table 3B below. 52H5D3B8 antibody showed higher cell binding efficiency than GC33 ( Figure 2B and Table 3B).

[0199] Table 3B. Binding activity to GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells and Huh-7 cells

[0200]

[0201] Cross-species activity

[0202] ELISA assays were performed to evaluate the binding of chimeric antibodies to human, mouse and cynomolgus monkey GPC3, respectively.

[0203] Briefly, microtiter plates were coated with 100 μL / well of PBS containing 1 μg / mL of human, mouse, and cynomolgus GPC3 proteins at 4°C overnight and then blocked with 150 μL / well of 1% BSA. Three-fold dilutions of chimeric antibodies starting from 9 μg / mL were added to each well and incubated at 37°C for 1 h. The plates were washed with PBS / Tween and then incubated with horseradish peroxidase (HRP)-conjugated mouse anti-human IgG Fc antibody at 37°C for 30 min. After washing, the plates were developed with TMB substrate and analyzed with a spectrophotometer at an OD wavelength of 450 nm. The 52H5D3B8 antibody binds to human GPC3, cynomolgus GPC3, and mouse GPC3 ( Figure 3 and Table 4).

[0204] Table 4A. Cross-species activity of 52H5D3B8 and human GPC3

[0205] people <![CDATA[EC of 52H5D3B8 50 > 17.03ng / mL

[0206] Table 4B. Cross-species activity of 52H5D3B8 against cynomolgus monkey and mouse GPC3

[0207] Cynomolgus monkey Mouse <![CDATA[EC of 52H5D3B8 50 > 15.25 ng / mL 4948ng / mL

[0208] Example 4: Optimization of 52H5D3B8 chimeric monoclonal antibody

[0209] This example describes the optimization of the 52H5D3B8 chimeric monoclonal antibody by mutation of CDR3 to enhance the binding activity of the 52H5D3B8 chimeric monoclonal antibody to human GPC3.

[0210] Mutation: Since the CDR3 domain of the antibody plays a vital role in the interaction with the antigen, the CDR3 domain of the 52H5D3B8 chimeric monoclonal antibody was mutated. Both CDRH3 and CDRL3 were designed to have two candidate mutation sites, respectively. The 15 candidate combinations of CDRH3 and CDRL3 sequences are listed together in Table 5 below.

[0211] Table 5. Variable region sequences of 52H5D3B8 mutants

[0212]

[0213]

[0214]

[0215] Table 5A. CDR sequences of 52H5D3B8-1#

[0216] 52H5D3B8-1# sequence SEQ ID NO: CDRH3 YYSYAY 15 CDRL3 SQSIHVPYT 22

[0217] Table 5B. CDR sequences of 52H5D3B8-2#

[0218] 52H5D3B8-2# sequence SEQ ID NO: CDRH3 FYSFAY 33 CDRL3 SQSIHVPYT 22

[0219] Table 5C. CDR sequences of 52H5D3B8-3#

[0220] 52H5D3B8-3# sequence SEQ ID NO: CDRH3 FYSYAY 20 CDRL3 FQSIHVPY 18

[0221] Table 5D. CDR sequences of 52H5D3B8-4#

[0222] 52H5D3B8-4# sequence SEQ ID NO: CDRH3 FYSYAY 20 CDRL3 SQSTHVPYT 28

[0223] Table 5E. CDR sequences of 52H5D3B8-5#

[0224] 52H5D3B8-5# sequence SEQ ID NO: CDRH3 YYSFAY 24 CDRL3 SQSIHVPYT 22

[0225] Table 5F. CDR sequences of 52H5D3B8-6#

[0226] 52H5D3B8-6# sequence SEQ ID NO: CDRH3 YYSYAY 15 CDRL3 FQSIHVPY 18

[0227] Table 5G.52H5D3B8-7# CDR sequence

[0228] 52H5D3B8-7# sequence SEQ ID NO: CDRH3 YYSYAY 15 CDRL3 SQSTHVPYT 28

[0229] Table 5H. CDR sequences of 52H5D3B8-8#

[0230] 52H5D3B8-8# sequence SEQ ID NO: CDRH3 FYSFAY 33 CDRL3 FQSIHVPY 18

[0231] Table 5I. CDR sequences of 52H5D3B8-9#

[0232] 52H5D3B8-9# sequence SEQ ID NO: CDRH3 FYSFAY 33 CDRL3 SQSTHVPYT 28

[0233] Table 5J. CDR sequences of 52H5D3B8-10#

[0234]

[0235]

[0236] Table 5K. CDR sequence of 52H5D3B8-11#

[0237] 52H5D3B8-11# sequence SEQ ID NO: CDRH3 FYSFAY 33 CDRL3 FQSTHVPYT 34

[0238] Table 5L. CDR sequences of 52H5D3B8-12#

[0239] 52H5D3B8-12# sequence SEQ ID NO: CDRH3 YYSYAY 15 CDRL3 FQSTHVPYT 34

[0240] Table 5M. CDR sequences of 52H5D3B8-13#

[0241] 52H5D3B8-13# sequence SEQ ID NO: CDRH3 YYSFAY 24 CDRL3 SQSTHVPYT 28

[0242] Table 5N. CDR sequences of 52H5D3B8-14#

[0243] 52H5D3B8-14# sequence SEQ ID NO: CDRH3 YYSFAY 24 CDRL3 FQSIHVPY 18

[0244] Table 50. CDR sequences of 52H5D3B8-15#

[0245] 52H5D3B8-15 sequence SEQ ID NO: CDRH3 YYSFAY 24 CDRL3 FQSTHVPYT 34

[0246] Optimizing antibody binding to GPC3-CHOK1 cells

[0247] Cell-based binding: FACS was used to evaluate the binding activity of 52H5D3B8, 52H5D3B8-1#, 52H5D3B8-2#, 52H5D3B8-3#, 52H5D3B8-3#, 52H5D3B8-4#, 52H5D3B8-5#, 52H5D3B8-6#, 52H5D3B8-7#, 52H5D3B8-8#, 52H5D3B8-10#, 52H5D3B8-11#, 52H5D3B8-12#, 52H5D3B8-13#, 52H5D3B8-14# and 52H5D3B8-15# chimeric monoclonal antibodies to CHOK1 cells overexpressing human GPC3.

[0248] Briefly, GPC3-CHOK1 cells were first incubated with Fc blocking agent at 4°C for 15 min. The cells were washed with FACS buffer and three-fold serial dilutions of 52H5D3B8, 52H5D3B8-1#, 52H5D3B8-2#, 52H5D3B8-3#, 52H5D3B8-4#, 52H5D3B8-5#, 52H5D3B8-6#, 52H5D3B8-7#, 52H5D3B8-8#, 52H5D3B8-9#, 52H5D3B8-10#, 52H5D3B8-11#, 52H5D3B8-12#, 52H5D3B8-13#, 52H5D3B8-14# and 52H5D3B8-15# chimeric monoclonal antibodies were dispensed into each well with an initial concentration of 30 μg / mL and kept at 4°C for 60 min. The cells were washed with FACS buffer and fixed with 2% PFA for 15 min at room temperature. After washing with FACS buffer, Alexa Fluor was added to each well. 647AffiniPure goat anti-human IgG and incubated at room temperature for 30 min. Samples were washed twice with FACS buffer. The mean fluorescence intensity (MFI) of 647 was measured by MACSQuant Analyzer 16. Figure 4 As shown, the affinity of 52H5D3B8-6# chimeric monoclonal antibody to GPC3-CHOK1 cells is higher than that of other monoclonal antibodies. Table 6 below lists the EC values ​​of each chimeric monoclonal antibody to GPC3-CHOK1 cells. 50 Table 6. Binding activity to GPC3-CHOK1 cells and affinity ranking determined by Biacore

[0249]

[0250] Affinity ranking of optimized antibodies using Biacore

[0251] The optimized antibody of 52H5D3B8 monoclonal antibody was tested for binding to human GPC3-his using the capture method using Biacore. Single or double doses of human GPC3-his tagged protein were injected onto the captured antibody. All experiments were performed on Biacore8K. Data analysis was performed using Biacore8K evaluation software.

[0252] As shown in the results of Table 6 above, the binding affinity of the optimized antibodies to the recombinant GPC3 protein is between 10-8 and 10-10. In addition, 52H5D3B8-6#mAb exhibits the highest affinity to the GPC3 protein.

[0253] Optimizing antibody binding to GPC3-CHOK1 cells

[0254] Cell-based binding: FACS was used to evaluate the binding activity of 52H5D3B8, 52H5D3B8-6# chimeric monoclonal antibody and GC33 antibody to CHOK1 cells overexpressing human GPC3, Hep3B cells and Huh-7 cells.

[0255] Briefly, GPC3-CHOK1 cells, Hep3B cells, or Huh-7 cells were first incubated with Fc blocking agent at 4°C for 15 min. The cells were washed with FACS buffer, and then 3-fold serial dilutions of 52H5D3B8, 52H5D3B8-6# chimeric monoclonal antibody, or GC33 monoclonal antibody were added to each well, starting at 30 μg / mL, and placed at 4°C for 60 min. The cells were washed with FACS buffer and fixed with 2% PFA for 15 min at room temperature. After washing with FACS buffer, Alexa Fluor was added to each well. 647AffiniPure goat anti-human IgG and incubated at room temperature for 30 min. Samples were washed twice with FACS buffer. The mean fluorescence intensity (MFI) of 647 was measured by MACSQuant Analyzer 16. Figure 5 As shown in Figure 2, the binding affinity of 52H5D3B8-6# chimeric monoclonal antibody to GPC3-CHOK1 cells, Hep3B cells, and Huh-7 cells was higher than that of 52H5D3B8 chimeric monoclonal antibody and GC33 antibody. 50 Listed in Table 7 below.

[0256] Table 7. Binding activity to GPC3-CHOK1 cells, Hep3B cells and Huh-7 cells

[0257]

[0258] Example 5: Humanization of chimeric antibody 52H5D3B8-6#

[0259] The variable regions of 52H5D3B8-6# were used to create humanized monoclonal antibodies. In the first step of this process, the amino acid sequences of the VH and VL of 52H5D3B8-6# were compared to an existing human Ig gene sequence database to find the best overall matching human germline Ig gene sequence.

[0260] For the light chain of 52H5D3B8-6#, IGKV2-29*02 was the most suitable germline, and for the heavy chain of 52H5D3B8-6#, IGHV1-46*01 was selected as the humanized framework. Then the humanized 52H5D3B8-6#CDR-grafted antibody was designed, in which CDRL1, CDRL2 and CDRL3 were grafted onto the framework sequence of IGKV2-29*02, and CDRH1, CDRH2 and CDRH3 were grafted onto the framework sequence of IGHV1-46*01. Then a 3D model was generated to determine the amino acids in the original mouse FR region sequence that are critical for antibody binding and conformation. Based on the 52H5D3B8-6#CDR-grafted antibody sequence, 3 additional humanized heavy chains and 3 additional light chains were created. For the heavy chain, L, K, I, A, F and T in the framework region participated in the back mutation. For the light chain, K and F in the framework region participated in the back mutation.

[0261] The amino acid sequences of the humanized antibodies are listed in Table 8 below.

[0262] Table 8A. Humanized antibody sequences (underlined CDRs; bold / italics indicate back mutations)

[0263] Table 8B. CDR sequences of 52H5D3B8-6#

[0264] 52H5D3B8-6 sequence SEQ ID NO: CDRH1 DYEM 13 CDRH2 AIHPGSGGTAYNQKFKG 19 CDRH3 YYSYAY 15 CDRL1 RSSQSLVHSNGNTYLQ 21 CDRL2 KVSNRFS 17 CDRL3 FQSIHVPY 18

[0265] Then 4 humanized heavy chains and 4 humanized light chains were synthesized and cloned into pcDNA3.4 vectors respectively. After plasmid extraction, 4 heavy chains and 4 light chains were paired and expressed in HEK293 cells. The pairing of human VH and human VL produced 16 kinds of humanized antibodies (see Table 9).

[0266] Table 9. Humanized antibodies and their VH and VL regions 52H5D3B8-6#

[0267]

[0268] Example 6: Antigen Binding Properties of Humanized Antibodies

[0269] Affinity ranking of humanized antibodies by Biacore

[0270] To evaluate antigen binding activity, the affinity of the chimeric antibodies was measured by Biacore 8K. Serial dilutions of human GPC3-his tag protein were injected onto the captured chimeric antibody 52H5D3B8-6#-VH+VL at a flow rate of 30 μL / min for 3 min. The antigen was allowed to dissociate for 720 s. Among the multiple test doses, the two concentrations closest to the affinity of the chimeric antibody were selected for affinity testing of 16 humanized antibodies. Figure 6 As shown in Table 10, all 16 humanized antibodies showed similar affinities, comparable to the chimeric antibodies.

[0271] Table 10. Affinity ranking of humanized antibodies

[0272]

[0273]

[0274] Binds to CHOK1 cells, HEPG2 cells, Hep3B cells, and Huh-7 cells overexpressing human GPC3

[0275] To evaluate the antigen binding properties, the binding of humanized antibodies to CHOK1 cells, HEPG2 cells, Hep3B cells, and Huh-7 cells overexpressing human GPC3 was analyzed by FACS. Briefly, GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells, and Huh-7 cells were first incubated with Fc blocking agent at 4°C for 15 min. The cells were washed with FACS buffer, and 4-fold serial dilutions of humanized antibodies (starting concentration of 10 μg / mL) were distributed to each well and placed at 4°C for 60 min. The cells were washed with FACS buffer and fixed with 2% PFA for 15 min at room temperature. After washing with FACS buffer, Alexa Fluor 500 was added to each well. 647AffiniPure goat anti-human IgG and incubate at room temperature for 30 min. The MFI of 647 was determined by MACSQuant Analyzer16. The 16 humanized antibodies were divided into two plates. Figure 7-8 As shown, the EC of each humanized antibody against GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells and Huh-7 cells 50 The EC values ​​of humanized antibodies and 52H5D3B8-6#-VH+VL were compared in the same plate. 50, all candidate humanized antibodies showed binding efficiencies comparable to those of chimeric antibodies.

[0276] Table 11. Binding activity to GPC3-CHOK1 cells, Hep3B cells and Huh-7 cells

[0277]

[0278] Table 12. Binding activity to GPC3-CHOK1 cells, Hep3B cells and Huh-7 cells

[0279]

[0280] Full kinetic affinity of humanized antibodies determined using Biacore

[0281] The binding of humanized antibodies to recombinant human GPC3 protein (human GPC3-his tag) was tested using the capture method using Biacore. 52H5D3B8-6#-VH+VL, 52H5D3B8-6#-VH1+VL1, 52H5D3B8-6#-VH1+VL3, 52H5D3B8-6#-VH1+VL4, 52H5D3B8-6#-VH2+VL4, 52H5D3B8-6#-VH2+VL1, 52H5D3B8-6#-VH3+VL3 monoclonal antibodies were captured using a CM5 sensor chip. Serially diluted human GPC3-his tag protein was injected onto the captured antibody at a flow rate of 30μL / min for 2min. The antigen was allowed to dissociate for 360s. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore 8K evaluation software. Fig. 9 and as shown in Table 13 below.

[0282] Table 13. Affinity determined using Biacore

[0283]

[0284] Binds to CHOK1 cells and HEPG2 cells overexpressing human GPC3

[0285] FACS was used to evaluate the binding activity of 52H5D3B8-6#-VH1+VL1 and 52H5D3B8-6#-VH2+VL3 with GC33 (Chugai Pharmaceutical) and Y035 (Carsgen Therapeutics, US patent application US20190046659) on CHOK1 cells and HEPG2 cells overexpressing human GPC3. The EC of each antibody on GPC3-CHOK1 cells and HEPG2 cells 50 Listed in Table 14 below.

[0286] like Fig.10 As shown, the binding affinities of 52H5D3B8-6#-VH1+VL1 and 52H5D3B8-6#-VH2+VL3 to GPC3-CHOK1 cells and HEPG2 cells were comparable, both higher than the benchmark monoclonal antibodies.

[0287] Table 14. Binding activity to GPC3-CHOK1 GPC3-CHOK1 cells

[0288]

[0289] Example 7: Affinity maturation of humanized antibody 52H5D3B8-6#

[0290] In order to enhance the affinity of the 52H5D3B8-6# humanized antibody, affinity maturation was performed. In brief, four phage libraries containing single or two-point saturation mutations in the CDR region were constructed. Three candidates with unique mutations in the CDRs were obtained through two rounds of solid or liquid panning screening. The CDRs of these candidates were transplanted into the 52H5D3B8-6#-VH2+VL3 framework to generate antibodies for further binding and functional validation.

[0291] The amino acid sequences of the variable regions of the framework-grafted affinity maturation candidates are listed in Table 15 below.

[0292] Table 15. Variable region sequences of 52H5D3B8-18#-VH2+VL3, 52H5D3B8-19#-VH2+VL3, 52H5D3B8-20#-VH2+VL3

[0293]

[0294]

[0295] Table 15A. CDR sequences of 52H5D3B8-18#-VH2+VL3

[0296] Table 15B. CDR sequences of 52H5D3B8-19#-VH2+VL3

[0297] Table 15C. CDR sequences of 52H5D3B8-20#-VH2+VL3

[0298]

[0299] Binding to CHOK1 cells overexpressing human GPC3

[0300] To assess antigen binding properties, affinity-matured antibodies were analyzed by FACS for binding to CHOK1 cells overexpressing human GPC3 as described previously. The EC of each antibody against GPC3-CHOK1 cells was 50 Listed in Table 16 below.

[0301] like Fig.11 As shown, all affinity matured antibodies had improved binding efficiency to GPC3-CHOK1 cells compared with the parental 52H5D3B8-6#-VH2+VL3 and the benchmark antibody GC33.

[0302] Table 16. Binding activity to GPC3-CHOK1 cells

[0303]

[0304] Example 8: ADCC signal transduction mediated by humanized GPC3 antibody

[0305] In order to evaluate the antibody-dependent cellular toxicity (ADCC) signal transduction mediated by antigen binding of 52H5D3B8-6#-VH1+VL1, 52H5D3B8-6#-VH2+VL3 and three affinity matured antibodies, and compared with GC33, an ADCC signal transduction reporter assay was established. In brief, a full-length human CD16a with a high affinity allotype (176V) bound to the Fc region was introduced into the Jurkat-NFAT luciferase reporter cell line (referred to as Jukrat-hCD16a-NFAT). In this cell line, the expression of the luciferase gene is controlled by the NFAT response element. The Jukrat-hCD16a-NFAT cell line enables us to evaluate the ADCC signal transduction mediated by GPC3 binding by examining downstream NFAT signal transduction. The reporter cell line is co-cultured with several GPC3-positive tumor cell lines, including HEPG2 or Huh-7 liver cancer cells with high or moderate GPC3 expression levels, respectively. Luciferase-based chemiluminescence can be detected by ONE-GloTM luciferase assay system (Promega, Cat. No E6130) and Envision multilabel plate readers (Perkin Elemer).

[0306] like Fig.12As shown in Table 17, 52H5D3B8-6#-VH1+VL1 and 52H5D3B8-6#-VH2+VL3 both showed effective GPC3 binding-mediated ADCC signal transduction on HEPG2 cells and Huh-7 cells, which was higher than GC33. In addition, the EC values ​​of 52H5D3B8-19#-VH2+VL3 and 52H5D3B8-20#-VH2+VL3 on ADCC signal transduction of HEPG2 cells were 50 It is better than the parent 52H5D3B8-6#-VH2+VL3, while 52H5D3B8-18#-VH2+VL3 shows a maximum ADCC signal transduction effect that is better than other antibodies. It is worth noting that in Huh-7 cells with moderate GPC3 expression levels, all humanized antibodies show stronger maximum ADCC signaling effects than GC33.

[0307] Table 17. ECs for ADCC signaling 50

[0308]

[0309] Example 9: Humanized GPC3 antibody mediates NK cell killing of tumors

[0310] In this example, the functional activity of the GPC3 humanized antibody was tested.

[0311] 9.1 GPC3 humanized antibody-mediated NK cell cytotoxicity against target cells

[0312] In order to evaluate whether GPC3-mediated ADCC signaling activation can be converted into NK cell cytotoxicity on target cells, a human primary NK cell-mediated cytotoxicity test was established. In short, fresh human primary NK cells (CD3-CD56+) were isolated from the buffy coat of healthy donors by magnetic beads (Miltenyi, catalog number 130-092-657) negative selection. The purity of the isolated NK cells was monitored by FACS analysis, usually exceeding 90%. The isolated NK cells were then left to stand in complete medium for 24 hours and used as effector cells. HEPG2 and Huh7 human hepatoma cell lines were used as target cells. NK cell-mediated cytotoxicity detection was performed according to the cytotoxicity detection kit (Roche, catalog number 11644793001) provided by the manufacturer. The effector cells were co-cultured with 1.0x104 target cells (E / T ratio = 5:1) at a density of 5.0x104 cells per well in a round-bottom 96-well microplate. The antibody was diluted 5 times from 50 nM and added to the corresponding wells. After incubation at 37 ° C for 5 h (HEPG2 cells) or 24 h (Huh-7 cells), lactate dehydrogenase (LDH) representing cell viability was detected using a cytotoxicity detection kit. The absorbance was measured at 492 nm. Calculation formula: Cytotoxicity (%) = (Effector cell-target cell mixture-Effector cell control-Low control) / (High control-Low control) × 100. Four-parameter nonlinear regression curve fitting was performed using GraphPad Prism 9.

[0313] like Fig.13 As shown in Table 18, both 52H5D3B8-6#-VH1+VL1 and 52H5D3B8-6#-VH2+VL3 showed effective GPC3 antibody-mediated NK cell cytotoxicity against HEPG2 cells and Huh-7 cells, which were higher than GC33.

[0314] In addition, both 52H5D3B8-19#-VH2+VL3 and 52H5D3B8-20#-VH2+VL3 exhibited GPC3 antibody-mediated NK cell cytotoxicity against HEPG2 cells comparable to that of the parental 52H5D3B8-6#-VH2+VL3. Interestingly, in Huh-7 cells with moderate GPC3 expression levels, the EC values ​​of 52H5D3B8-19#-VH2+VL3 and 52H5D3B8-20#-VH2+VL3 were significantly higher than those of the parental 52H5D3B8-6#-VH2+VL3. 50 The values ​​were better than those of the parent 52H5D3B8-6#-VH2+VL3.

[0315] Table 18. EC of ADCC cytotoxicity 50

[0316]

[0317] 9.2 NK cell degranulation stimulated by GPC3 humanized antibody

[0318] To investigate the ability of GPC3 humanized antibody to stimulate NK cell degranulation, the expression level of the degranulation marker CD107a in NK cells was analyzed. Briefly, primary human NK cells generated as described above were used as effector cells and cultured at 2.5 × 10 cells per well. 4 The cell density was 2.5×10 4 Target cells including HEPG2 (E / T ratio = 1:1) were co-incubated in a round-bottom 96-well microplate. The antibody was serially diluted 5 times starting from 50nM and added to the corresponding wells. After incubation at 37°C for 4h, the cells were harvested and washed with ice-cold staining buffer (0.5% BSA in PBS). Anti-CD107a antibody (BioLegend, catalog number 328608) was diluted in staining buffer, 1μL was added to each well, and incubated with the cells at 4°C in the dark for 40min. The samples were then washed and fixed with 2% PFA. In a flow cytometer Cells were analyzed on a Analyzer 16 (Miltenyi Biotec). Data were analyzed using Flowjo 10.0 software. Four-parameter nonlinear regression curve fitting was performed using GraphPad Prism 9.

[0319] like Fig.14 As shown, all GPC3 humanized antibodies stimulated CD107a upregulation more efficiently in HEPG2 cells compared to GC33.

[0320] 9.3GPC3 humanized antibody stimulates the production of NK cytokines

[0321] To investigate the ability of humanized antibodies to stimulate NK cells to produce cytokines in the presence of target cells, intercellular IFN-γ and TNF-α in NK cells were analyzed by FACS staining. Briefly, human primary NK cells were prepared as described above and plated at a density of 2.5 × 10 4 Effector cells and 2.5×10 4Target cells including HEPG2 (E / T ratio = 1:1) were co-cultured in round-bottom 96-well microwells. The protein transport inhibitor Brefeldin A (BFA) was added to the co-culture system to prevent cytokine secretion into the supernatant during NK cell activation. The antibodies were serially diluted 5-fold starting from 50nM and added to the corresponding wells. After incubation at 37°C for 4h, the samples were washed and intracellular staining was performed according to the steps of the Foxp3 / Transcription Factor Staining Buffer Set (ThermoFisher, Catalog No.: 00-5523-00). Anti-IFN-γ antibody (BioLegend, Catalog No.: 506510) and anti-TNF-α antibody (BD Biosciences, Catalog No.: 562783) were diluted in the recommended staining buffer, 1μL per well. On a flow cytometer Cells were analyzed on a Analyzer 16 (Miltenyi Biotec). Data were analyzed using Flowjo 10.0 software. Four-parameter nonlinear regression curve fitting was performed using GraphPad Prism 9.

[0322] like Fig.14 As shown, in HEPG2 cells, all GPC3 humanized antibodies induced more IFN-γ and TNF-α production in NK cells than GC33.

[0323] Example 10: Humanization of chimeric antibody 52H5D3B8

[0324] The 52H5D3B8 variable region genes were used to prepare humanized monoclonal antibodies. In the first step of this process, the amino acid sequences of 52H5D3B8 VH and VL or VK were compared with the existing human Ig gene sequence database to find the best overall matching human germline Ig gene sequence.

[0325] Briefly, IGKV2-29*02 and IGHV1-2*02 were used as the backbones for the heavy and light chains, respectively. A 3D model was then generated to determine the amino acids in the original mouse FR region sequence that are critical for antibody binding and conformation. Based on the 52H5D3B8 CDR-grafted antibody sequence, 5 additional humanized heavy chains and 1 additional light chain were created. For the heavy chain, I, K, A, L, and T in the framework involved back mutations. For the light chain, F in the framework involved back mutations.

[0326] The amino acid and nucleotide sequences of some humanized antibodies are listed in Table 19 below.

[0327] Table 19. Humanized antibody sequences (underlined CDRs; bold / italics indicate back mutations)

[0328]

[0329]

[0330] Then 6 humanized heavy chains and 2 humanized light chains were synthesized and cloned into pcDNA3.4 vectors respectively. After plasmid extraction, 6 heavy chains and 2 light chains were paired and expressed in HEK293 cells. The pairing of human VH and human VL produced 12 kinds of humanized antibodies (see Table 20).

[0331] Table 20. Humanized antibodies and their VH and VL regions A, 52H5D3B8

[0332]

[0333] Example 11: Antigen Binding Properties of Humanized Antibodies

[0334] Affinity ranking of humanized antibodies using Biacore

[0335] To evaluate antigen binding activity, the affinity of the chimeric antibody was measured using Biacore 8K. Two concentrations closest to the affinity of the chimeric antibody were selected for affinity testing of 12 humanized antibodies. Human GPC3-his tag protein was injected onto the captured antibody at a flow rate of 30 μL / min for 3 min. The antigen was allowed to dissociate for 720 s. As shown in Table 21, all 12 humanized antibodies showed similar affinity to the chimeric antibody.

[0336] Table 21. Affinity ranking of humanized antibodies

[0337]

[0338]

[0339] Binding to CHOK1 cells and Huh-7 cells overexpressing human GPC3

[0340] To evaluate antigen binding properties, the binding of humanized antibodies to CHOK1 cells and Huh-7 cells overexpressing human GPC3 was analyzed by FACS. Briefly, GPC3-CHOK1 cells and Huh-7 cells were first incubated with Fc blocking agent at 4°C for 15 min. The cells were washed with FACS buffer, and 4-fold serial dilutions of humanized antibodies (starting at a concentration of 10 μg / mL) were added to each well and placed at 4°C for 60 min. The cells were washed with FACS buffer and fixed with 2% PFA for 15 min at room temperature. After washing with FACS buffer, Alexa Fluor 500 was added to each well. 647AffiniPure goat anti-human IgG and incubate at room temperature for 30 min. The MFI of 647 was determined by MACSQuant Analyzer 16. Different plates may affect the EC value of 52H5D3B8-VH+VL. 50 Therefore, the 12 humanized antibodies were divided into two panels. The EC of each humanized antibody against GPC3-CHOK1 cells and Huh-7 cells 50 Listed in Tables 22-23 below. Fig.16 As shown, all candidate humanized antibodies exhibited comparable binding efficiencies to the chimeric antibodies for GPC3-CHOK1 cells and had better binding efficiencies to Huh-7 cells than the chimeric antibodies.

[0341] Table 22. Binding activity to GPC3-CHOK1 and Huh-7 cells

[0342]

[0343] Table 23. Binding activity to GPC3-CHOK1 and Huh-7 cells

[0344]

[0345]

[0346] Binds to CHOK1 cells, Huh-7 cells, and HEPG2 cells overexpressing human GPC3

[0347] FACS was used to evaluate the binding activity of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 to human GPC3 expressed on CHOK1 cells, Huh-7 cells, and HEPG2 cells with benchmark antibodies GC33 and Y035. In order to evaluate the characteristics of the CDR and framework of Hu 52H5D3B8-7 Ab, two chimeric antibodies were produced. The antibody called "Hu 52H5D3B8-7 / 8CDR with Y035 FR" contains the CDR of Hu52H5D3B8-7 and the FR of Y035. The antibody called "Y035 CDR with Hu 52H5D3B8-7 FR" represents a chimeric antibody with the Y035 CDR transplanted to the Hu 52H5D3B8-7 FR. The EC of each antibody to GPC3-CHOK1 cells, Huh-7 cells, and HEPG2 cells was evaluated. 50 Listed in Table 24 below.

[0348] like Fig.17As shown, Hu 52H5D3B8-7 and Hu 52H5D3B8-8 both bound to GPC3-CHOK1 cells, Huh-7 cells, and HEPG2 cells with comparable affinity, which was higher than the benchmark antibodies GC33 and Y035. More importantly, Hu52H5D3B8-7 / 8 CDR with Y035 FR and Y035 CDR with Hu 52H5D3B8-7 FR both showed higher affinity than Y035, indicating that the CDR and framework of Hu 52H5D3B8-7 contribute to the excellent binding ability to Y035.

[0349] Table 24. Binding activity to GPC3-CHOK1, Huh-7 cells and HEPG2 cells

[0350]

[0351] Example 12: Internalization activity of humanized GPC3 antibodies on tumor cell lines expressing GPC3

[0352] The internalization activity of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 with Y035 on HEPG2 cells was evaluated by cellular internalization assay. To evaluate the CDR and framework properties of Hu 52H5D3B8-7 Ab, two antibodies (Hu52H5D3B8-7 / 8 CDR with Y035 FR and Y035 CDR with Hu 52H5D3B8-7 FR) were also evaluated.

[0353] PHAb thiol dye is a pH sensor dye with very low fluorescence at pH>7. When it is internalized into endosomes or lysosomes with pH values ​​of approximately 6.3 or 4.7, respectively, the fluorescence increases dramatically. Briefly, pHAb thiol dye-labeled α-hIgG secondary antibody (10 μg / mL) was incubated with antibody (20 μg / mL) for 30 min. After incubation, the mixture antibody was serially diluted 2-fold. The diluted mixture was then added to each well pre-seeded with 2×10 4 HEPG2 cells were plated in a 96-well assay plate. After 48 h of incubation, the cells were plated in a multifunctional microplate reader ( 2105) to capture the fluorescence signal.

[0354] like Fig.18 As shown, both Hu 52H5D3B8-7 and Hu 52H5D3B8-8 showed higher internalization efficacy than other monoclonal antibodies. In addition, Hu 52H5D3B8-7 / 8CDR with Y035 FR showed higher internalization efficacy than Y035 (higher peak and comparable EC 50), indicating that the CDRs of Hu-52H5D3B8-7 contribute to enhanced target-mediated internalization compared with Y035. The EC of each monoclonal antibody 50 The and peak values ​​are listed in Table 25 below.

[0355] Table 25. Internalization activity of HEPG2 cells

[0356] Monoclonal antibodies <![CDATA[EC 50 ]]> Peak Hu 52H5D3B8-7 0.7195 μg / mL 58555 Hu 52H5D3B8-8 0.7805μg / mL 62378 Y035 1.078 μg / mL 51973 Hu 52H5D3B8-7 / 8CDR with Y035FR 1.142 μg / mL 59223 Y035CDR with Hu 52H5D3B8-7FR 1.107 μg / mL 52157

[0357] The present disclosure is not limited to the scope of the specific embodiments described, which are intended to be a single illustration of the various aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It is apparent to those skilled in the art that various modifications and variations may be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to cover modifications and variations of the present disclosure as long as they fall within the scope of the appended claims and their equivalents.

[0358] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An anti-GPC3 antibody or an antigen-binding fragment thereof, which is specific to human GPC3 protein and comprises a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VLCDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 are the following amino acid sequences: SEQ ID NO: 13, 19, 15, 21, 17 and 18, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The VH is an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-39; the VL is an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-44.

3. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: (a) the VH is the amino acid sequence shown in SEQ ID NO: 36, and the VL is the amino acid sequence shown in SEQ ID NO: 41; (b) the VH has the amino acid sequence shown in SEQ ID NO: 37, and the VL has the amino acid sequence shown in SEQ ID NO: 43; (c) the VH is the amino acid sequence shown in SEQ ID NO: 36, and the VL is the amino acid sequence shown in SEQ ID NO: 42; (d) the VH has the amino acid sequence shown in SEQ ID NO: 36, and the VL has the amino acid sequence shown in SEQ ID NO: 43; (e) the VH is the amino acid sequence shown in SEQ ID NO: 36, and the VL is the amino acid sequence shown in SEQ ID NO: 44; (f) the VH has the amino acid sequence shown in SEQ ID NO: 37, and the VL has the amino acid sequence shown in SEQ ID NO: 41; (h) the VH is the amino acid sequence shown in SEQ ID NO: 37, and the VL is the amino acid sequence shown in SEQ ID NO: 42; (i) the VH has the amino acid sequence shown in SEQ ID NO: 37, and the VL has the amino acid sequence shown in SEQ ID NO: 44; (j) the VH has the amino acid sequence shown in SEQ ID NO: 38, and the VL has the amino acid sequence shown in SEQ ID NO: 41; (k) the VH is the amino acid sequence shown in SEQ ID NO: 38, and the VL is the amino acid sequence shown in SEQ ID NO: 42; (1) the VH is the amino acid sequence shown in SEQ ID NO: 38, and the VL is the amino acid sequence shown in SEQ ID NO: 43; (m) the VH is the amino acid sequence shown in SEQ ID NO: 38, and the VL is the amino acid sequence shown in SEQ ID NO: 44; (n) the VH is the amino acid sequence shown in SEQ ID NO: 39, and the VL is the amino acid sequence shown in SEQ ID NO: 41; (o) the VH has the amino acid sequence shown in SEQ ID NO: 39, and the VL has the amino acid sequence shown in SEQ ID NO: 42; (p) the VH is the amino acid sequence shown in SEQ ID NO: 39, and the VL is the amino acid sequence shown in SEQ ID NO: 43; or (q) the VH has the amino acid sequence shown in SEQ ID NO: 39, and the VL has the amino acid sequence shown in SEQ ID NO:

44.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that The antibody or antigen-binding fragment thereof is a bivalent Fab antibody, or a fragment selected from the group consisting of F(ab')2, F(ab)2, Fab', Fab, Fv and scFv.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The antibody or antigen-binding fragment thereof is humanized. 6 . A multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and one or more antibodies or antigen-binding fragments having binding specificity to a target antigen other than GPC3.

7. A chimeric antigen receptor (CAR), comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, a transmembrane domain, a co-stimulatory domain and a CD3ξ intracellular domain.

8. One or more polynucleotides encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1-3 or the CAR as described in claim 7.

9. A cell comprising the polynucleotide of claim 8.

10. An antibody-drug conjugate comprising: (a) the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; and (b) a conjugated moiety conjugated to the antibody or antigen-binding fragment thereof, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

11. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the CAR according to claim 7 or the antibody-drug conjugate according to claim 10, and a pharmaceutically acceptable carrier.

12. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the CAR according to claim 7, or the antibody-drug conjugate according to claim 10 in the preparation of a drug for treating GPC3-positive liver cancer.

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