Anti-glypican 3 antibody

Abstract

Anti-GPC3 antibodies are provided, including mouse antibodies, humanized antibodies, and those with further optimized CDR sequences. The antibodies showed higher cell-based binding efficiency than leading anti-GPC3 antibody candidates in clinical development. The present disclosure provides, in various embodiments, antibodies and antigen-binding fragments specific for human GPC3 protein. Experimental tests show that these newly identified antibodies outperformed the benchmark GC33 antibody. Methods and uses for treating cancer with the above antibodies or fragments thereof are also provided.

Description

[Background technology]

[0001] background 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, the N-terminal subunit has a size of approximately 40 kDa and the C-terminal subunit is approximately 30 kDa. Six glypicans (GPC1-6) have been identified in mammals. GPC3 plays an important role in regulating cell proliferation, differentiation, adhesion and migration. GPC3 interacts with both Wnt and Frizzled (FZD) to form complexes and induce downstream signaling. The core protein of GPC3 may act as a co-receptor or receiver of Wnt.

[0002] Glypican 3 immunostaining can be used to distinguish between hepatocellular carcinoma (HCC) and dysplastic changes during liver cirrhosis. GPC3 protein expression is found 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.

[0003] GPC3 is a promising therapeutic target for the treatment of cancers such as liver cancer. Several therapeutic anti-GPC3 antibodies have been developed, including GC33 and YP7. Some of these antibodies inhibit Wnt signaling in liver cancer cells. Chimeric antigen receptor (CAR) T cell immunotherapy is also in various stages of development to treat cancer. In mice with xenograft or orthotopic 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 [Means for solving the problem]

[0004] Abstract The present disclosure provides, in various embodiments, antibodies and antigen-binding fragments specific for human GPC3 protein. Experimental testing shows that these newly identified antibodies outperform the benchmark GC33 antibody.

[0005] Therefore, according to one embodiment of the present disclosure, there is provided an anti-glypican 3 (GPC3) antibody or a fragment thereof having specificity for human glypican 3 protein, comprising 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 comprise the amino acid sequences of SEQ ID NOs: 71 to 76, respectively.

[0006] 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.

[0007] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are, respectively: SEQ ID NOs: 13, 19, 15, 21, 17 and 18; SEQ ID NOs: 13, 19, 20, 21, 17 and 22; SEQ ID NOs: 13 to 18; SEQ ID NOs: 13, 23, 24, 21, 17 and 18; SEQ ID NOs: 25, 26, 20, 27, 17 and 28; SEQ ID NOs: 25, 29, 20, 27, 17 and 28; SEQ ID NOs: 25, 30, 15, 31, 17 and 32; SEQ ID NOs: 59, 60, 15, 21, 61, and 62; SEQ ID NOs: 63, 19, 15, 21, 64, and 62; or SEQ ID NOs: 13, 65, 15, 21, 66, and 62 The amino acid sequence of

[0008] 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 of SEQ ID NO: 15; the VL CDR1 comprises the amino acid sequence of 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.

[0009] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 respectively comprise the amino acid sequences of SEQ ID NOs: 13, 19, 15, 21, 17, and 18. In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-39, and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-44.

[0010] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 36, and the VL comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 37, and the VL comprises the amino acid sequence of SEQ ID NO: 43.

[0011] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:36 and the VL comprises the amino acid sequence of SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:36 and the VL comprises the amino acid sequence of SEQ ID NO:43. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:36 and the VL comprises the amino acid sequence of SEQ ID NO:44. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:37 and the VL comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:37 and the VL comprises the amino acid sequence of SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:37 and the VL comprises the amino acid sequence of SEQ ID NO:44. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:38 and the VL comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:38 and the VL comprises the amino acid sequence of SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:38 and the VL comprises the amino acid sequence of SEQ ID NO:43. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:38 and the VL comprises the amino acid sequence of SEQ ID NO:44. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:39 and the VL comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:39 and the VL comprises the amino acid sequence of SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:39 and the VL comprises the amino acid sequence of SEQ ID NO:43. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:39 and the VL comprises the amino acid sequence of SEQ ID NO:44.

[0012] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequence of SEQ ID NO: 59, 60, 15, 21, 61, and 62, respectively. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 53, and the VL comprises the amino acid sequence of SEQ ID NO: 54.

[0013] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequence of SEQ ID NO: 63, 19, 15, 21, 64, and 62, respectively. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 55, and the VL comprises the amino acid sequence of SEQ ID NO: 56.

[0014] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequence of SEQ ID NO: 13, 65, 15, 21, 66, and 62, respectively. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 57, and the VL comprises the amino acid sequence of SEQ ID NO: 58.

[0015] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of 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 of SEQ ID NO: 48, and the VL comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 48, and the VL comprises the amino acid sequence of SEQ ID NO: 52.

[0016] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:45 and the VL comprises the amino acid sequence of SEQ ID NO:51. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:45 and the VL comprises the amino acid sequence of SEQ ID NO:52. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:46 and the VL comprises the amino acid sequence of SEQ ID NO:51. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:46 and the VL comprises the amino acid sequence of SEQ ID NO:52. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:47 and the VL comprises the amino acid sequence of SEQ ID NO:51. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:47 and the VL comprises the amino acid sequence of SEQ ID NO:52. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:49 and the VL comprises the amino acid sequence of SEQ ID NO:51. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:49 and the VL comprises the amino acid sequence of SEQ ID NO:52. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:50 and the VL comprises the amino acid sequence of SEQ ID NO:51. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:50, and the VL comprises the amino acid sequence of SEQ ID NO:52.

[0017] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 13 to 18. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 1, and VL comprises the amino acid sequence of SEQ ID NO: 2.

[0018] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 23, 24, 21, 17, and 18, respectively. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 5, and the VL comprises the amino acid sequence of SEQ ID NO: 6.

[0019] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 25, 26, 20, 27, 17, and 28, respectively. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 7, and the VL comprises the amino acid sequence of SEQ ID NO: 8.

[0020] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequence of SEQ ID NO: 25, 29, 20, 27, 17, and 28, respectively. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 9, and the VL comprises the amino acid sequence of SEQ ID NO: 10.

[0021] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequence of SEQ ID NO: 25, 30, 15, 31, 17, and 32, respectively. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 11, and the VL comprises the amino acid sequence of SEQ ID NO: 12.

[0022] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 13; the VH CDR2 comprises the amino acid sequence of 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 VL CDR1 comprises the amino acid sequence of SEQ ID NO: 21; the VL CDR2 comprises the amino acid sequence of 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.

[0023] In some embodiments, the antibody or fragment thereof is humanized.

[0024] Also provided in one embodiment is an antibody-drug conjugate comprising: (a) an antibody of the present disclosure or a fragment thereof; and (b) a conjugated moiety conjugated to the antibody or fragment thereof, wherein the conjugated moiety is selected from a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0025] In one embodiment, a multispecific antibody is also provided that comprises an antibody of the present disclosure and one or more secondary antibodies or antigen-binding fragments that have binding specificity for a target antigen that is not GPC3.

[0026] Also provided is a chimeric antigen receptor (CAR) comprising an antibody or fragment thereof of the present disclosure, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.

[0027] In another embodiment, a polynucleotide encoding the antibody or fragment thereof of the present disclosure, a cell comprising the polynucleotide, and a composition comprising the antibody or fragment thereof and a pharma- ceutically acceptable carrier are provided. Methods and uses for treating cancer using the antibody or fragment thereof are also provided. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 shows the binding affinity of the 52H5D3B8 antibody to the GPC3 protein (human GPC3-his tag) as measured by Biacore.

[0029] [Figure 2A] 2A-B show that the affinity of the 52H5D3B8 antibody is higher than that of GC33 for binding to GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells or Huh-7 cells. [Figure 2B] 2A-B show that the affinity of the 52H5D3B8 antibody is higher than that of GC33 for binding to GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells or Huh-7 cells.

[0030] [Diagram 3] FIG. 3 shows the ELISA binding activity of 52H5D3B8 bound to human, mouse and cynomolgus GPC3.

[0031] [Figure 4] FIG. 4 shows that the affinity of 52H5D3B8-6# chimeric monoclonal antibody (mAb) for binding to GPC3-CHOK1 cells is higher than other optimized 52H5D3B8 chimeric mAbs.

[0032] [Diagram 5] FIG. 5 shows that the affinity of 52H5D3B8-6# chimeric mAb for binding to GPC3-CHOK1 cells, Hep3B cells and Huh-7 cells is the highest among 52H5D3B8 chimeric mAb and GC33 antibody.

[0033] [Figure 6] FIG. 6 shows the binding affinity of 52H5D3B8-6# antibody to GPC3 protein (human GPC3-his tag) as measured by Biacore 8K for the chimeric antibody.

[0034] [Figure 7] FIG. 7 shows the EC50 of each humanized antibody against GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells, and Huh-7 cells.

[0035] [Figure 8] FIG. 8 shows the EC50 of each humanized antibody against GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells, and Huh-7 cells.

[0036] [Figure 9]FIG. 9 shows binding of 52H5D3B8-6#-VH+VL, 52H5D3B8-6#-VH1+VL1, 52H5D3B8-6#-VH1+VL3, 52H5D3B8-6#-VH1+VL4, 52H5D3B8-6#-VH2+VL4, 52H5D3B8-6#-VH3+VL1 to recombinant GPC3 protein (human GPC3-his tag) when tested by Biacore using the capture method.

[0037] [Figure 10] FIG. 10 shows the results of antibody binding to human GPC3-overexpressing CHOK1 cells and HEPG2 cells.

[0038] [Figure 11] FIG. 11 shows the results of binding to human GPC3-overexpressing CHOK1 cells for affinity matured antibodies.

[0039] [Figure 12] FIG. 12 shows the results of antibodies inducing GPC3 antibody-mediated ADCC signaling.

[0040] [Figure 13] FIG. 13 shows that the antibodies activated NK cell function.

[0041] [Figure 14] FIG. 14 shows degranulation of antibody stimulated NK cells.

[0042] [Figure 15] FIG. 15 shows cytokine production in stimulated NK cells.

[0043] [Figure 16] FIG. 16 shows that the binding efficiency of all candidate humanized antibodies is comparable to that of the chimeric antibody on GPC3-CHOK1 cells and better than that of the chimeric antibody on Huh-7 cells.

[0044] [Figure 17]FIG. 17 shows the results of antibody binding to human GPC3-overexpressing CHOK1 cells, Huh-7 cells and HEPG2 cells.

[0045] [Figure 18] FIG. 18 shows the results of antibody internalization into HEPG2 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Detailed Description definition It should be noted that the term "a" or "an" entity refers to one or more of that entity, e.g., "an antibody" is understood to represent one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0047] As used herein, "antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen-binding fragment or single chain. Thus, the term "antibody" includes any protein or peptide containing molecule that includes at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, the complementarity determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, 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 a portion of a binding protein.

[0048] The term "antibody fragment" or "antigen-binding fragment" as used herein refers to a portion of an antibody, such as an F(ab') 2 , F(ab) 2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term "antibody fragment" also includes aptamers, spiegelmers, 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.

[0049] The term antibody encompasses a wide variety of classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses within these (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, such as IgG, IgM, IgA, IgG, or IgE, respectively.

[0050] Immunoglobulin subclass (isotype), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgG 5 and the like are well characterized and known to provide functional specialization. Modified versions of each of these classes and isotypes are readily discernible to one of skill in the art in light of the present disclosure and are therefore within the scope of the present disclosure. All immunoglobulin classes are expressly within the scope of the present disclosure, and the following discussion will generally refer to the IgG class of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides of approximately 23,000 daltons molecular weight and two identical heavy chain polypeptides of 53,000-70,000 molecular weight. The four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains on either side, beginning at the mouth of the "Y" and continuing through the variable region.

[0051] Antibodies, antigen-binding polypeptides thereof, variants or derivatives of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, epitope-binding fragments such as Fab, Fab' and F(ab')2, Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments comprising the VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies disclosed herein). Immunoglobulin or antibody molecules of the disclosure can be of 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 molecule.

[0052] As used herein, the term "chimeric antibody" should be taken to mean any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial, or modified, according to the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.

[0053] The antibody disclosed herein can be of any animal origin, including birds and mammals.Preferably, the antibody is human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibody.In some embodiments, the variable region can be of chondrichthyan origin (e.g., from shark).

[0054] As used herein, the term "recombinant" when referring to a polypeptide or polynucleotide, refers to a form of a polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which may be made by combining polynucleotides that do not normally occur together.

[0055] Hybridoma techniques can be carried out under different "stringency" conditions. Generally, low stringency hybridization reactions are carried out at about 40°C in about 10xSSC or equivalent ionic strength / temperature solutions. Medium stringency hybridization is typically carried out at about 50°C in about 6xSSC, and high stringency hybridization reactions are generally carried out at about 60°C in about 1xSSC. Hybridization reactions can also be carried out under "physiological conditions", which are well known to those skilled in the art. Non-limiting examples of physiological conditions are the temperature, ionic strength, pH and Mg2+ concentration normally found in cells. GPC-3 antibody

[0056] As demonstrated in the attached experimental examples, the present 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 showed higher cell-based binding efficiency than GC33, a potent anti-GPC3 antibody in clinical development. In addition, experimental data show that these new antibodies have higher activity in inducing internalization into cells. Due to such properties, these new antibodies are particularly suitable for use in, for example, antibody-drug conjugates (ADCs).

[0057] In addition, the sequence of the 52H5D3B8 antibody was further optimized by amino acid substitutions in the VH and VL CDR3. One such optimized antibody, for example, 52H5D3B8-6#, bound to GPC3-CHOK1 cells, Hep3B cells and Huh-7 cells with higher affinity than both the parent 52H5D3B8 chimeric mAb and the reference GC33 antibody.

[0058] Humanized versions of the 52H5D3B8-6# antibody were generated and compared to GC33 as well as another benchmark antibody, Y035 (as disclosed in US Patent Publication No. 20190046659). Interestingly, as shown in Figure 10 and Table 14, the humanized antibodies 52H5D3B8-6#-VH1+VL1 and 52H5D3B8-6#-VH2+VL3 retained the high affinity of the parent antibodies and significantly outperformed both GC33 and Y035.

[0059] The humanized antibody was subjected to further affinity maturation as part of efforts to further improve binding efficiency.As shown in Figure 11 and Table 16, encouragingly, all of the affinity matured antibodies showed improved binding efficiency compared with parent antibody (52H5D3B8-6#-VH2+VL3) and benchmark antibody GC33.And surprisingly, when targeting cells with moderate GPC3 expression level, the humanized antibody showed significantly higher ADCC signaling potency than GC33 (Figure 12 and Table 17).

[0060] These humanized and affinity matured antibodies were further tested for their ability to activate NK cells in the presence of GPC3 expressing cells. As shown in Figure 13 and Table 18, all of these new antibodies had higher activity than GC33. They induced higher production of IFN-γ and TNF-α in NK cells than GC33 (Figure 15).

[0061] The parent antibody 52H5D3B8 was also subjected to humanization, and two of the humanized versions, Hu 52H5D3B8-7 and Hu 52H5D3B8-8, were tested for their binding and internalization activities using GC33 and Y035 as references. As shown in Figures 17-18 and Tables 24-25, these humanized antibodies outperformed both references. Very interestingly, when the CDRs of Hu 52H5D3B8-7 / 8 were inserted into the framework of Y035, or when the framework regions of Hu 52H5D3B8-7 / 8 were used to support the CDRs of Y035, all the resulting grafted antibodies performed better than Y035 (Table 24). This clearly indicates the superiority of both the CDRs and framework sequences of these new antibodies.

[0062] These antibodies and their CDR sequences were found to be highly homologous to each other, suggesting that the CDR sequences may be interchangeable (see Table A). [Table A-1] [Table A-2]

[0063] Thus, in one embodiment, the 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 comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, e.g., those shown in Tables A, 1A-1F, 5A-5O, 8A, and 8B.

[0064] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 71, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 72, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 73, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 74, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 75, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 76.

[0065] SEQ ID NO: 71 is X 1 YE X 2 H, where X 1 can be D, R or G, and X 2 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, and X 2 can be A, E or G, and X 3 can be S or T, and X 4 can be D or G, and X 5 can be D, G, N or S, and X 6 can be N, S or T, and X 7 can be Q or S, and X 8 can be R, K or L. SEQ ID NO: 73 is X 1 YS X 2 AY, where X 1 can be F or Y, X 2 can be F or Y. SEQ ID NO: 74 is RS X 1 QS X 2 VH X 3 NG X 4 T.Y.L. X 5 where X 1 can be R or S, and X 2 can be L or P, and X 3 can be S, R or T, and X 4can be H or N, and X 5 can be H or Q. SEQ ID NO: 75 is KVSNRX 1 X 2 where X 1 can be F or Y, X 2 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, and X 2 can be Q or E, and X 3 can be S or T, and X 4 can be I or T.

[0066] 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 of 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.

[0067] 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.

[0068] In some embodiments, anti-GPC3 antibodies and antigen-binding fragments are also provided that compete with any of the antibodies disclosed herein in binding to human GPC3.In some embodiments, anti-GPC3 antibodies and antigen-binding fragments are also provided that bind to the same epitope as any of the antibodies disclosed herein.In some embodiments, anti-GPC3 antibodies and antigen-binding fragments are also provided that include the VH and VL CDR1, CDR2 and CDR3 of the antibodies disclosed herein.

[0069] In some embodiments, an antibody or antigen-binding fragment is provided that comprises the CDRs of antibody 52H5D3B8-6# or a humanized / affinity matured version thereof. In some embodiments, VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 59, and 63; VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 60, and 65; VH CDR3 comprises an amino acid sequence of SEQ ID NO: 15; VL CDR1 comprises an amino acid sequence of SEQ ID NO: 21; VL CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 61, 64, and 66; VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 62. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 19, 15, 21, 17, and 18, respectively. 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.

[0070] Examples of humanized antibodies or antigen-binding fragments derived from 52H5D3B8-6# include those shown in Table 8A. Examples also include those having 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.

[0071] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 36, and the VL comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 37, and the VL comprises the amino acid sequence of SEQ ID NO: 43.

[0072] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:36 and the VL comprises the amino acid sequence of SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:36 and the VL comprises the amino acid sequence of SEQ ID NO:43. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:36 and the VL comprises the amino acid sequence of SEQ ID NO:44. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:37 and the VL comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:37 and the VL comprises the amino acid sequence of SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:37 and the VL comprises the amino acid sequence of SEQ ID NO:44. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:38 and the VL comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:38 and the VL comprises the amino acid sequence of SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:38 and the VL comprises the amino acid sequence of SEQ ID NO:43. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:38 and the VL comprises the amino acid sequence of SEQ ID NO:44. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:39 and the VL comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:39 and the VL comprises the amino acid sequence of SEQ ID NO:42. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:39 and the VL comprises the amino acid sequence of SEQ ID NO:43. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:39 and the VL comprises the amino acid sequence of SEQ ID NO:44.

[0073] 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 of SEQ ID NOs: 59, 60, 15, 21, 61, and 62, respectively. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 53, and the VL comprises the amino acid sequence of SEQ ID NO: 54.

[0074] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequence of SEQ ID NO: 63, 19, 15, 21, 64, and 62, respectively. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 55, and the VL comprises the amino acid sequence of SEQ ID NO: 56.

[0075] In some embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequence of SEQ ID NO: 13, 65, 15, 21, 66, and 62, respectively. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 57, and the VL comprises the amino acid sequence of SEQ ID NO: 58.

[0076] In some embodiments, an antibody or antigen-binding fragment is provided that comprises the CDRs of antibody 52H5D3B8. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 19, 20, 21, 17, and 22, respectively. In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 45-50, and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 51, and 52.

[0077] Examples of humanized antibodies or antigen-binding fragments derived from 52H5D3B8 include those shown in Table 19. Examples also include those having 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.

[0078] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 48, and the VL comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 48, and the VL comprises the amino acid sequence of SEQ ID NO: 52.

[0079] In some embodiments, antibodies or antigen-binding fragments derived from antibody 52H5D3B8 are provided, such as those having alternative VH and VL CDR3s as shown in Tables 5A-5O. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 13; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 19; VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 20, 24, and 33; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 21; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 17; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 22, 28, 32, and 34.

[0080] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:35 and the VL comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:67 and the VL comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:3 and the VL comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:3 and the VL comprises the amino acid sequence of SEQ ID NO:68. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:69 and the VL comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:35 and the VL comprises the amino acid sequence of SEQ ID NO:68. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:67 and the VL comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:67 and the VL comprises the amino acid sequence of SEQ ID NO:68. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:3 and the VL comprises the amino acid sequence of SEQ ID NO:70. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:67 and the VL comprises the amino acid sequence of SEQ ID NO:70. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:35 and the VL comprises the amino acid sequence of SEQ ID NO:70. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:69 and the VL comprises the amino acid sequence of SEQ ID NO:68. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:69 and the VL comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:69 and the VL comprises the amino acid sequence of SEQ ID NO:70.

[0081] In some embodiments, an antibody or antigen-binding fragment is provided that comprises the CDRs of antibody 18C1D9. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequence of SEQ ID NO: 13-18. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 1, and VL comprises the amino acid sequence of SEQ ID NO: 2.

[0082] In some embodiments, an antibody or antigen-binding fragment is provided that comprises the CDRs of antibody 163E7D12. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 23, 24, 21, 17, and 18, respectively. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 5, and VL comprises the amino acid sequence of SEQ ID NO: 6.

[0083] In some embodiments, an antibody or antigen-binding fragment is provided that comprises the CDRs of antibody 172B4E9. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO: 25, 26, 20, 27, 17, and 28, respectively. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 7, and VL comprises the amino acid sequence of SEQ ID NO: 8.

[0084] In some embodiments, an antibody or antigen-binding fragment is provided that comprises the CDRs of antibody 152E9F7. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO: 25, 29, 20, 27, 17, and 28, respectively. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 9, and VL comprises the amino acid sequence of SEQ ID NO: 10.

[0085] In some embodiments, an antibody or antigen-binding fragment is provided that comprises the CDRs of antibody 171F9C5. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO: 25, 30, 15, 31, 17, and 32, respectively. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 11, and VL comprises the amino acid sequence of SEQ ID NO: 12. Antibody-drug conjugates

[0086] Preliminary data indicates that these new antibodies have higher activity in inducing internalization into cells. Such properties make these new antibodies particularly suitable for use, for example, in antibody-drug conjugates (ADCs).

[0087] In some embodiments, the antibody or fragment may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

[0088] In one embodiment, the antibody or fragment of the present disclosure is covalently linked to a drug moiety. The drug moiety may be a reactive group that has a conjugation point on the antibody, or may be modified to include the group. For example, the drug moiety may be attached by alkylation (e.g., at the epsilon-amino lysine or N-terminus of the antibody), reductive amination of oxidized carbohydrates, transesterification of hydroxyl and carboxyl groups, amidation of amino or carboxyl groups, and conjugation to thiols.

[0089] In some embodiments, the number of conjugated drug moieties per antibody molecule, p, ranges on average 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, p ranges on average from 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p ranges on average from 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, p ranges on average from about 1 to about 20, from about 1 to about 10, from about 2 to about 10, from about 2 to about 9, from about 1 to about 8, from about 1 to about 7, from about 1 to about 6, from about 1 to about 5, from about 1 to about 4, from about 1 to about 3, or from about 1 to about 2. In some embodiments, p ranges from about 2 to about 8, from about 2 to about 7, from about 2 to about 6, from about 2 to about 5, from about 2 to about 4, or from about 2 to about 3.

[0090] For example, if the chemical activation of the protein results in the formation of free thiol groups, the protein can be conjugated with a sulfhydryl reactive substance. In one embodiment, the agent is one that is substantially specific for free thiol groups. Such agents include, for example, maleimides, haloacetamides (e.g., iodo, bromo, or chloro), haloesters (e.g., iodo, bromo, or chloro), halomethylketones (e.g., iodo, bromo, or chloro), benzyl halides (e.g., iodide, bromide, or chloride), vinylsulfones, and pyridylthios.

[0091] The drug may be linked to the antibody or fragment by a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. Cleavable linkers are typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that can be cleaved by intracellular proteases, such as lysosomal or endosomal proteases. In exemplary embodiments, the linker may be a dipeptide linker, such as a valine-citrulline (val-cit), phenylalanine-lysine (phe-lys) linker, or a maleimidocapronic-valine-citrulline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PABA) linker. Another linker is sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (smcc). Sulfo-smcc conjugation occurs through a maleimide group, which reacts with sulfhydryls (thiols, -SH), while its sulfo-NHS ester is reactive to primary amines (such as those found in lysine and protein or peptide N-termini). Yet another linker is maleimidocaproyl (mc). Other suitable linkers include linkers that are hydrolyzable at a particular pH or pH range, such as hydrazone linkers. Additional suitable cleavable linkers include disulfide linkers. Linkers, such as mc linkers, can be covalently attached to antibodies to such an extent that the antibody must be degraded intracellularly to release the drug.

[0092] The linker may contain a group for linking to an antibody. For example, the linker may contain an amino, hydroxyl, carboxyl, or sulfhydryl reactive group (e.g., maleimide, haloacetamide (e.g., iodo, bromo, or chloro), haloester (e.g., iodo, bromo, or chloro), halomethylketone (e.g., iodo, bromo, or chloro), benzyl halide (e.g., iodide, bromide, or chloride), vinylsulfone, and pyridylthio).

[0093] In some embodiments, the drug moiety is a cytotoxic or cytostatic agent, an immunosuppressant, a radioisotope, a toxin, etc. The conjugate can be used to inhibit the growth of tumor or cancer cells, to cause apoptosis in tumor or cancer cells, or to treat cancer in patients. Thus, the conjugate can be used in various situations for the treatment of cancer in animals. The conjugate can be used to deliver drugs to tumor or cancer cells. Without being bound by theory, in some embodiments, the conjugate can bind to or associate with cancer cells that express GPC3, and the conjugate and / or drug can be taken up into tumor or cancer cells by receptor-mediated endocytosis.

[0094] Once inside the cell, one or more specific peptide sequences in the conjugate (e.g., in the linker) are hydrolytically cleaved by one or more tumor or cancer cell-associated proteases, resulting in the release of the drug. The released drug is then free to migrate into the cell and induce cytotoxic or cytostatic or other activity. In some embodiments, the drug is cleaved from the antibody outside the tumor or cancer cell, and then the drug penetrates the cell or acts on the cell surface.

[0095] Examples of drug moieties or payloads are DM1 (maytansine, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)- or N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), mc-MMAD (6-maleimidocaproyl-monomethylauristatin-D or N-methyl-L-valyl-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1 S)-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidinyl]-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valinamide), mc-MMAF (maleimidocaproyl-monomethylauristatin F or N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl mc-Val-Cit-PABA-MMAE (6-Maleimidocaproyl-ValcCit-(p-aminobenzyloxycarbonyl)-monomethylauristatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-valyl-N5-(αR,βR,2S)-β-methoxy-α-methyl-2-pyrrolidinepropanoyl-L-phenylalanine) DM1 is a derivative of the tubulin 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 maytansinoid or an auristatin.

[0096] The antibody or fragment may be conjugated or fused to a therapeutic agent which may include a detectable label, e.g., a radioactive label, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, combinations thereof, and other such agents known in the art.

[0097] An antibody can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

[0098] Metals that emit fluorescence, e.g. 152Antibodies can also be detectably labeled using Eu, or others of the lanthanide series. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).Techniques for conjugating various moieties to antibodies are well known and are described, for example, in 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 Developments in Antibodies See, "Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. (52:119-58 (1982)). multifunctional molecules

[0099] Antibodies or antigen-binding fragments specific for GPC3, as a molecule preferentially expressed on tumor cells, can be combined with second or higher ordinal fragments specific for immune cells to generate bispecific or multispecific antibodies.

[0100] In some embodiments, the immune cell is selected from the group consisting of a T cell, a B cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a phagocyte, a natural killer cell, an eosinophil, a basophil, and a mast cell.

[0101] 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.

[0102] Different types of bispecific antibodies are also provided. In some embodiments, each of the anti-GPC3 fragment and the second fragment is 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.

[0103] Bifunctional molecules are also provided that do not simply comprise antibodies or antigen-binding fragments.As tumor antigen targeting molecules, antibodies or antigen-binding fragments specific to GPC3, such as those described herein, can be combined with immunocytokines or ligands, optionally via peptide linkers.Linked immunocytokines 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.Such bifunctional molecules can combine immune checkpoint blocking effect with tumor site local immune regulation. Methods for Preparing Chimeric Antigen Receptors, Polynucleotides, and Antibodies

[0104] In one embodiment, a chimeric antigen receptor (CAR) is also provided, comprising the antibody or fragment thereof of the present disclosure as a targeting unit. In some embodiments, the CAR comprises the antibody or fragment thereof of the present disclosure, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.

[0105] The transmembrane domain can be designed to be fused to the extracellular domain, including antibody or fragment, optionally via hinge domain.It can also be fused to an intracellular domain, such as a costimulatory domain.In some embodiments, the transmembrane domain can include the natural transmembrane region of the costimulatory domain (e.g., the TM region of CD28T or 4-IBB used as costimulatory domain) or the natural transmembrane domain of the hinge region (e.g., the TM region of CD8alpha or CD28T used as hinge domain).

[0106] In some embodiments, a transmembrane domain may comprise a sequence that spans a cell membrane, but extends into the cytoplasm of the cell and / or into the extracellular space. For example, a transmembrane may comprise a membrane-spanning sequence that itself may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids that extend into the cytoplasm of the cell and / or into the extracellular space. Thus, a transmembrane domain may comprise a region that spans the membrane, and may further comprise amino acids that extend beyond the inner or outer surface of the membrane itself, and still be considered to be a "transmembrane domain".

[0107] In some embodiments, the transmembrane domain is fused to the cytoplasmic domain via a short linker. Optionally, a short peptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. A glycine-serine doublet (GS), a glycine-serine-glycine triplet (GSG), or an alanine-alanine-alanine triplet (AAA) would be a suitable linker.

[0108] In some embodiments, the CAR further comprises a costimulatory domain. In some embodiments, the costimulatory domain is located between the transmembrane domain and the activation domain. Examples of costimulatory domains include CD2, CD3 delta, CD3 epsilon, CD3 gamma, 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(Tactile), CD100(SEMA4D), CD103 (ITGAE), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD158A(KIR2DL1), CD158B1(KI R2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), C D158F2(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 costimulatory factor (ICOS), LFA-1 (CD 11a / CD 18), KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptor, Toll ligand receptor, and fragments or combinations thereof.

[0109] In some embodiments, the cytoplasmic portion of the CAR also comprises a signaling / activation domain. In one embodiment, the signaling / activation domain is a CD3 zeta domain or an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a CD3 zeta domain.

[0110] The present disclosure also provides an isolated polynucleotide or nucleic acid molecule that encodes the antibody, its variant or derivative, or CAR of the present disclosure.The polynucleotide of the present disclosure can encode the entire heavy and light chain variable region of the antigen-binding polypeptide, its variant or derivative on the same polynucleotide molecule or on separate polynucleotide molecules.In addition, the polynucleotide of the present disclosure can encode the part of the heavy and light chain variable region of the antigen-binding polypeptide, its variant or derivative on the same polynucleotide molecule or on separate polynucleotide molecules.

[0111] Methods for making antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptide of the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as 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 antigen administration but has an inactive endogenous locus. Exemplary techniques that can be used to make such antibodies are described in U.S. Patent Nos. 6,150,584, 6,458,592, and 6,420,140, ​​which are incorporated herein by reference in their entirety. Treatment and Use

[0112] As described herein, the antibodies, variants, derivatives, or antibody-drug conjugates of the disclosure can be used in certain treatment and diagnostic methods.

[0113] The present disclosure further relates to antibody-based therapy, including administering the disclosed antibodies, fragments, or antibody-drug conjugates to patients, such as animals, mammals, and humans, to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, the disclosed antibodies (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding the disclosed antibodies (including variants and derivatives thereof as described herein).

[0114] The antibody of the present disclosure can also be used to treat or inhibit cancer.As provided above, GPC3 can be overexpressed in tumor cells, particularly in liver, stomach, pancreas, esophagus, ovary and lung tumors.Inhibition of GPC3 has been shown to be useful in tumor treatment.

[0115] Thus, in some embodiments, a method for treating cancer in a patient in need thereof is provided.In one embodiment, the method entails administering to the patient an effective amount of the antibody, fragment, or antibody-drug conjugate of the present disclosure.In some embodiments, at least one of the cancer cells (e.g., stromal cells) in the patient overexpresses GPC3.

[0116] Cell therapy, for example, chimeric antigen receptor (CAR) T cell therapy, is also provided in the present disclosure. Suitable cells can be used that are transduced with a vector that encodes or contacts a CAR that includes (or alternatively is engineered to express) the anti-GPC3 antibody of the present disclosure. Thus, by such contact or manipulation, the cells can be introduced into a cancer patient that requires treatment. The cancer patient can have any type of cancer as disclosed herein. The cells (e.g., T cells) can be, for example, but are not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof.

[0117] In some embodiments, the cells are isolated from the cancer patient himself or herself. In some embodiments, the cells are provided by a donor or from a cell bank. If the cells are isolated from the cancer patient, unwanted immune responses can be minimized.

[0118] Non-limiting examples of cancer include bladder cancer, breast cancer, colon 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, pancreatic, esophageal, ovarian, and lung cancer.

[0119] Additional diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed and / or prognosed by the disclosed antibodies or variants, or derivatives thereof, include, but are not limited to, progression and / or metastasis of malignancies and related disorders, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, and solid tumors, including, but not limited to, sarcomas and Carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, 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, These include, but are not limited to, alveolar carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, 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.

[0120] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, its variant or derivative used, the patient's age, weight, general health, sex and diet, as well as the number of administrations, excretion rate, drug combinations, and the severity of the particular disease being treated.The judgment of such factors by medical practitioners is within the ordinary skill of the art.The amount will also depend 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 amount used can be determined by the principles of pharmacology and pharmacokinetics well known in the art.

[0121] Methods of administration of antibodies, fragments, or antibody-drug conjugates include, but are 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, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Thus, pharmaceutical compositions containing antigen-binding polypeptides of the present disclosure can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as powders, ointments, drops, or transdermal patches), bucally, or as oral sprays or nasal drops.

[0122] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0123] Administration can be systemic or local.In addition, it may be desirable to introduce the antibody of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection, and intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir such as an Ommaya reservoir.Pulmonary administration can also be utilized, for example, by using an inhaler or nebulizer, and a formulation containing an aerosolizing agent.

[0124] It may be desirable to administer an antigen-binding polypeptide or composition of the disclosure locally to the area in need of treatment, which can be achieved by, for example and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with wound dressing after surgery, by injection, by catheter, by suppository, or by a deposit, said deposit being of a porous, nonporous or gelatinous material, including membranes, e.g., sialastic membranes, or fibers. Preferably, when administering proteins, including antibodies, of the disclosure, care should be taken to use materials to which the proteins do not absorb.

[0125] The amount of the antibody, fragment, or antibody-drug conjugate of the present disclosure that will be effective in treating, inhibiting and preventing inflammatory, immune or malignant diseases, disorders or conditions can be determined by standard clinical techniques.In addition, in vitro assays can be used as necessary to help identify optimal dosage ranges.The exact dose to be used in the formulation will also depend on the route of administration and the severity of disease, disorder or condition, and should be determined according to the judgment of the practitioner and each patient's circumstances.Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0126] As a general proposition, the dosage of the antibody, fragment, or antibody-drug conjugate of the present disclosure administered to a patient is typically between 0.001 mg and 100 mg per kg of the patient's body weight, between 0.01 mg and 20 mg per kg of the patient's body weight, or between 0.5 mg and 10 mg per kg of the patient's body weight. Generally, human antibodies have a longer half-life in the human body than antibodies from other animal species due to the immune response to the foreign polypeptides. Thus, lower dosages and less frequent administration of human antibodies are often possible. Additionally, the dosage and frequency of administration of the antibodies of the present disclosure can be reduced by enhancing antibody uptake and tissue penetration (e.g., into the brain) by modifications such as lipidation.

[0127] In additional embodiments, the compositions of the present disclosure are administered in combination with cytokines. Cytokines that may 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.

[0128] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy. composition

[0129] The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of antibody, fragment, or antibody-drug conjugate and acceptable carrier. In some embodiments, the composition further comprises a second anti-cancer agent (e.g., immune checkpoint inhibitor).

[0130] In specific embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, for use in animals, and more particularly in humans. Moreover, a "pharmaceutically acceptable carrier" will generally be a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid of any type.

[0131] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be utilized as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates, if desired. 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 osmotic tonicity, such as sodium chloride or dextrose, are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions can be formulated as suppositories using traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of 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 the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation must be suitable for the method of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0132] In an embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Optionally, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, these ingredients are supplied either separately or mixed in unit dosage form, for example as a dry frozen powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent. If the composition is to be administered by injection, it can be dispensed using an injection bottle containing sterile water or saline of pharmaceutical grade. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0133] The compounds of the present disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. EXAMPLES

[0134] Example 1 Generation of mouse monoclonal antibodies against human GPC3 This example describes the generation of anti-human GPC3 mouse monoclonal antibodies using hybridoma technology.

[0135] Antigen: human GPC3-His protein.

[0136] Immunization: To generate mouse monoclonal antibodies targeting human GPC3, Balb / c and C57BL / 6 mice were first immunized with GPC3-His protein. The immunized mice were then boosted with GPC3-His protein. To select mice producing antibodies that bind to GPC3 protein, the sera of the immunized mice were subjected to antibody titer evaluation by ELISA. Briefly, microtiter plates were coated with 0.5 μg / ml human GPC3 protein in ELISA coating buffer, 100 μl / well, overnight at 4°C, and then blocked with 150 μl / well 1% BSA. Dilutions of serum from immunized mice were added to each well and incubated at 37°C for 1-2 hours. The plates were washed with PBS / Tween® and then incubated with anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) for 1 hour at 37°C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. After several rounds of immunization, the immune response was also tested by serum FACS against GPC3-CHOK1 cell line, and CHOK1 parental cell line served as a negative control. The resulting mice were used for fusion. Hybridoma supernatants were screened by ELISA.

[0137] Cell fusion: Fusion was performed by electrofusion. Fused cells were plated in 50 96-well plates per fusion.

[0138] Screening: Supernatants were screened by ELISA against recombinant human (rh) GPC3-His protein and the counterscreening antigen. Positive supernatants were then subjected to a confirmatory screen by cell-based binding to the GPC3-CHOK1 cell line.

[0139] Subcloning and screening: Positive primary clones from each fusion were subcloned by limiting dilution to ensure that the subclones were derived from a single parent cell. The subclones were screened using the same approach as the primary clones, and culture supernatants of positive clones were subjected to additional confirmatory screening by affinity ranking.

[0140] 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. [Table 1] [Table 1A] [Table 1B] [Table 1C] [Table 1D] [Table 1E] [Table 1F]

[0141] Example 2 Binding affinities of mouse and chimeric mAbs Affinity ranking of mouse monoclonal antibodies Binding of mouse monoclonal antibodies to human GPC3-his was tested on Biacore using the capture method. Antibodies were captured using a CM5 sensor chip. A single dose of human GPC3-his tagged protein was injected over the captured antibody for 2 minutes at a flow rate of 30 μl / min. The antigen was left to dissociate for 360 seconds. All experiments were performed on a Biacore 8K. Data analysis was performed using the Biacore 8K evaluation software.

[0142] As shown by the results in Table 2A, all mouse monoclonal antibodies showed high binding affinity to the recombinant GPC3 protein. [Table 2A]

[0143] The variable regions of murine antibodies 18C1D9, 52H5D3B8, 163E7D12, 172B4E9, 152E9F7 and 171F9C5 were then fused to human IgG1 to generate chimeric mAbs. Full kinetic affinity of 52H5D3B8 by Biacore

[0144] Binding of chimeric 52H5D3B8 mAb to human GPC3-his was tested on Biacore using the capture method. 52H5D3B8 mAb was captured using a CM5 sensor chip. Serial dilutions of human GPC3-his tagged protein were injected over the captured antibody for 2 min at a flow rate of 30 μl / min. Antigen was left to dissociate for 360 s. All experiments were performed on a Biacore 8K. Data analysis was performed using the Biacore 8K evaluation software.

[0145] As shown by the results in FIG. 1 and Table 2B below, the 52H5D3B8 antibody showed high binding affinity to the recombinant GPC3 protein. [Table 2B]

[0146] Example 3 Binding activity to GPC3 antigen FACS testing of chimeric antibodies Cell-based binding: Using FACS, the binding activity of chimeric mAbs (18C1D9, 52H5D3B8, 163E7D12, 172B4E9, 152E9F7 and 171F9C5) to CHOK1 cells overexpressing human GPC3 was assessed in comparison with the known GPC3 antibody GC33 (Chugai Pharmaceutical).

[0147] Briefly, GPC3-CHOK1 cells were washed with FACS buffer and divided into wells with 3-fold serial dilutions of chimeric mAb or GC33 mAb starting at 5 μg / mL for 40 minutes at 4° C. After washing with FACS buffer, Alexa Fluor® 647 AffiniPure goat anti-human IgG (H+L) was added to each well and incubated for 30 minutes at 4° C. Samples were washed twice with FACS buffer. Mean fluorescence intensity (MFI) of Alexa Fluor® 647 was evaluated. The EC50 of each mAb against GPC3-CHOK1 cells is listed in Table 3A below. The 52H5D3B8 chimeric antibody showed higher cell-based binding efficiency than GC33 (FIG. 2A and Table 3A). [Table 3A] FACS testing of 52H5D3B8 chimeric antibody

[0148] Cell-based binding: Using FACS, the binding activity of 52H5D3B8 chimeric mAb to CHOK1, HEPG2, Hep3B and Huh-7 cells in which human GPC3 was overexpressed was assessed in comparison with the known GPC3 antibody GC33 (Chugai Pharmaceutical).

[0149] Briefly, GPC3-CHOK1, HEPG2, Hep3B or Huh-7 cells were first incubated with Fc blocking reagent for 15 min at 4°C. The cells were washed with FACS buffer and split into wells with 52H5D3B8 chimeric mAb or GC33 mAb, 3-fold serially diluted starting at 30 μg / mL, for 60 min at 4°C. The cells were washed with FACS buffer and fixed with 2% PFA for 15 min at RT. After washing with FACS buffer, Alexa Fluor® 647 AffiniPure goat anti-human IgG was added to each well and incubated for 30 min at RT. The samples were washed twice with FACS buffer. The mean fluorescence intensity (MFI) of Alexa Fluor® 647 was evaluated by a MACSQuant Analyzer 16. As shown in Figure 2B, 52H5D3B8 bound to GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells or Huh-7 cells with comparable binding efficiency, which is higher than GC33. The EC50 of each mAb against GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells and Huh-7 cells is listed in Table 3B below. The 52H5D3B8 antibody showed a higher cell-based binding efficiency than GC33 (Figure 2B and Table 3B). [Table 3B] Cross-species activity

[0150] ELISA studies were performed to assess the binding of the chimeric antibodies to human, mouse and cynomolgus GPC3, respectively.

[0151] Briefly, microtiter plates were coated with 1 μg / ml human, mouse and cynomolgus GPC3 proteins in PBS, 100 μl / well, overnight at 4° C., then blocked with 150 μl / well 1% BSA. Three-fold dilutions of chimeric antibodies starting at 9 μg / ml were added to each well and incubated for 1 hour at 37° C. Plates were washed with PBS / Tween and then incubated with mouse anti-human IgG Fc antibody conjugated with horseradish peroxidase (HRP) for 30 minutes at 37° C. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. 52H5D3B8 antibody bound to human, cynomolgus GPC3 and mouse GPC3 (FIG. 3 and Table 4). [Table 4A] [Table 4B]

[0152] Example 4 Optimization of 52H5D3B8 chimeric mAb This example describes the optimization of 52H5D3B8 chimeric mAb by mutation of CDR3 to enhance the binding activity of the 52H5D3B8 chimeric mAb to human GPC3.

[0153] Mutation: Because the CDR3 domain of an antibody plays a crucial role in interacting with an antigen, mutations were added to the CDR3 domain of the 52H5D3B8 chimeric mAb. Both CDRH3 and CDRL3 were designed with two potential mutation sites each. Together, 15 potential combinations of CDRH3 and CDRL3 sequences are listed in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3]

Table 5A

Table 5B

Table 5C

Table 5D

Table 5E

Table 5F

Table 5H

Table 5I

Table 5J

Table 5K

Table 5L

Table 5M

Table 5N

Table 5O

[0154] Cell-based binding: FACS was used to assess the binding activity 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 mAbs to CHOK1 cells overexpressing human GPC3.

[0155] Briefly, GPC3-CHOK1 cells were first incubated with Fc blocking reagent for 15 min at 4°C. Cells were washed with FACS buffer and split into wells containing 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 mAbs in 3-fold serial dilutions starting at 30 μg / mL for 60 min at 4° C. The cells were washed with FACS buffer and fixed with 2% PFA for 15 minutes at RT. After washing with FACS buffer, Alexa Fluor® 647 AffiniPure goat anti-human IgG was added to each well and incubated for 30 minutes at RT. The samples were washed twice with FACS buffer. The mean fluorescence intensity (MFI) of Alexa Fluor® 647 was evaluated by MACSQuant Analyzer 16. As shown in Figure 4, 52H5D3B8-6# chimeric mAb bound to GPC3-CHOK1 cells with higher affinity than the others. The EC50 of each chimeric mAb against GPC3-CHOK1 cells is listed in Table 6 below. [Table 6-1] [Table 6-2] Affinity ranking of optimized antibodies by Biacore

[0156] The binding of the optimized antibody of 52H5D3B8 mAb to human GPC3-his was tested on Biacore using the capture method. A single dose or two doses of human GPC3-his tagged protein were injected onto the captured antibody. All experiments were performed on a Biacore 8K. Data analysis was performed using the Biacore 8K evaluation software.

[0157] As shown in the results in Table 6 above, the optimized antibody showed 10 -8 ~10 -10 In addition, 52H5D3B8-6# mAb showed the highest affinity for GPC3 protein. Optimized antibody cell binding to GPC3-CHOK1 cells

[0158] Cell-based binding: FACS was used to assess the binding activity of 52H5D3B8, 52H5D3B8-6# chimeric mAb, and GC33 antibody to CHOK1, Hep3B, and Huh-7 cells in which human GPC3 was overexpressed.

[0159] Briefly, GPC3-CHOK1 cells, Hep3B cells or Huh-7 cells were first incubated with Fc blocking reagent for 15 min at 4°C. Cells were washed with FACS buffer and split into wells with 52H5D3B8, 52H5D3B8-6# chimeric mAb or GC33 antibody, 3-fold serially diluted starting at 30 μg / mL, for 60 min at 4°C. Cells were washed with FACS buffer and fixed with 2% PFA for 15 min at RT. After washing with FACS buffer, Alexa Fluor® 647 AffiniPure goat anti-human IgG was added to each well and incubated for 30 min at RT. Samples were washed twice with FACS buffer. Mean fluorescence intensity (MFI) of Alexa Fluor® 647 was evaluated by MACSQuant Analyzer 16. As shown in Figure 5, 52H5D3B8-6#chimeric mAb bound to GPC3-CHOK1 cells, Hep3B cells and Huh-7 cells with higher affinity than both 52H5D3B8 chimeric mAb and GC33 antibody. The EC50 of each mAb against GPC3-CHOK1 cells, Hep3B cells and Huh-7 cells is listed in Table 7 below. [Table 7]

[0160] Example 5 Humanization of chimeric antibody 52H5D3B8-6# The 52H5D3B8-6# variable regions were utilized to generate a humanized mAb. In the first step of this process, the amino acid sequences of the 52H5D3B8-6# VH and VL were compared to available databases of human Ig gene sequences to find the best overall match to the human germline Ig gene sequence.

[0161] For the light chain of 52H5D3B8-6#, IGKV2-29 * 02 was the best matched germline, and for the heavy chain of 52H5D3B8-6#, IGHV1-46 *01 was selected as the humanized backbone. Then, a humanized 52H5D3B8-6# CDR-grafted antibody was designed, with CDRL1, L2 and L3 substituted for IGKV2-29. * 02 framework sequence, and CDRH1, H2 and H3 were grafted onto IGHV1-46 * 01 framework sequence. A 3D model was then generated to determine the amino acids in the original mouse FR region sequence that are essential for antibody binding and conformation. Based on the 52H5D3B8-6# CDR-grafted antibody sequence, three additional humanized heavy chains and three additional humanized light chains were created. For the heavy chain, L, K, I, A, F and T in the framework were involved in back mutations. For the light chain, K and F in the framework were involved in back mutations.

[0162] The amino acid sequences of the humanized antibodies are listed in Table 8 below. [Table 8A] [Table 8B]

[0163] Then, four humanized heavy chains and four humanized light chains were synthesized and cloned into pcDNA3.4 vector respectively. After extracting plasmid, four heavy chains and four light chains were paired and expressed in HEK293 cells. By pairing human VH and human VL, 16 humanized antibodies were produced (see Table 9). [Table 9]

[0164] Example 6 Antigen-binding properties of humanized antibodies Affinity ranking of humanized antibodies by Biacore To evaluate the antigen binding activity, the affinity of the chimeric antibody was measured by Biacore 8K. Serial dilutions of human GPC3-his tagged protein were injected over the captured chimeric antibody 52H5D3B8-6#-VH+VL for 3 minutes at a flow rate of 30 μl / min. The antigen was allowed to dissociate for 720 seconds. Among the multiple detection doses, the two concentrations with the closest affinity to that of the chimeric antibody were selected for the affinity detection of the 16 humanized antibodies. As shown in Figure 6 and Table 10, all 16 humanized antibodies showed similar affinities, which are comparable to the chimeric antibodies. [Table 10-1] [Table 10-2] Binding to CHOK1, HEPG2, Hep3B and Huh-7 cells overexpressing human GPC3

[0165] To evaluate the antigen-binding properties, the humanized antibodies were analyzed by FACS for their binding to CHOK1, HEPG2, Hep3B and Huh-7 cells in which human GPC3 was overexpressed. Briefly, GPC3-CHOK1, HEPG2, Hep3B and Huh-7 cells were first incubated with Fc blocking reagent for 15 min at 4°C. The cells were washed with FACS buffer and divided into wells with humanized antibodies serially diluted 4-fold starting at 10 μg / mL for 60 min at 4°C. The cells were washed with FACS buffer and fixed with 2% PFA for 15 min at RT. After washing with FACS buffer, Alexa Fluor® 647 AffiniPure goat anti-human IgG was added to each well and incubated for 30 min at RT. The MFI of Alexa Fluor® 647 was evaluated by MACSQuant Analyzer 16. The 16 humanized antibodies were divided into two plates. As shown in Figures 7-8, the EC50 of each humanized antibody against GPC3-CHOK1 cells, HEPG2 cells, Hep3B cells and Huh-7 cells is listed in Tables 11-12 below. By comparing the EC50 of 52H5D3B8-6#-VH+VL and the humanized antibodies in the same plate, all the humanized antibody candidates showed binding efficiency equivalent to that of the chimeric antibody. [Table 11-1] [Table 11-2] [Table 12] Full kinetic affinity of humanized antibodies by Biacore

[0166] Binding of humanized antibodies to recombinant GPC3 protein (human GPC3-his tag) was tested on Biacore using the capture method. 52H5D3B8-6#-VH+VL, 52H5D3B8-6#-VH1+VL1, 52H5D3B8-6#-VH1+VL3, 52H5D3B8-6#-VH1+VL4, 52H5D3B8-6#-VH2+VL4, 52H5D3B8-6#-VH3+VL1, 52H5D3B8-6#-VH3+VL3 mAbs were captured using a CM5 sensor chip. Serial dilutions of human GPC3-his tag protein were injected over the captured antibody for 2 minutes at a flow rate of 30 μl / min. Antigen was left to dissociate for 360 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore 8K Evaluation software. Results are shown in Figure 9 and Table 13 below. [Table 13] Binding to CHOK1 and HEPG2 cells overexpressing human GPC3

[0167] Using FACS, the binding activity of 52H5D3B8-6#-VH1+VL1 and 52H5D3B8-6#-VH2+VL3 against CHOK1 cells and HEPG2 cells overexpressing human GPC3 was evaluated in comparison with GC33 (Chugai Pharmaceutical) and Y035 (Carsgen Therapeutics, US Patent Publication No. 20190046659). The EC50 of each antibody against GPC3-CHOK1 cells and HEPG2 cells is listed in Table 14 below.

[0168] As shown in Figure 10, both 52H5D3B8-6#-VH1+VL1 and 52H5D3B8-6#-VH2+VL3 bound to GPC3-CHOK1 and HEPG2 cells with comparable affinities, which were higher than those of the benchmark mAbs. [Table 14]

[0169] Example 7 Affinity maturation of humanized antibody 52H5D3B8-6# To enhance the affinity of the humanized antibody of 52H5D3B8-6#, affinity maturation was performed. Briefly, four phage libraries containing single or two saturation mutations in the CDR region were constructed. Three candidates with unique mutations in the CDR were obtained by two rounds of screening using solid or liquid panning. The CDRs of these candidates were grafted into the 52H5D3B8-6#-VH2+VL3 framework to generate antibodies for further binding and functional evaluation.

[0170] The amino acid sequences of the variable regions of the affinity maturation candidates grafted into the framework are listed in Table 15 below. [Table 15] [Table 15A] [Table 15B] [Table 15C] Binding to CHOK1 cells overexpressing human GPC3

[0171] To evaluate the antigen-binding properties, affinity matured antibodies were analyzed for their binding to CHOK1 cells overexpressing human GPC3 by FACS as previously described. The EC50 of each antibody against GPC3-CHOK1 cells is listed in Table 16 below.

[0172] As shown in FIG. 11, all affinity matured antibodies showed improved binding efficiency on GPC3-CHOK1 cells compared to the parental 52H5D3B8-6#-VH2+VL3 and the benchmark antibody GC33. [Table 16]

[0173] Example 8 Humanized GPC3 antibody mediated ADCC signaling To evaluate the antigen binding-mediated antibody-dependent cellular cytotoxicity (ADCC) signaling of 52H5D3B8-6#-VH1+VL1, 52H5D3B8-6#-VH2+VL3 and three affinity matured antibodies compared with GC33, ADCC signaling reporter assay was constructed. Briefly, full-length human CD16a with high affinity allotype (176V) binding to Fc region was introduced into Jurkat-NFAT luciferase reporter cell line (referred to as Jurkat-hCD16a-NFAT). The expression of luciferase gene is under the control of NFAT response element in this cell line. Jurkat-hCD16a-NFAT cell line allowed us to evaluate GPC3 binding-mediated ADCC signaling by examining downstream NFAT signaling. This reporter cell line was co-cultured with several GPC3-positive tumor cell lines, including HEPG2 or Huh-7 hepatocellular carcinoma cells, which have high or moderate GPC3 expression levels, respectively. Luciferase-based chemiluminescence could be detected by ONE-Glo™ Luciferase Assay System (Promega, Cat. No. E6130) and Envision multilabel plate reader (PerkinElemer).

[0174] As shown in FIG. 12 and Table 17, both 52H5D3B8-6#-VH1+VL1 and 52H5D3B8-6#-VH2+VL3 showed efficient GPC3 binding-mediated ADCC signaling to HEPG2 cells and Huh-7 cells, which are higher than GC33. In addition, both 52H5D3B8-19#-VH2+VL3 and 52H5D3B8-20#-VH2+VL3 showed better EC50 of ADCC signaling to HEPG2 cells than parent 52H5D3B8-6#-VH2+VL3, while 52H5D3B8-18#-VH2+VL3 showed better maximum ADCC signaling potency than other antibodies. Notably, in Huh-7 cells, which showed moderate GPC3 expression level, all humanized antibodies showed enhanced maximum ADCC signaling potency than GC33. [Table 17]

[0175] Example 9 Humanized GPC3 antibody mediated tumor killing by NK cells In this example, the functional activity of a GPC3 humanized antibody was tested. 9.1 NK cytotoxicity to target cells mediated by GPC3 humanized antibody

[0176] To evaluate whether GPC3-mediated ADCC signaling activation leads to the cytotoxic effect of NK cells against target cells, a human primary NK cell-mediated cytotoxicity assay was configured. Briefly, fresh human primary NK cells (CD3-CD56+) were isolated from buffy coats of healthy donors by negative selection with magnetic beads (Miltenyi, Cat. No. 130-092-657). The purity of the isolated NK cells was monitored by FACS analysis, and was typically higher than 90%. The isolated NK cells were then rested in complete culture medium for 24 h and used as effector cells. HEPG2 or Huh-7 human hepatocellular carcinoma cell lines were used as target cells. The NK cell-mediated cytotoxicity assay was performed by following the manufacturer's protocol of the Cytotoxicity Detection Kit (Roche, Cat. No. 11644793001). Effector cells were added at 5.0 × 10 cells per well. 4 1.0 x 10 cells in a round-bottom 96-well microplate at a density of 4 The cells were co-cultured with target cells (E / T ratio = 5:1). Antibodies were serially diluted 5-fold starting at 50 nM and added to the corresponding wells. After 5 h (for HEPG2 cells) or 24 h (for Huh-7 cells) incubation at 37°C, lactate dehydrogenase (LDH), which represents cell viability in each well, was detected by a cytotoxicity detection kit. The absorbance was measured at 492 nm. Calculation formula: cytotoxicity (%) = (effector-target cell mix-effector cell control-low control) / (high control-low control) x 100. Four-parameter nonlinear regression curve fitting was performed by GraphPad Prism 9.

[0177] As shown in Figure 13 and Table 18, both 52H5D3B8-6#-VH1+VL1 and 52H5D3B8-6#-VH2+VL3 showed efficient GPC3 antibody-mediated NK cytotoxicity to HEPG2 cells and Huh-7 cells, which is higher than that of GC33.

[0178] In addition, both 52H5D3B8-19#-VH2+VL3 and 52H5D3B8-20#-VH2+VL3 showed comparable GPC3 antibody-mediated NK cytotoxicity to HEPG2 cells as the parental 52H5D3B8-6#-VH2+VL3. Interestingly, in Huh-7 cells, which showed moderate GPC3 expression levels, both 52H5D3B8-19#-VH2+VL3 and 52H5D3B8-20#-VH2+VL3 showed better EC50 than the parental 52H5D3B8-6#-VH2+VL3. [Table 18] 9.2 Degranulation of NK cells stimulated by GPC3 humanized antibody

[0179] To investigate the ability of the GPC3 humanized antibody to stimulate NK cell degranulation, the expression level of the degranulation marker CD107a was analyzed in NK cells. Briefly, 2.5×10 cells per well were cultured. 4 Human primary NK cells generated as described above as effector cells at a density of 2.5 × 10 cells / well were cultured in round-bottom 96-well microplates containing HEPG2. 4 The cells were co-incubated with the target cells (E / T ratio = 1:1). Antibodies were serially diluted 5-fold starting at 50 nM and added to the corresponding wells. After 4 hours of incubation at 37°C, the cells were harvested and washed with ice-cold staining buffer (0.5% BSA in PBS). Anti-CD107a antibody (BioLegend, Cat. No. 328608) was diluted in staining buffer at 1 μL per well and incubated with the cells for 40 minutes at 4°C in the dark. The samples were then washed and fixed with 2% PFA. The cells were analyzed with a flow cytometer MACSQuant® Analyze 16 (Miltenyi Biotech). The data were analyzed with Flowjo 10.0 software. Four-parameter nonlinear regression curve fitting was performed by GraphPad Prism 9.

[0180] As shown in FIG. 14, all GPC3 humanized antibodies stimulated more effective CD107a upregulation in the presence of HEPG2 cells than GC33. 9.3 NK cell cytokine production stimulated by humanized GPC3 antibody

[0181] To investigate the ability of the humanized antibodies to stimulate NK cell cytokine production in the presence of target cells, intracellular IFN-γ and TNF-α in NK cells were analyzed by FACS staining. Briefly, human primary NK cells were cultured at 2.5 × 10 cells per well. 4 2.5 x 10 cells containing HEPG2 as effector cells in a round-bottom 96-well microplate prepared as above at a density of 1 x 10 cells. 4 The NK cells were co-cultured with target cells (E / T ratio = 1:1). 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. Antibodies were serially diluted 5-fold starting at 50 nM and added to the corresponding wells. After 4 h of incubation at 37 °C, the samples were washed and intracellularly stained according to the procedure in the Foxp3 / Transcription Factor Staining Buffer Set (ThermoFisher, Cat. No. 00-5523-00). Anti-IFN-γ antibody (BioLegend, Cat. No. 506510) and anti-TNF-α antibody (BD Biosciences, Cat. No. 562783) were diluted in the recommended staining buffer at 1 μL per well. The cells were analyzed with a flow cytometer MACSQuant® Analyze 16 (Miltenyi Biotech). Data were analyzed with Flowjo 10.0 software. Four-parameter nonlinear regression curve fitting was performed with GraphPad Prism 9.

[0182] As shown in FIG. 15, all of the GPC3 humanized antibodies induced greater IFN-γ and IFN-α production in NK cells than GC33 in the presence of HEPG2 cells.

[0183] Example 10 Humanization of chimeric antibody 52H5D3B8 The 52H5D3B8 variable region genes were utilized to generate humanized mAbs. In the first step of this process, the 52H5D3B8 VH and VL or VK amino acid sequences were compared to available databases of human Ig gene sequences to find the best overall match to the human germline Ig gene sequence.

[0184] In short, IGKV2-29 * 02 and IGHV1-2 * 02 was used as the framework for the heavy and light chains, respectively. Then, a 3D model was generated to determine the amino acids in the original mouse FR region sequence that are essential for antibody binding and conformation. Based on the 52H5D3B8 CDR-grafted antibody sequence, five additional humanized heavy chains and one additional light chain were created. For the heavy chain, I, K, A, L, and T in the framework were involved in back mutations. For the light chain, F in the framework was involved in back mutations.

[0185] The amino acid and nucleotide sequences for portions of the humanized antibodies are listed in Table 19 below. [Table 19-1] [Table 19-2]

[0186] Then, six humanized heavy chains and two humanized light chains were synthesized and cloned into pcDNA3.4 vector respectively. After extracting plasmid, six heavy chains and two light chains were combined and expressed in HEK293 cells. By combining human VH and human VL, 12 humanized antibodies were produced (see Table 20). [Table 20]

[0187] Example 11 Antigen-binding properties of humanized antibodies Affinity ranking of humanized antibodies by Biacore To evaluate the antigen binding activity, the affinity of the chimeric antibody was measured by Biacore 8K. The two concentrations with the closest affinity to that of the chimeric antibody were selected for the affinity detection of the 12 humanized antibodies. Human GPC3-his tag protein was injected over the captured antibody for 3 minutes at a flow rate of 30 μl / min. The antigen was allowed to dissociate for 720 seconds. As shown in Table 21, all 12 humanized antibodies showed similar affinity to the chimeric antibody. [Table 21] Binding to CHOK1 and Huh-7 cells overexpressing human GPC3

[0188] To evaluate the antigen-binding properties, the humanized antibodies were analyzed by FACS for their binding to CHOK1 and Huh-7 cells in which human GPC3 was overexpressed. Briefly, GPC3-CHOK1 and Huh-7 cells were first incubated with Fc blocking reagent for 15 min at 4°C. The cells were washed with FACS buffer and divided into wells with humanized antibodies serially diluted 4-fold starting at 10 μg / mL for 60 min at 4°C. The cells were washed with FACS buffer and fixed with 2% PFA for 15 min at RT. After washing with FACS buffer, Alexa Fluor® 647 AffiniPure goat anti-human IgG was added to each well and incubated for 30 min at RT. The MFI of Alexa Fluor® 647 was evaluated by MACSQuant Analyzer 16. Because different plates may affect the EC50 of 52H5D3B8-VH+VL, the 12 humanized antibodies were divided into two plates. The EC50 of each humanized antibody for GPC3-CHOK1 cells and Huh-7 cells is listed in Tables 22-23 below. As shown in Figure 16, all humanized antibody candidates showed binding efficiency equivalent to that of the chimeric antibody for GPC3-CHOK1 cells and better than that of the chimeric antibody for Huh-7 cells. [Table 22] [Table 23] Binding to CHOK1, Huh-7 and HEPG2 cells overexpressing human GPC3

[0189] The binding activity of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 to human GPC3 expressed on CHOK1, Huh-7 and HEPG2 cells was evaluated in comparison with the benchmark antibodies GC33 and Y035 using FACS. To evaluate the CDR and framework properties of Hu 52H5D3B8-7 Ab, two chimeric antibodies were generated. The antibody named "Hu 52H5D3B8-7 / 8 CDR with Y035 FR" contains Hu 52H5D3B8-7 CDR and Y035 FR. The antibody named "Y035 CDR with Hu 52H5D3B8-7 FR" represents the chimeric antibody with Y035 CDR grafted to Hu 52H5D3B8-7 FR. The EC50 of each antibody against GPC3-CHOK1 cells, Huh-7 cells and HEPG2 cells is listed in Table 24 below.

[0190] As shown in Figure 17, both Hu 52H5D3B8-7 and Hu 52H5D3B8-8 bound to GPC3-CHOK1 cells, Huh-7 cells and HEPG2 cells with comparable affinity, which is higher than benchmark antibodies GC33 and Y035.More importantly, both Hu 52H5D3B8-7 / 8 CDR with Y035 FR and Y035 CDR with Hu 52H5D3B8-7 FR showed higher affinity than Y035, demonstrating that the CDR and framework of Hu 52H5D3B8-7 contributed to the superior binding ability of Y035. [Table 24]

[0191] Example 12 Internalization activity of humanized GPC3 antibody against tumor cell lines expressing GPC3 Using cell-based internalization, the internalization activity of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 was evaluated in comparison to Y035 on HEPG2 cells. To evaluate the CDR and framework properties of the Hu 52H5D3B8-7 Ab, the two antibodies (Hu 52H5D3B8-7 / 8 CDRs with Y035 FR, and Y035 CDRs with Hu 52H5D3B8-7 FR) were also evaluated.

[0192] PHAb thiol dye is a pH sensor dye with very low fluorescence at pH>7, resulting in a dramatic increase in fluorescence when / if it is internalized into endosomes or lysosomes where the pH is approximately 6.3 or 4.7, respectively. Briefly, pHAb thiol dye-labeled α-hIgG secondary antibody (10 μg / mL) was incubated with Ab (20 μg / mL) for 30 min. After incubation, the mixed Ab was serially diluted 2-fold. The diluted mixture was then diluted with 2 × 10 4 HEPG2 cells were added to a 96-well assay plate pre-seeded with each well. After 48 hours of incubation, the fluorescent signal was captured with a Multimode plate reader (Envision® 2105).

[0193] As shown in Figure 18, both Hu 52H5D3B8-7 and Hu 52H5D3B8-8 showed higher internalization efficacy than other mAbs.In addition, Hu 52H5D3B8-7 / 8 CDRs with Y035 FR showed higher internalization efficacy (higher top and similar EC50) than Y035, indicating that the CDRs of Hu-52H5D3B8-7 contribute to enhanced target-mediated internalization compared to Y035.The EC50 and top of each mAb are listed in Table 25 below. [Table 25] * * *

[0194] The present disclosure is not limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit and scope of the present disclosure. Thus, the present disclosure encompasses modifications and variations of the present disclosure, provided that they fall within the scope of the appended claims and their equivalents.

[0195] 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-glypican 3 (GPC3) antibody or a fragment thereof having specificity for human glypican 3 protein, comprising a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, and a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 71 to 76, respectively.

2. 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; the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 22, 28, 32, 34 and 62; The antibody or fragment thereof according to claim 1.

3. the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are, respectively: SEQ ID NOs: 13, 19, 15, 21, 17 and 18; SEQ ID NOs: 13, 19, 20, 21, 17 and 22; SEQ ID NOs: 13-18; SEQ ID NOs: 13, 23, 24, 21, 17 and 18; SEQ ID NOs: 25, 26, 20, 27, 17 and 28; SEQ ID NOs: 25, 29, 20, 27, 17 and 28; SEQ ID NOs: 25, 30, 15, 31, 17 and 32; SEQ ID NOs: 59, 60, 15, 21, 61, and 62; SEQ ID NOs: 63, 19, 15, 21, 64, and 62; or SEQ ID NOs: 13, 65, 15, 21, 66, and 62 The antibody or fragment thereof according to claim 2, comprising the amino acid sequence:

4. 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 of SEQ ID NO: 15; the VL CDR1 comprises the amino acid sequence of 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; the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 62; The antibody or fragment thereof according to claim 1.

5. The antibody or fragment thereof of claim 4, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 19, 15, 21, 17, and 18, respectively.

6. The antibody or fragment thereof according to claim 5, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35 to 39, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 40 to 44.

7. (a) the VH comprises the amino acid sequence of SEQ ID NO: 36 and the VL comprises the amino acid sequence of SEQ ID NO: 41; (b) the VH comprises the amino acid sequence of SEQ ID NO: 37 and the VL comprises the amino acid sequence of SEQ ID NO: 43; (c) the VH comprises the amino acid sequence of SEQ ID NO: 36 and the VL comprises the amino acid sequence of SEQ ID NO: 42; (d) the VH comprises the amino acid sequence of SEQ ID NO: 36 and the VL comprises the amino acid sequence of SEQ ID NO: 43; (e) the VH comprises the amino acid sequence of SEQ ID NO: 36 and the VL comprises the amino acid sequence of SEQ ID NO: 44; (f) the VH comprises the amino acid sequence of SEQ ID NO: 37 and the VL comprises the amino acid sequence of SEQ ID NO: 41; (g) the VH comprises the amino acid sequence of SEQ ID NO: 37 and the VL comprises the amino acid sequence of SEQ ID NO: 42; (h) the VH comprises the amino acid sequence of SEQ ID NO: 37 and the VL comprises the amino acid sequence of SEQ ID NO: 44; (i) the VH comprises the amino acid sequence of SEQ ID NO: 38 and the VL comprises the amino acid sequence of SEQ ID NO: 41; (j) the VH comprises the amino acid sequence of SEQ ID NO: 38 and the VL comprises the amino acid sequence of SEQ ID NO: 42; (k) the VH comprises the amino acid sequence of SEQ ID NO: 38 and the VL comprises the amino acid sequence of SEQ ID NO: 43; (l) the VH comprises the amino acid sequence of SEQ ID NO: 38 and the VL comprises the amino acid sequence of SEQ ID NO: 44; (m) the VH comprises the amino acid sequence of SEQ ID NO: 39 and the VL comprises the amino acid sequence of SEQ ID NO: 41; (n) the VH comprises the amino acid sequence of SEQ ID NO: 39 and the VL comprises the amino acid sequence of SEQ ID NO: 42; (o) the VH comprises the amino acid sequence of SEQ ID NO: 39 and the VL comprises the amino acid sequence of SEQ ID NO: 43; or (p) the VH comprises the amino acid sequence of SEQ ID NO: 39 and the VL comprises the amino acid sequence of SEQ ID NO: 44; The antibody or fragment thereof according to claim 5.

8. The antibody or fragment thereof of claim 4, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 59, 60, 15, 21, 61, and 62, respectively.

9. The antibody or fragment thereof of claim 8, wherein the VH comprises the amino acid sequence of SEQ ID NO: 53 and the VL comprises the amino acid sequence of SEQ ID NO:

54.

10. The antibody or fragment thereof of claim 4, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 63, 19, 15, 21, 64, and 62, respectively.

11. The antibody or fragment thereof of claim 10, wherein the VH comprises the amino acid sequence of SEQ ID NO: 55 and the VL comprises the amino acid sequence of SEQ ID NO:

56.

12. The antibody or fragment thereof of claim 4, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 65, 15, 21, 66, and 62, respectively.

13. The antibody or fragment thereof of claim 12, wherein the VH comprises the amino acid sequence of SEQ ID NO: 57 and the VL comprises the amino acid sequence of SEQ ID NO:

58.

14. The antibody or fragment thereof of claim 3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 19, 20, 21, 17, and 22, respectively.

15. The antibody or fragment thereof of claim 14, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 45 to 50, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 51 and 52.

16. (a) the VH comprises the amino acid sequence of SEQ ID NO: 48 and the VL comprises the amino acid sequence of SEQ ID NO: 51; (b) the VH comprises the amino acid sequence of SEQ ID NO: 48 and the VL comprises the amino acid sequence of SEQ ID NO: 52; (c) the VH comprises the amino acid sequence of SEQ ID NO: 45 and the VL comprises the amino acid sequence of SEQ ID NO: 51; (d) the VH comprises the amino acid sequence of SEQ ID NO: 45 and the VL comprises the amino acid sequence of SEQ ID NO: 52; (e) the VH comprises the amino acid sequence of SEQ ID NO: 46 and the VL comprises the amino acid sequence of SEQ ID NO: 51; (f) the VH comprises the amino acid sequence of SEQ ID NO: 46 and the VL comprises the amino acid sequence of SEQ ID NO: 52; (g) the VH comprises the amino acid sequence of SEQ ID NO: 47 and the VL comprises the amino acid sequence of SEQ ID NO: 51; (h) the VH comprises the amino acid sequence of SEQ ID NO: 47 and the VL comprises the amino acid sequence of SEQ ID NO: 52; (i) the VH comprises the amino acid sequence of SEQ ID NO: 49 and the VL comprises the amino acid sequence of SEQ ID NO: 51; (j) the VH comprises the amino acid sequence of SEQ ID NO: 49 and the VL comprises the amino acid sequence of SEQ ID NO: 52; (k) the VH comprises the amino acid sequence of SEQ ID NO: 50 and the VL comprises the amino acid sequence of SEQ ID NO: 51; or (l) the VH comprises the amino acid sequence of SEQ ID NO: 50, and the VL comprises the amino acid sequence of SEQ ID NO: 52; The antibody or fragment thereof described in claim 14.

17. The antibody or fragment thereof according to claim 3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 13 to 18, respectively.

18. The antibody or fragment thereof of claim 17, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1 and the VL comprises the amino acid sequence of SEQ ID NO:

2.

19. The antibody or fragment thereof of claim 3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 23, 24, 21, 17, and 18, respectively.

20. The antibody or fragment thereof of claim 19, wherein the VH comprises the amino acid sequence of SEQ ID NO: 5 and the VL comprises the amino acid sequence of SEQ ID NO:

6.

21. The antibody or fragment thereof of claim 3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 25, 26, 20, 27, 17, and 28, respectively.

22. The antibody or fragment thereof of claim 21, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

23. The antibody or fragment thereof of claim 3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 25, 29, 20, 27, 17, and 28, respectively.

24. The antibody or fragment thereof of claim 23, wherein the VH comprises the amino acid sequence of SEQ ID NO: 9 and the VL comprises the amino acid sequence of SEQ ID NO:

10.

25. The antibody or fragment thereof of claim 3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 25, 30, 15, 31, 17, and 32, respectively.

26. The antibody or fragment thereof of claim 25, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO:

12.

27. the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 13; the VH CDR2 comprises the amino acid sequence of 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 of SEQ ID NO:21; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 17; the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 22, 28, 32 and 34; The antibody or fragment thereof according to claim 2.

28. (a) the VH comprises the amino acid sequence of SEQ ID NO: 35 and the VL comprises the amino acid sequence of SEQ ID NO: 4; (b) the VH comprises the amino acid sequence of SEQ ID NO: 67 and the VL comprises the amino acid sequence of SEQ ID NO: 4; (c) the VH comprises the amino acid sequence of SEQ ID NO: 3 and the VL comprises the amino acid sequence of SEQ ID NO: 40; (d) the VH comprises the amino acid sequence of SEQ ID NO: 3 and the VL comprises the amino acid sequence of SEQ ID NO: 68; (e) the VH comprises the amino acid sequence of SEQ ID NO: 69 and the VL comprises the amino acid sequence of SEQ ID NO: 4; (f) the VH comprises the amino acid sequence of SEQ ID NO: 35 and the VL comprises the amino acid sequence of SEQ ID NO: 68; (g) the VH comprises the amino acid sequence of SEQ ID NO: 67 and the VL comprises the amino acid sequence of SEQ ID NO: 40; (h) the VH comprises the amino acid sequence of SEQ ID NO: 67 and the VL comprises the amino acid sequence of SEQ ID NO: 68; (i) the VH comprises the amino acid sequence of SEQ ID NO: 3 and the VL comprises the amino acid sequence of SEQ ID NO: 70; (j) the VH comprises the amino acid sequence of SEQ ID NO: 67 and the VL comprises the amino acid sequence of SEQ ID NO: 70; (k) the VH comprises the amino acid sequence of SEQ ID NO: 35 and the VL comprises the amino acid sequence of SEQ ID NO: 70; (l) the VH comprises the amino acid sequence of SEQ ID NO: 69 and the VL comprises the amino acid sequence of SEQ ID NO: 68; (m) the VH comprises the amino acid sequence of SEQ ID NO: 69 and the VL comprises the amino acid sequence of SEQ ID NO: 40; or (n) the VH comprises the amino acid sequence of SEQ ID NO: 69, and the VL comprises the amino acid sequence of SEQ ID NO: 70; 28. The antibody or fragment thereof of claim 27.

29. The antibody or fragment thereof according to claim 1, which 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.

30. The antibody or fragment thereof of claim 1, which is humanized.

31. A multispecific antibody comprising the antibody or fragment thereof described in claim 1 and one or more antibodies or antigen-binding fragments having binding specificity for a target antigen other than GPC3.

32. A chimeric antigen receptor (CAR) comprising the antibody or fragment thereof according to claim 1, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.

33. One or more polynucleotides encoding the antibody or fragment thereof of claim 1 or the CAR of claim 31.

34. A cell comprising the polynucleotide of claim 33.

35. (a) the antibody or fragment thereof according to claim 1; and (b) a conjugation moiety conjugated to the antibody or fragment thereof. wherein said conjugation moiety is selected from a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

36. A composition comprising the antibody or fragment thereof according to any one of claims 1 to 30, the CAR according to claim 32, or the antibody-drug conjugate according to claim 35, and a pharmaceutically acceptable carrier.

37. A composition for treating cancer in a patient in need thereof, comprising the antibody or fragment thereof according to any one of claims 1 to 30, the CAR according to claim 32, or the antibody-drug conjugate according to claim 35.

38. Use of the antibody or fragment thereof according to any one of claims 1 to 30, the CAR according to claim 32, or the antibody-drug conjugate according to claim 35, for the preparation of a medicament for treating cancer.

Images