Human monoclonal antibodies against phosphatidylinositol proteoglycan-3

Abstract

We describe an antibody or antigen binding fragment thereof comprising a cloned heavy chain variable region (VH) sequence and a light chain variable region (VL) sequence selected from the group consisting of 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1, said antibody or antigen binding fragment thereof being capable of binding specifically to glypican-3 (GPC-3) (Genbank accession number: NP004475.1), or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

Description

[0001] This divisional patent application is a divisional application of the patent application for invention titled "Composition" with the application number 202080036994.6, which was filed on March 22, 2020. Technical Field

[0002] The present invention relates to the fields of medicine, cell biology, molecular biology, and genetics. The present invention relates to the field of medicine. Background Art

[0003] Unmet need for new treatment strategies for hepatocellular carcinoma (HCC)

[0004] Hepatocellular carcinoma (HCC) is the sixth most common cancer globally, with a reported global incidence of 854,000 in 2015.

[0005] More than half of the global HCC cases occur in developing countries due to the prevalent chronic hepatitis B virus (HBV) infection. HCC is also the fourth leading cause of cancer-related mortality.

[0006] The vast majority of patients are usually not suitable for curative treatment after diagnosis due to their advanced stage of the disease, and the systemic treatment outcomes for HCC are disappointing.

[0007] The main targeted therapeutic agent for HCC is Nexavar (Sorafenib, Bayer), a small molecule inhibitor for multiple protein kinases. However, even with extensive use in HCC treatment, Sorafenib only shows negligible clinical benefits and a low response rate, and the improvement in overall survival is very limited. Since the launch of Sorafenib in 2007, no new drugs have entered the market, indicating that it is very difficult to develop new treatment methods for HCC.

[0008] Therefore, there is still an urgent medical need to discover new treatment strategies for HCC. Summary of the Invention

[0009] According to a first aspect of the present invention, we provide an antibody or an antigen-binding fragment thereof, which comprises a cloned heavy chain variable region (V H ) sequence selected from the following and a light chain variable region (V L)Sequences: 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1. The antibody or antigen-binding fragment thereof is capable of specifically binding to glypican-3 (GPC-3) (Genbank accession number: NP_004475.1). It can be a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with such a sequence.

[0010] The antibody or antigen-binding fragment can be such that V H and V L sequences are selected from: SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO:9 and SEQ ID NO:10; SEQ ID NO:11 and SEQ ID NO:12; SEQ ID NO:13 and SEQ ID NO:14; SEQ ID NO:15 and SEQ ID NO:16; SEQ ID NO:17 and SEQ ID NO:18; SEQ ID NO:19 and SEQ ID NO:20; SEQ ID NO:21 and SEQ ID NO:22; SEQ ID NO:23 and SEQ ID NO:24; SEQ ID NO:25 and SEQ ID NO:26; SEQ ID NO:27 and SEQ ID NO:28; SEQ ID NO:29 and SEQ ID NO:30; SEQ ID NO:31 and SEQ ID NO:32; SEQ ID NO:33 and SEQ ID NO:34; SEQ ID NO:35 and SEQ ID NO:36; SEQ ID NO:37 and SEQ ID NO:38; SEQ ID NO:39 and SEQ ID NO:40; SEQ ID NO:41 and SEQ ID NO:42; SEQ ID NO:43 and SEQ ID NO:44; SEQ ID NO:45 and SEQ ID NO:46; and SEQ ID NO:47 and SEQ ID NO:48.

[0011] The antibody or antigen-binding fragment thereof is capable of binding to an epitope of human glypican-3 (GPC-3) with an affinity of 55 nM or less, 50 nM or less, 45 nM or less, 40 nM or less, 35 nM or less, 30 nM or less, 25 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less. The affinity can be measured by EC 50 determined by ELISA.

[0012] The antibody or antigen-binding fragment thereof is capable of binding to a cell line selected from the group consisting of HepG2 (GPC-3 高 ) and Hep3B (GPC-3 中 ).

[0013] The antibody or antigen-binding fragment thereof is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) of GPC-3-expressing cells in the presence of natural killer (NK) cells.

[0014] The antibody or antigen-binding fragment thereof may be a humanized antibody comprising a human constant region.

[0015] The antibody or antigen-binding fragment thereof may include a monoclonal antibody, a humanized monoclonal antibody, Fv, F(ab’), F(ab’) 2 or single-chain Fv (scFv) fragment. It may comprise a single-chain Fv fragment that contains a V H sequence and a V L sequence.

[0016] The antibody or antigen-binding fragment thereof may be a monoclonal antibody selected from the group consisting of: 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1.

[0017] The antibody or antigen-binding fragment thereof further comprises a heavy-chain variable region (V H ) sequence and a light-chain variable region (V L ) sequence capable of binding to CD3 (Genbank accession number: NM_000733.4).

[0018] The antibody or antigen-binding fragment is also capable of specifically binding to CD-3 or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0019] The antibody or antigen-binding fragment can be a heavy chain variable region (V H ) sequence and a light chain variable region (V L ) sequence capable of binding to CD3, which comprise the sequences shown in SEQ ID NO:97 and SEQ ID NO:98.

[0020] The antibody or antigen-binding fragment is capable of activating T cells.

[0021] According to a second aspect of the present invention, there is provided a chimeric antigen receptor (CAR) comprising: (a) an antigen-binding fragment as described above; (b) a transmembrane domain; and (c) a co-stimulatory intracellular signaling domain. We also provide chimeric antigen receptor T cells (CAR T) expressing such chimeric antigen receptor.

[0022] According to a third aspect of the present invention, we provide a nucleic acid capable of encoding the antibody or antigen-binding fragment as described above. The nucleic acid may comprise a sequence selected from the group consisting of: SEQ ID NO:49 and SEQ ID NO:50; SEQ ID NO:51 and SEQ ID NO:52; SEQ ID NO:53 and SEQ ID NO:54; SEQ ID NO:55 and SEQ ID NO:56; SEQ ID NO:57 and SEQ ID NO:58; SEQ ID NO:59 and SEQ ID NO:60; SEQ ID NO:61 and SEQ ID NO:62; SEQ ID NO:63 and SEQ ID NO:64; SEQ ID NO:65 and SEQ ID NO:66; SEQ ID NO:67 and SEQ ID NO:68; SEQ ID NO:69 and SEQ ID NO:70; SEQ ID NO:71 and SEQ ID NO:72; SEQ ID NO:73 and SEQ ID NO:74; SEQ ID NO:75 and SEQ ID NO:76; SEQ ID NO:77 and SEQ ID NO:78; SEQ ID NO:79 and SEQ ID NO:80; SEQ ID NO:81 and SEQ ID NO:82; SEQ ID NO:83 and SEQ ID NO:84; SEQ ID NO:85 and SEQ ID NO:86; SEQ ID NO:87 and SEQ ID NO:88; SEQ ID NO:89 and SEQ ID NO:90; SEQ ID NO:91 and SEQ ID NO:92; SEQ ID NO:93 and SEQ ID NO:94; and SEQ ID NO:95 and SEQ ID NO:96. The nucleic acid may be comprised in an expression vector.

[0023] As a fourth aspect of the present invention, there is provided a host cell comprising such a nucleic acid. The host cell may include Chinese hamster ovary (CHO) or HEK293 cells.

[0024] According to a fifth aspect of the present invention, we provide a pharmaceutical composition comprising: the antibody or antigen-binding fragment as described above, CAR, CAR T, nucleic acid or host cell, and a pharmaceutically acceptable excipient, diluent or carrier.

[0025] A sixth aspect of the present invention provides a compound comprising the antibody or its antigen-binding fragment as described above, wherein the antibody or its antigen-binding fragment is linked to a cytotoxic agent. The linkage may be through a cleavable linker.

[0026] The compound may comprise an antibody-drug conjugate.

[0027] In a seventh aspect of the invention, there is provided an antibody, antigen-binding fragment, CAR, CAR T, nucleic acid, host cell, pharmaceutical composition, compound or antibody-drug conjugate as described above, for use in a method of treating, preventing or alleviating cancer such as hepatocellular carcinoma (HCC).

[0028] According to an eighth aspect of the invention, there is provided a method for preparing an antibody or antigen-binding fragment, the method comprising expressing a nucleic acid as described above in a host cell (such as the host cell described above), and optionally isolating the expressed antibody or antigen-binding fragment.

[0029] According to a ninth aspect of the invention, there is provided the use of such an antibody, antigen-binding fragment, CAR, CAR T, nucleic acid, pharmaceutical composition, compound or antibody-drug conjugate in the preparation of a medicament for treating, preventing or alleviating cancer such as hepatocellular carcinoma (HCC).

[0030] According to a tenth aspect of the invention, there is provided a method for detecting hepatocellular carcinoma (HCC) cells, the method comprising detecting the regulation of the expression, amount or activity of glypican-3 (GPC-3) in or of the cells with an antibody or antigen-binding fragment as described above.

[0031] As an eleventh aspect of the invention, there is provided a method for diagnosing hepatocellular carcinoma (HCC) in an individual, wherein the method comprises: detecting the regulated expression level of glypican-3 (GPC-3) in the cells of the individual by contacting the cells with an antibody or antigen-binding fragment as described above; comparing with the expression level of glypican-3 in the cells of an individual known not to have hepatocellular carcinoma (HCC); wherein an increase in the expression level of glypican-3 indicates that the individual has or may have hepatocellular carcinoma (HCC).

[0032] According to a twelfth aspect of the invention, there is provided a diagnostic kit for hepatocellular carcinoma (HCC), the kit comprising an antibody or antigen-binding fragment or nucleic acid as described above and instructions for use.

[0033] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, and immunology, which are within the capabilities of those of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O’D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies: A Laboratory Manual: Portable Protocol NO.I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-314-2), 1855. Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, NY, Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3. Each of these routine texts is incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A to Figure 1 D are graphs showing the binding ELISA of 24 anti-GPC-3 IgG 1 clones. The binding ELISA experiments were performed in 3 batches ( Figure 1 A, Figure 1 B, Figure 1 C), and humanized GC33 IgG 1 was used as a reference control. The calculated EC 50 values are shown in the table ( Figure 1 D).

[0035] Figure 2 A to Figure 2 D are graphs showing the binding of 24 anti-GPC-3 clones to GPC-3 高 HepG2 cells, GPC-3 中 Hep3B cells, and GPC-3 阴性 SK-Hep1 cells at different antibody concentrations. Flow cytometry analysis was performed in 4 batchesFigure 2 A, Figure 2 B, Figure 2 C, Figure 2 D), with humanized GC33 IgG 1 as a reference control.

[0036] Figure 3 is a graph showing the cross-reactive binding ELISA of anti-GPC-3 IgG 1 clones with human GPC-3 recombinant protein, cynomolgus monkey GPC-3 recombinant protein, and mouse GPC-3 recombinant protein. Humanized GC33 IgG 1 was used as a plotting control.

[0037] Figure 4 A and Figure 4 B are graphs showing the binding of 24 anti-GPC-3 clones at an antibody concentration of 20 nM to mouse GPC-3-transduced Hepa1-6 cells (Hepa1-6-mGPC-3) and parental Hepa1-6 cells, with humanized GC33 IgG 1 as a reference control.

[0038] Figure 4 A is a histogram of fluorescence intensity showing the mGPC-3 expression levels on Hepa1-6-mGPC-3 cells and parental Hepa1-6 cells.

[0039] Figure 4 B is a graph in which cells positively stained with different anti-GPC-3 antibodies are gated and the percentage (%) is calculated.

[0040] Figure 5 is a graph showing the specific killing of GPC-3 1 HepG2 cells in an ADCC assay in the presence of different anti-GPC-3 IgG 高 clones. The shown is the mean cytotoxicity ± SD of triplicate measurements. Humanized GC33 IgG 1 was used as a reference control.

[0041] Figure 6 A to Figure 6 C are graphs showing the specific cell lysis of anti-GPC-3 CAR-T cells against GPC-3 高 HepG2 cells and GPC-3 阴性 SK-Hep1 cells, with a 48-hour time-course measurement using the xCelligence impedance assay. Detection was performed in 3 batches using CAR-T cells generated from different donors ( Figure 6 A, Figure 6 B, Figure 6C). The average percent cell lysis ± SD for three replicates is shown. Anti-GPC-3 CAR-T cells generated from the scFv sequence using the murine GC33 clone were used as a reference control.

[0042] Figure 7 A to Figure 7 C are graphs showing ELISA measurements of interferon-γ and IL-2 secreted by anti-GPC-3 CAR-T cells after co-culture with GPC-3 高 HepG2 cells or GPC-3 阴性 SK-Hep1 cells for 24 hours and 48 hours. CAR-T cells generated from different donors were tested in 3 batches ( Figure 7 A, Figure 7 B, Figure 7 C). Anti-GPC-3 CAR-T cells generated from the scFv sequence using the murine GC33 clone were used as a reference control.

[0043] Figure 8 A shows a schematic diagram of an antibody in which a knob-in-hole mutation was introduced to promote correct heavy chain pairing and a LALA mutation was introduced to remove Fcγ receptor binding; Figure 8 B to Figure 8 D are graphs showing 5 anti-GPC-3 bispecific T cell engager antibodies binding to ( Figure 8 B) GPC-3 阳性 HepG2 cells, ( Figure 8 C) GPC-3 阴性 SK-Hep1 cells, and ( Figure 8 D) CD3 阳性 human T cells at different antibody concentrations. The top subgraph shows the mean fluorescence intensity of anti-GPC-3 bispecific T cell engager antibodies binding to different cells, while the bottom subgraph shows the percentage of cells positively stained with anti-GPC-3 bispecific T cell engager antibodies.

[0044] Figure 9 A to Figure 9 H are graphs showing activation of human T cells by anti-GPC-3 bispecific T cell engager antibodies when co-cultured with GPC-3 阳性 HepG2 cells. In the presence of different anti-GPC-3 bispecific T cell engager antibodies at 3 different concentrations, naive human T cells were co-cultured with ( Figure 9 A) HepG2 cells or ( Figure 9 B) SK-Hep1 cells for 24 hours, and then the T cells were harvested and stained with antibodies against T cell activation markers CD25 and CD69 for flow cytometry analysis. The T cell activator TransAct TMT cells processed by (Miltenyi Biotec) were used as positive controls.

[0045] Figure 9 C and Figure 9 D are graphs showing the percentage of T cells activated by different anti-GPC-3 bispecific T cell engaging antibodies at 3 different concentrations, calculated and expressed as the total percentage of CD25+CD69- cells, CD25-CD69+ cells, and CD25+CD69+ cells, after co-culture with ( Figure 9 C)GPC-3 阳性 HepG2 cells or ( Figure 9 D)GPC-3 阴性 SK-Hep1 cells.

[0046] Figure 9 E and Figure 9 F are graphs showing the ELISA measurement of interferon-γ secreted by human T cells after co-culture with ( Figure 9 E)GPC-3 阳性 HepG2 cells or ( Figure 9 F)GPC-3 阴性 SK-Hep1 cells for 24 hours and addition of different anti-GPC-3 bispecific T cell engaging antibodies at 3 different concentrations.

[0047] Figure 9 G and Figure 9 H are graphs showing the ELISA measurement of IL-2 secreted by human T cells after co-culture with ( Figure 9 G)GPC-3 阳性 HepG2 cells or ( Figure 9 H)GPC-3 阴性 SK-Hep1 cells for 24 hours and addition of different anti-GPC-3 bispecific T cell engaging antibodies at 3 different concentrations.

[0048] Figure 10 A and Figure 10 B are graphs showing T cell-mediated cytotoxicity of GPC-3 阳性 HepG2 cells by anti-GPC-3 bispecific T cell engaging antibodies.

[0049] Figure 10 A is a graph showing the percentage of lysis of GPC-3 阳性 HepG2 cells mediated by activated human T cells after treatment with 5 different anti-GPC-3 bispecific T cell engaging antibodies (clone 1C4, 1D2, 2B5, 4H1, and 5C4), with time-course measurement up to 72 hours using xCelligence impedance detection. Shown is the mean % ± SD of lysis in two wells.

[0050] Figure 10 Panel B shows the percentage of lysis of 5 different anti-GPC-3 bispecific T cell engager antibodies at different concentrations at the detection endpoint (72 hours).

[0051] Figure 11 A through Figure 11 Panel C shows the anti-GPC-3 CAR T cells blocking GPC-3 阳性 in the xenograft model. Two million GPC-3 阳性 Hep3B cells were subcutaneously injected into the right flanks of NSG mice. Four days later, the mice were randomly grouped (4-5 mice per group), and 10 million anti-GPC-3 CAR T cells (clone 5C4 scFv) or mock T cells were intravenously injected into these mice via the tail vein. Eighty-four days after the initial tumor inoculation, all tumor-free mice from the CAR T group were re-challenged subcutaneously with 1 million Hep3B cells. The tumor size ( Figure 11 A) and body weight ( Figure 11 B) of each mouse were measured and recorded every 3-7 days. ( Figure 11 C) Six days after injection of CAR T cells, the sera of mock T group and CAR T group mice were analyzed using the Luminex assay, and the levels of representative cytokines were shown.

[0052] Figure 12 A through Figure 12 Panel D shows the anti-GPC-3 CAR T cells inhibiting the growth of Hep3B xenografts in vivo.

[0053] Figure 12 Panel A shows the percentage of CAR expression on different anti-GPC-3 CAR T cells detected by flow cytometry analysis.

[0054] Figure 12 Panel B shows the percentage of lysis of GPC-3 阳性 Hep3B cells and GPC-3 阴性 SK-Hep1 cells mediated by anti-GPC-3 CAR T cells, and time-course measurements were performed for up to 72 hours using the xCelligence impedance assay.

[0055] Figure 12 Panels C and Figure 12 D show the injection of 2 million GPC-3 阳性Figure of Hep3B cells injected subcutaneously into the right flank of NSG mice. Twenty-six days later, the mice were grouped according to tumor size (3 - 4 mice per group). Ten million different anti-GPC-3 CAR T cells (clone 1D2, clone 4H1, and clone 5C4) or mock T cells were injected into these mice via the tail vein. The tumor size of each mouse was measured and recorded every 3 - 4 days ( Figure 12 C) and body weight ( Figure 12 D).

[0056] Figure 13 A to Figure 13 F are figures showing the use of anti-GPC-3 CAR T cells in combination with anti-PD1 antibody in vivo to eradicate large Hep3B xenografts.

[0057] On day 0, two million GPC-3 阳性 Hep3B cells were injected subcutaneously into the right flank of NSG mice. Twenty-four days later, the mice were grouped according to measurable tumor size (4 mice per group). Ten million anti-GPC-3 CAR T cells (clone 5C4) or mock T cells were injected into these mice via the tail vein. Ten days after CAR T cell injection (day 34), anti-PD1 IgG 4 antibody was injected intraperitoneally into all mice, for a total of 5 injections, at intervals of 3 - 5 days. The tumor size of each mouse was measured and recorded every 3 - 4 days ( Figure 13 A) and body weight ( Figure 13 B).

[0058] Figure 13 C is a survival curve graph showing mice from the mock T group (dashed line) and the CAR T group. Death was defined when the mice died spontaneously or when the tumor size in the mice exceeded 2000 mm 3 .

[0059] Figure 13 D is a figure showing the analysis of peripheral immune cells by flow cytometry 16 days after CAR T cell injection and the population of human T cells boxed.

[0060] Figure 13 E is a graph calculating the circulating concentrations of total peripheral T cells (CD3+), peripheral CAR T cells (CAR+CD3+), peripheral CD4 T cells (CD4+CD3+), and peripheral CD8 T cells (CD8+CD3+) and comparing mice from the mock T group and the CAR T group.

[0061] Figure 13 F is a figure showing the analysis of sera from mice in the mock T group and the CAR T group by Luminex assay and showing representative cytokine levels.

[0062] Figure 14 A to Figure 14 K are the figures showing that anti-GPC-3 CAR T cells did not induce toxicity in vivo. Anti-GPC-3 CAR T cells were generated using 9 different anti-GPC-3 antibody scFv sequences. On day 0, 10 million mock T cells ( Figure 14 B) or different anti-GPC-3 CAR T cells ( Figure 14 C to Figure 14 K) were intravenously injected into mixed-sex NSG mice (M: male, F: female). A group of mice that received no treatment ( Figure 14 A) was also included as healthy controls. The body weight of each mouse was measured and recorded every 3 - 7 days.

[0063] Figure 15 A to Figure 15 F are the figures showing that anti-GPC-3 murine CAR T cells inhibited the growth of MC38-mGPC-3 xenografts in vivo.

[0064] Figure 15 A is the figure showing the establishment of MC38 cells stably expressing murine GPC-3 protein (MC38-mGPC-3) by transducing MC38 parental cells with a lentivirus-based vector. The expression of murine GPC-3 protein was verified by staining with anti-GPC-3 IgG (clone 5C4) followed by flow cytometry analysis.

[0065] Figure 15 B is the figure showing the percentage of murine CAR expression on anti-GPC-3 murine CAR T cells detected by flow cytometry analysis.

[0066] Figure 15 C is the figure showing the percentage of cell lysis of MC38-mGPC-3 cells mediated by anti-GPC-3 murine CAR T cells (clone 5C4), with time-course measurements up to 96 hours using xCelligence impedance detection.

[0067] Figure 15 D and Figure 15 E are the figures showing the subcutaneous injection of 800,000 MC38-mGPC-3 cells into the right flanks of WT C57BL / 6 mice. After 12 days, the mice were grouped according to the measurable tumor size (4 - 5 mice per group). 8 million anti-GPC-3 murine CAR T cells (clone 5C4) or mock T cells were intravenously injected into the tail veins of these mice. The tumor size ( Figure 15 D) and body weight ( Figure 15 E) of each mouse were measured and recorded every 3 - 4 days.

[0068] Figure 15 Figure F shows that anti-GPC-3 mouse CAR T cells did not induce toxicity in WT mice. On day 0, 8 million anti-GPC-3 mouse CAR T cells (clone 5C4) or mock T cells were intravenously injected into WT C57BL / 6 mice. The body weight of each mouse was measured and recorded every 3 - 7 days.

[0069] Sequence

[0070] SEQ ID NO:1 shows the amino acid sequence of the heavy chain (V H ) of monoclonal antibody 5C4. SEQ ID NO:2 shows the amino acid sequence of the light chain (V L ) of monoclonal antibody 5C4.

[0071] SEQ ID NO:3 shows the amino acid sequence of the heavy chain (V H ) of monoclonal antibody 4H1. SEQ ID NO:4 shows the amino acid sequence of the light chain (V L ) of monoclonal antibody 4H1.

[0072] SEQ ID NO:5 shows the amino acid sequence of the heavy chain (V H ) of monoclonal antibody 1D2. SEQ ID NO:6 shows the amino acid sequence of the light chain (V L ) of monoclonal antibody 1D2.

[0073] SEQ ID NO:7 shows the amino acid sequence of the heavy chain (V H ) of monoclonal antibody 1C4. SEQ ID NO:8 shows the amino acid sequence of the light chain (V L ) of monoclonal antibody 1C4.

[0074] SEQ ID NO:9 shows the amino acid sequence of the heavy chain (V H ) of monoclonal antibody 2B5. SEQ ID NO:10 shows the amino acid sequence of the light chain (V L ) of monoclonal antibody 2B5.

[0075] SEQ ID NO:11 shows the amino acid sequence of the heavy chain (V H ) of monoclonal antibody 1F1. SEQ ID NO:12 shows the amino acid sequence of the light chain (V L ) of monoclonal antibody 1F1.

[0076] SEQ ID NO:13 shows the amino acid sequence of the heavy chain (V H) amino acid sequence. SEQ ID NO:14 shows the light chain (V L ) amino acid sequence.

[0077] SEQ ID NO:15 shows the heavy chain (V H ) amino acid sequence of monoclonal antibody 4A5. SEQ ID NO:16 shows the light chain (V L ) amino acid sequence of monoclonal antibody 4A5.

[0078] SEQ ID NO:17 shows the heavy chain (V H ) amino acid sequence of monoclonal antibody 1D8. SEQ ID NO:18 shows the light chain (V L ) amino acid sequence of monoclonal antibody 1D8.

[0079] SEQ ID NO:19 shows the heavy chain (V H ) amino acid sequence of monoclonal antibody 1D3. SEQ ID NO:20 shows the light chain (V L ) amino acid sequence of monoclonal antibody 1D3.

[0080] SEQ ID NO:21 shows the heavy chain (V H ) amino acid sequence of monoclonal antibody 2F1. SEQ ID NO:22 shows the light chain (V L ) amino acid sequence of monoclonal antibody 2F1.

[0081] SEQ ID NO:23 shows the heavy chain (V H ) amino acid sequence of monoclonal antibody 3C6. SEQ ID NO:24 shows the light chain (V L ) amino acid sequence of monoclonal antibody 3C6.

[0082] SEQ ID NO:25 shows the heavy chain (V H ) amino acid sequence of monoclonal antibody 3D12. SEQ ID NO:26 shows the light chain (V L ) amino acid sequence of monoclonal antibody 3D12.

[0083] SEQ ID NO:27 shows the heavy chain (V H ) amino acid sequence of monoclonal antibody 3A9. SEQ ID NO:28 shows the light chain (V L ) amino acid sequence of monoclonal antibody 3A9.

[0084] SEQ ID NO:29 shows the heavy chain (V H) amino acid sequence. SEQ ID NO:30 shows the light chain (V L ) amino acid sequence.

[0085] SEQ ID NO:31 shows the heavy chain (V H ) amino acid sequence. SEQ ID NO:32 shows the light chain (V L ) amino acid sequence.

[0086] SEQ ID NO:33 shows the heavy chain (V H ) amino acid sequence. SEQ ID NO:34 shows the light chain (V L ) amino acid sequence.

[0087] SEQ ID NO:35 shows the heavy chain (V H ) amino acid sequence. SEQ ID NO:36 shows the light chain (V L ) amino acid sequence.

[0088] SEQ ID NO:37 shows the heavy chain (V H ) amino acid sequence. SEQ ID NO:38 shows the light chain (V L ) amino acid sequence.

[0089] SEQ ID NO:39 shows the heavy chain (V H ) amino acid sequence. SEQ ID NO:40 shows the light chain (V L ) amino acid sequence.

[0090] SEQ ID NO:41 shows the heavy chain (V H ) amino acid sequence. SEQ ID NO:42 shows the light chain (V L ) amino acid sequence.

[0091] SEQ ID NO:43 shows the heavy chain (V H ) amino acid sequence. SEQ ID NO:44 shows the light chain (V L ) amino acid sequence.

[0092] SEQ ID NO:45 shows the heavy chain (VH ) amino acid sequence. SEQ ID NO:46 shows the light chain (V L ) amino acid sequence.

[0093] SEQ ID NO:47 shows the heavy chain (V H ) amino acid sequence of monoclonal antibody 1E1. SEQ ID NO:48 shows the light chain (V L ) amino acid sequence of monoclonal antibody 1E1. Detailed implementation mode

[0094] Glypican-3, an excellent target for HCC immunotherapy

[0095] Glypican-3 (GPC-3) is a member of the heparan sulfate proteoglycan family and is a molecule attached to the cell surface through a glycosyl-phosphatidylinositol (GPI) anchor. In recent years, GPC-3 has been found to be a highly specific biomarker for HCC diagnosis and progression because it is overexpressed almost exclusively on HCC cells and not on normal adult hepatocytes or other normal tissues.

[0096] In addition, it has been demonstrated that GPC-3 can serve as a co-receptor for multiple growth factors, which in turn stimulate oncogenic signaling pathways to promote HCC growth.

[0097] Therefore, as a specific HCC surface marker, GPC-3 is an excellent target for HCC antibody-based immunotherapy.

[0098] We disclose the nucleotide sequences and amino acid sequences of 24 antibodies or their antigen-binding portions that specifically target glypican-3 both in solution and on the cell surface.

[0099] We describe monoclonal antibodies produced against glypican-3 (GPC-3). We refer to these monoclonal antibodies as 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1.

[0100] These antibodies are capable of specifically binding to glypican-3 (GPC-3) expressed in solution and on the cell surface. The antibodies are also capable of cross-reacting with glypican-3 (GPC-3) from many other species. The antibodies are capable of inducing antibody-dependent cell-mediated cytotoxicity. The antibodies can be used to construct anti-GPC-3 CAR T cells that efficiently kill tumor cells.

[0101] We disclose the heavy chain variable region (V H ) sequences and the light chain variable region (V L ) sequences of each of the following clones: 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1.

[0102] We found that Fab fragments and single-chain Fv fragments containing these variable regions have the same cross-reactivity and strong neutralizing activity.

[0103] Accordingly, we provide polypeptides containing these variable regions for the treatment and detection of cancer, such as hepatocellular carcinoma.

[0104] Anti-glypican-3 (GPC-3) antibody

[0105] The Examples describe the production and preparation of antibodies produced by and reactive with the GPC-3 protein.

[0106] We disclose the functional and structural characteristics of murine monoclonal antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1 against glypican-3 (GPC-3). We found that these anti-GPC-3 antibodies are capable of killing tumor cells.

[0107] Accordingly, we broadly provide anti-GPC-3 antibodies. The anti-GPC-3 antibodies can comprise the heavy chain variable region and the light chain variable region of 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, or 1E1.

[0108] The anti-GPC-3 antibodies can be used to target hepatocellular carcinoma.

[0109] The anti-tumor antibodies may be capable of binding to GPC-3 polypeptides from many different species.

[0110] The antibody may have cross-reactivity, i.e., be able to bind more than one polypeptide. For example, the antibody may be able to bind two or more variants of a specific polypeptide within a defined polypeptide group. Thus, two or more variants may comprise homologous or orthologous polypeptides from different types of polypeptides in the group. The antibody may be able to bind substantially all variants of a specific polypeptide in the group.

[0111] The disclosure herein enables the preparation of these antibodies and their fragments and variants, including humanized antibodies and chimeric antibodies, which have one or more properties similar or identical to those of anti-GPC-3 antibodies (such as 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1).

[0112] These properties may include binding affinity, binding specificity, cross-reactivity, binding affinity, neutralizing activity, etc., as described in further detail below. The specific antibodies and their variants described herein can be prepared by those skilled in the art based on the information disclosed herein and using the molecular biology techniques that we also describe in detail.

[0113] Anti-GPC-3 antibody

[0114] The polypeptide to which the anti-GPC-3 antibody binds may comprise a glypican protein. We specifically disclose anti-GPC-3 antibodies.

[0115] Accordingly, the term "anti-GPC-3 antibody" should be understood to include each of the monoclonal antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1 (and their humanized counterparts). Also included are polypeptides and their variants, homologs, fragments, and derivatives comprising the variable region of any one of the antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1. Where the context permits, the term should also include variants, homologs, fragments, and derivatives of the anti-GPC-3 antibodies as described below.

[0116] Anti-GPC-3 antibodies can be generated against glypican-3 from any species, such as human glypican-3. They can be used to bind to or target tumor cells, such as hepatocellular carcinoma cells.

[0117] Therefore, anti-GPC-3 antibodies can also be regarded as anti-tumor antibodies or anti-hepatocellular carcinoma antibodies.

[0118] In the examples, for instance, monoclonal antibodies and their variants including Fab, scFv, etc., as well as humanized monoclonal antibodies and their properties are disclosed. The examples also describe Fab fragments and single-chain Fvs from monoclonal antibodies. Other variants including humanized versions of each of these antibodies are also disclosed.

[0119] We disclose the variable region sequences of monoclonal antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1. We also disclose variants, homologs, fragments and derivatives of these variable regions. Using this sequence information, those skilled in the art can prepare antibodies comprising these variable regions or their variants, homologs, fragments and derivatives.

[0120] We also disclose the sequences of nucleic acid constructs for expressing these monoclonal antibodies. The sequences of these constructs are capable of preparing monoclonal antibodies having the same sequences as antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1. We also disclose variants, homologs, fragments and derivatives of antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1.

[0121] We disclose the sequences of constructs capable of expressing humanized monoclonal antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1. We describe methods for expressing the desired antibodies from cells transfected with the said constructs (as well as variants, homologs, fragments and derivatives of these humanized constructs).

[0122] Using such sequences and expression methods, a person skilled in the art can easily transfect relevant host cells and make them express a complete monoclonal or humanized anti-GPC-3 antibody, or its variants, homologs, fragments, and derivatives.

[0123] The monoclonal antibodies and their variants may comprise the variable regions of antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1, and also comprise the variable regions of other anti-GPC-3 antibodies known in the art. The anti-GPC-3 antibodies may comprise the same or different variable regions in a single antibody molecule. They may comprise one variable region, or more than one variable region. Thus, we provide a person skilled in the art with the ability to prepare any number of antibodies that comprise binding reactivity that is the same as or similar to that of antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1 or other anti-GPC-3 antibodies.

[0124] Such antibodies may comprise the complete sequence or substantially complete sequence (i.e., heavy and light chains) of the antibody, or they may comprise fragments of the complete antibody (e.g., Fv, F(ab’), and F(ab’) 2 fragment or single-chain antibody (scFv)). The antibody may also comprise a fusion protein or synthetic protein that comprises the antigen-binding site of the antibody as described in detail below.

[0125] It is also obvious that such antibodies can be engineered to obtain desired properties, such as reduced host reactivity, reduced rejection, etc.

[0126] The engineering may include "humanization", by which we mean the inclusion (or replacement) of one or more human residues or sequences in an antibody sequence, such as a mouse antibody sequence. "Humanization" in the context of this article includes "chimeric" antibodies, in which the antibody comprises discrete portions of mouse and human sequences, e.g., where one or both variable regions comprise mouse sequences and the remainder of the antibody molecule (e.g., the constant region) comprises human sequences. In such chimeric antibodies, for example, the entire variable region of a mouse or rat antibody can be expressed together with a human constant region. This provides such chimeric antibodies with human effector functions and also reduces the immunogenicity (HAMA) caused by the murine Fc region.

[0127] Humanization of the 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1 antibodies can be performed by any suitable means, for example, the method described in Hanson BJ, Boon AC, Lim AP, Webb A, Ooi EE, Webby RJ. Passive immunoprophylaxis and therapy with humanized monoclonal antibody specific for influenza A H5 hemagglutinin in mice. Respir Res 17:126, 2006.

[0128] Generally, a "chimeric antibody" may refer to an antibody having either heavy chains and light chains encoded by nucleotide sequences derived from mouse immunoglobulin genes and having either heavy chains and light chains encoded by nucleotide sequences derived from human immunoglobulin genes.

[0129] "Humanization" also includes antibodies with CDR grafts or remodeling. Therefore, it includes more discrete levels of engineering, for example, antibodies in which mouse variable regions have been mutated to include human residues to reduce immunogenicity. In such antibodies, only the complementary determining regions from the rodent antibody V region can be combined with the framework regions from the human V region. Compared with chimeric antibodies, such antibodies should be more human and less immunogenic.

[0130] For the avoidance of doubt, when specific antibody names are mentioned herein, they should be understood to include murine monoclonal antibodies (secreted by hybridomas) and their humanized versions, unless the context dictates otherwise. Thus, for example, when referring to antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 or 1E1, this includes monoclonal antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1 (i.e., derived from mice), as well as humanized monoclonal antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1.

[0131] GPC-3 binding polypeptide

[0132] We also provide polypeptides that generally have GPC-3 protein binding activity. Such polypeptides include anti-GPC-3 antibodies. The GPC-3 binding polypeptides can comprise one or more properties that are the same as or similar to those of monoclonal antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1. For convenience, the polypeptides can generally be referred to as "anti-GPC-3 antibodies".

[0133] Constructing binding molecules is within the skill of the reader. The binding molecules may not be (or may not be described as) antibodies or immunoglobulins, but contain anti-GPC-3 binding activity as described herein. Thus, where the context permits, the term "anti-GPC-3 antibody" should be understood to include any molecule that is capable of binding GPC-3. Such molecules can include polypeptides, small molecules, as well as antibodies and immunoglobulins, and can be identified by a variety of methods known in the art, such as by screening libraries for GPC-3 binding activity.

[0134] The GPC-3 binding polypeptide (including anti-GPC-3 antibody) may have properties similar to or the same as those of monoclonal antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1. Such similar or identical properties may particularly include binding properties. The GPC-3 binding polypeptide is generally capable of binding to GPC-3 polypeptides, such as GPC-3 from humans, GPC-3 from mice, and the like.

[0135] GPC-3 epitope

[0136] The anti-GPC-3 antibody may have the same or similar binding specificity, binding affinity, and / or avidity as antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1. The anti-GPC-3 antibody may specifically bind to the epitopes bound by antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1.

[0137] Methods for determining the epitopes that bind to a specific antibody are known in the art. Such epitope mapping methods are described, for example, in Hanson et al., (2006). Respiratory Research, 7:126. In addition, those skilled in the art will be able to produce antibodies and screen them for specific properties.

[0138] Therefore, those skilled in the art will be able to easily identify anti-GPC-3 antibodies that bind to the same epitopes as monoclonal antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1.

[0139] Antibody binding

[0140] The anti-GPC-3 antibody is capable of binding to human glypican-3. It is also capable of binding to mouse glypican-3.

[0141] The binding between the anti-GPC-3 antibody and its target can be more or less, stronger or weaker, transient, semi-permanent or permanent. The antibody can bind to its target with an EC of 1 μM or lower (e.g., 100 nM or lower). 50 bind to its target with a binding affinity.

[0142] It can bind with an affinity of 90 nM or lower, 80 nM or lower. It can bind with an affinity of 70 nM or lower, 60 nM or lower, 50 nM or lower, 40 nM or lower, 30 nM or lower, 20 nM or lower, 10 nM or lower, 5 nM or lower, 4 nM or lower, 3 nM or lower, 2 nM or lower, 1 nM or lower. It can bind with an affinity of 0.5 nM or lower, 0.4 nM or lower, 0.3 nM or lower or 0.2 nM or lower.

[0143] The antibody can bind to its target with a K in the μM or nM range. d It can bind to its target. It can bind with a K of 10 -7 M or lower, 10 -7 M or lower, 10 -8 M or lower, 10 -9 M or lower or 10 -10 M or lower and bind. d The binding can be measured by any means known in the art, such as ELISA or surface plasmon resonance, both of which are described in detail in the Examples.

[0144] The binding can occur intracellularly or extracellularly between the anti-GPC-3 antibody and the GPC-3 polypeptide. This binding can inactivate, inhibit or reduce the activity of the GPC-3 polypeptide. The binding can neutralize the activity of GPC-3.

[0145] The activity can include any biological activity caused by or associated with the GPC-3 polypeptide. The activity can include binding to another protein, such as a receptor, a downstream protein or a downstream factor. The other protein can include GPC-3 itself. The activity can include a polymerization activity, such as a trimerization activity or a homotrimerization activity. The binding between the anti-GPC-3 antibody and the GPC-3 polypeptide can inactivate, inhibit or reduce the activity of the receptor, the downstream protein or the downstream factor. The activity can include a biochemical activity or a pathogenic activity.

[0146] The binding can inactivate, inhibit or reduce the activity of cells expressing GPC-3. It can inactivate or neutralize or kill cells expressing GPC-3. The binding between the anti-GPC-3 antibody and GPC-3 can kill tumor cells expressing GPC-3.

[0147] The binding can inactivate, inhibit or reduce the activity of cells expressing GPC-3. It can inactivate or neutralize or kill cells expressing GPC-3. The binding between the anti-GPC-3 antibody and GPC-3 can kill tumor cells expressing GPC-3.

[0148] antibody

[0149] Where context permits, the terms "antibody" and "immunoglobulin" as used herein may be used interchangeably. These terms include fragments of antibody molecules prepared by proteolytic cleavage or recombinantly, which are capable of selectively reacting with or recognizing GPC-3 or its epitopes, such as the epitopes of GPC-3 that bind to antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1

[0150] Non-limiting examples of such proteolytic fragments and / or recombinant fragments include Fab, F(ab’) 2 、Fab’, Fv fragments and single-chain antibodies (scFv) containing V L and V H domains linked by a peptide linker. These Fvs can be linked covalently or non-covalently to form antibodies with two or more binding sites.

[0151] An "ScFv molecule" refers to a molecule in which the V H and V L partner domains are linked by a flexible oligopeptide. A general review of the techniques involved in synthesizing antibody fragments that retain their specific binding sites can be found in Winter & Milstein (1991) Nature 349, 293-299.

[0152] Intact antibodies and F(ab’) 2 fragments are "bivalent". "Bivalent" means that the antibody and F(ab’) fragments have two antigen-binding sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen-binding site.

[0153] Anti-GPC-3 antibodies may include high-affinity antibodies with a binding affinity of 1 μm or lower (e.g., 100 nm or lower, e.g., 0.17 nM to 84 nM).

[0154] The term "binding affinity" as used herein refers to the EC 50 binding affinity of the antibodies disclosed herein (e.g., anti-GPC-3 antibodies). It can be measured using ELISA or surface plasmon resonance. High EC 50 binding affinity is desirable as it reflects the affinity of the Fab fragment for the antigen.

[0155] The term "affinity" can also be defined according to the dissociation rate or dissociation rate (k off ) of an antibody (e.g., an anti-GPC-3 antibody). The lower the dissociation rate, the higher the affinity of the antibody (e.g., an anti-GPC-3 antibody) for the antigen.

[0156] The anti-GPC-3 antibody may comprise the peptide itself or form part of a fusion protein.

[0157] The anti-GPC-3 antibodies described herein include any antibody having GPC-3 binding activity, such as the ability to bind to GPC-3 or the ability to bind to the same epitope as bound by antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1.

[0158] The anti-GPC-3 antibody also includes whole antibodies or intact antibodies, whether murine, humanized or human, derivatives and bioactive fragments of such antibodies. These may include antibody fragments having GPC-3 binding activity, said antibody fragments having amino acid substitutions or sugars or other molecules attached to amino acid functional groups, etc.

[0159] The anti-GPC-3 antibody may include an isolated antibody or a purified antibody. The antibody can be obtained from any suitable source or produced by any suitable source, whether natural or not, or it can be a synthetic anti-GPC-3 antibody, a semi-synthetic anti-GPC-3 antibody, a derived anti-GPC-3 antibody, or a recombinant anti-GPC-3 antibody.

[0160] When the anti-GPC-3 antibody is a non-natural anti-GPC-3 antibody, it may include at least a portion of an antibody prepared by recombinant DNA technology, or an anti-GPC-3 antibody produced by chemical synthesis technology, or a combination thereof.

[0161] The term "derivative" as used herein includes chemical modifications of the anti-GPC-3 antibody. By way of example, such modifications can replace hydrogen with an alkyl, acyl or amino group. The sequence of the anti-GPC-3 antibody may be the same as the naturally occurring form or it may be a variant, homolog, fragment or derivative thereof.

[0162] Antibody variable region

[0163] The term "variable region" as used herein refers to the light chain (V L ) and the heavy chain (V H) that contain the determinants of binding recognition specificity and overall affinity of the antibody for GPC-3 (or variant, homologue, fragment or derivative), as the case may be.

[0164] Each pair of light chains (V L ) and heavy chain (V H ) participate in antigen recognition and form antigen binding sites. The domains of the light and heavy chains have the same basic structure, with each domain having four framework (FR) regions whose sequences are relatively conserved and connected by three complementarity determining regions (CDRs). The FR regions maintain the structural integrity of the variable domains. CDRs are polypeptide segments within the variable domains that mediate antigen binding.

[0165] As used herein, the term "constant region" refers to domains of the light (CL) and heavy (CH) chains of an antibody (or variant, homolog, fragment or derivative) that provide structural stability and other biological functions, such as antibody chain association, secretion, transplacental mobility and complement binding, but are not involved in binding to the GPC-3 epitope. The amino acid sequence and corresponding exon sequence in the constant region gene depends on the species from which it is derived. However, the amino acid sequence variation that leads to allotypes is relatively limited for a particular constant region within a species. "Allotype" is an antigenic determinant (or epitope) that distinguishes alleles.

[0166] The variable region of each chain is connected to the constant region by a connecting polypeptide sequence. The connecting sequence is encoded by the "J" sequence in the light chain gene and the combination of the "D" sequence and the "J" sequence in the heavy chain gene.

[0167] Antibody variable region sequences

[0168] According to the methods and compositions described herein, anti-GPC-3 antibodies can be generated from these variable region sequences by methods known in the art.

[0169] For example, the heavy and light chain sequences can be recombined into the constant sequence of a selected antibody by recombinant genetic engineering techniques known to those skilled in the art.

[0170] Constant region sequences are known in the art and can be obtained from a number of databases, such as the IMGT / LIGM-DB database (described in Giudicelli et al, 2006, Nucleic Acids Research 34 (Database Issue): D781-D784 and LeFranc et al (1995) LIGM-DB / IMGT: An Integrated Database of Ig and TcR, Part of the Immunogenetics Database. Annals of the New York Academy of Sciences 764 (1), 47–47 doi: 10.1111 / j.1749-6632.1995.tb55805.x) and the IMGT / GENE-DB database (described in Giudicelli et al, 2005, Nucleic Acids Res. 2005 Jan 1; 33 (Database issue): D256-61). IMGT / LIGM-DB and IMGT / GENE-DB are part of the ImMunoGeneTics database at www.ebi.ac.uk / imgt / .

[0171] Methods for combining variable regions of a given sequence and constant regions to produce intact antibodies are known in the art and described in Hanson et al., (2006). Respiratory Research, 7:126.

[0172] Antibody scFv sequence

[0173] Fragments of intact antibodies, such as Fv, F(ab') and F(ab') can be generated by means known in the art. 2 Fragment or single chain antibody (scFv).

[0174] Using the published sequences and methods described in the literature, for example, the heavy and light chains of the variable regions of antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1 having the sequences shown herein can be transgenically fused to mouse IgG constant region sequences to generate mouse monoclonal anti-GPC-3 antibodies. The variable regions of antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1 can be engineered with mouse or human IgG constant regions to generate mouse monoclonal antibodies or humanized antibodies capable of binding to GPC-3 polypeptides.

[0175] Peptide sequence

[0176] It should be understood that the polypeptide sequences disclosed herein are not limited to the specific sequences described herein, but also include homologous sequences obtained from any source, such as related cell homologs, homologs from other species, and variants or derivatives thereof, provided that they have at least one biological activity of an anti-GPC-3 antibody (as the case may be).

[0177] Thus, the present disclosure includes variants, homologues or derivatives of the amino acid sequences described herein, as well as variants, homologues or derivatives of the amino acid sequences encoded by the nucleotide sequences disclosed herein. Such sequences are generally referred to as "anti-GPC-3 antibody" sequences.

[0178] Biological Activity

[0179] In some embodiments, the sequence comprises at least one biological activity of an anti-GPC-3 antibody, as appropriate.

[0180] The biological activity may include immunological activity. The anti-GPC-3 antibody may have the same or similar immunological activity as antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1 or humanized versions thereof. "Immunological activity" refers to the ability of an anti-GPC-3 antibody to induce a specific immune response in an appropriate animal or cell when bound to a GPC-3 antigen.

[0181] The biological activity may include antigen binding activity. The anti-GPC-3 antibody may bind to GPC-3 or an epitope thereof. The anti-GPC-3 antibody may bind to the same epitope as antibody 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, or 1E1.

[0182] The anti-GPC-3 antibody may bind to the antigen or epitope with the same, reduced or increased affinity or avidity. For example, the anti-GPC-3 antibody may bind to the antigen or epitope with at least 10%, such as 20%, such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more affinity or avidity compared to a homologous antibody or its humanized counterpart, as the case may be.

[0183] The activity may include inhibition of the activity of Glypican-3. Glypican-3, as a coreceptor of growth factors (eg, BMP, FGF, hedgehog), is reported to be involved in the pathogenesis, development and metastasis of HCC.

[0184] The anti-GPC-3 antibody may have the same, reduced, or increased inhibitory activity as a homologous antibody. For example, the effectiveness of the anti-GPC-3 antibody may be at least 10%, such as 20%, such as 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the homologous antibody (e.g., antibody 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, or 1E1, or a humanized counterpart thereof, as the case may be. By this we mean that if a homologous antibody is able to reduce GPC-3 activity, etc. by, for example, 90%, then an anti-GPC-3 antibody may be able to reduce GPC-3 activity, etc. by less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, etc., compared to an untreated animal or cell.

[0185] Other assays that detect antibody events may also be used instead of or in addition to the assays described.

[0186] Homologs

[0187] The disclosed anti-GPC-3 antibody polypeptides include homologous sequences obtained from any source, such as related viral / bacterial proteins, cellular homologs, and synthetic peptides and variants or derivatives thereof. Thus, the polypeptides also include those encoding homologs of anti-GPC-3 antibodies from other species, including other animals, such as mammals (e.g., mice, rats, or rabbits) or humans.

[0188] In the context of this article, homologous sequences or homologues are considered to include amino acid sequences that are at least 60%, 70%, 80% or 90% (e.g., at least 95% or 98%) identical to the sequence of a related polypeptide at the amino acid level over at least 30 amino acids (e.g., 50, 70, 90 or 100 amino acids), for example as shown in the sequence listing herein. In the context of this article, homologous sequences are considered to include amino acid sequences that are at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% (e.g., at least 95% or 98%) identical to the sequence of a related polypeptide at the amino acid level over, for example, at least 15, 25, 35, 50 or 100 amino acids (e.g., 200, 300, 400 or 500 amino acids). Although homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of this article, homology can be expressed in terms of sequence identity. Sequence identity can be determined over the entire length of the related sequences (i.e., for example, over the entire length or full-length sequence of the related genes).

[0189] Homology comparisons can be performed by eye, or more usually, with the aid of existing sequence comparison programs. These commercially available computer programs can calculate the % homology between two or more sequences.

[0190] Homology % can be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called a "non-gap" alignment. Typically, such non-gap alignments are performed only over a relatively small number of residues (e.g., less than 50 contiguous amino acids).

[0191] While this is a very simple and consistent approach, it fails to take into account that, for example, one insertion or deletion in an otherwise identical pair of sequences will cause subsequent amino acid residues to fail to align, and thus may result in a large reduction in % homology when a global alignment is performed. Therefore, most sequence comparison methods aim to produce an optimal alignment that takes possible insertions and deletions into account without overly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local homology.

[0192] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible (reflecting a higher relatedness between the two compared sequences) will receive a higher score than a sequence with many gaps. An "affine gap cost" is often used to charge a relatively high cost for the presence of a gap, while charging a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, when using such software for sequence comparisons, the default values ​​may be used. For example, when using the GCG Wisconsin Bestfit software package (see below), the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0193] Therefore, calculating the maximum homology % first requires generating the best alignment taking into account the gap penalty. A suitable computer program for performing this alignment is the GCG Wisconsin Bestfit software package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12: 387). Examples of other software that can perform sequence alignments include, but are not limited to, the BLAST software package (see Ausubel et al., 1999ibid–Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS comparison tool suite. Both BLAST and FASTA can be used for offline and online searches (see Ausubel et al., 1999ibid, pages 7-58 to 7-60). The GCG Bestfit program can be used.

[0194] Although the final homology % can be measured based on identity, the alignment process itself is usually not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​usually used, which assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). The GCG Wisconsin program usually uses public default values ​​or a custom symbol comparison table (if provided) (see the user manual for more details). For the public default values ​​of the GCG software package, or in the case of other software, a default matrix such as BLOSUM62 can be used.

[0195] Once the software has produced an optimal alignment, % homology, e.g. % sequence identity, can be calculated. The software typically does this as part of the sequence comparison and generates a numerical result.

[0196] Variants and derivatives

[0197] The term "variant" or "derivative" in relation to the amino acid sequences described herein includes any substitution, variation, modification, replacement, deletion or addition of one (or more) amino acids in the sequence. The resulting amino acid sequence may retain substantially the same activity as the unmodified sequence, e.g., having at least the same activity as the anti-GPC-3 antibody polypeptides set forth herein (e.g., in the sequence listing). Thus, the key features of the sequence, i.e., the ability to bind to a GPC-3 polypeptide or the ability to reduce viral infectivity, homotrimerization, viral uptake, etc., as described elsewhere, may be retained.

[0198] The polypeptides or fragments or homologues thereof having the amino acid sequences shown in the examples may be modified for use in the methods and compositions described herein. Typically, the modifications are made to maintain the biological activity of the sequence. Amino acid substitutions may be made, for example, 1, 2 or 3 to 10, 20 or 30 substitutions, as long as the modified sequence retains the biological activity of the unmodified sequence. Amino acid substitutions may include the use of non-naturally occurring analogs, for example, for increasing the plasma half-life of therapeutically administered polypeptides.

[0199] Natural variants of anti-GPC-3 antibodies may contain conservative amino acid substitutions. For example, conservative substitutions may be defined according to the following table. Amino acids in the same box in the second column, such as amino acids in the same row in the third column, may be substituted for each other:

[0200]

[0201] Snippet

[0202] The polypeptides disclosed herein and useful as markers also include fragments of the above-mentioned full-length polypeptides and variants thereof, including fragments of the sequences listed in the sequence listing.

[0203] Polypeptides also include fragments of the full-length sequence of any anti-GPC-3 antibody polypeptide. Fragments may contain at least one epitope. Methods for identifying epitopes are well known in the art. Fragments typically contain at least 6 amino acids, such as at least 10, 20, 30, 50 or 100 or more amino acids.

[0204] The polypeptide fragments of the anti-GPC-3 antibody protein and allelic variants and species variants thereof may contain one or more (e.g., 5, 10, 15 or 20) substitutions, deletions or insertions, including conservative substitutions. When substitutions, deletions and / or insertions occur, for example, less than 50%, 40% or 20% of the amino acid residues described in the sequence listing are changed in different species.

[0205] Anti-GPC-3 antibodies and fragments, homologues, variants and derivatives thereof can be prepared by recombinant means. However, they can also be prepared by synthetic means (e.g., solid phase synthesis) using techniques well known to those skilled in the art. The protein can also be produced as a fusion protein, for example to aid extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and / or transcriptional activation domain) and beta-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the target protein sequence to allow for removal of the fusion protein sequence. The fusion protein can be a fusion protein that does not hinder the function of the target protein sequence. The protein can also be obtained by purifying cell extracts from animal cells.

[0206] The anti-GPC-3 antibody polypeptides, variants, homologues, fragments and derivatives disclosed herein may be in substantially isolated form. It should be understood that such polypeptides may be mixed with carriers or diluents that do not interfere with the intended purpose of the protein and still be considered substantially isolated. The anti-GPC-3 antibody variants, homologues, fragments or derivatives may also be in substantially purified form, in which case they typically comprise a protein in a preparation wherein more than 90% (e.g., 95%, 98% or 99%) of the protein in the preparation is one protein.

[0207] The anti-GPC-3 antibody polypeptides, variants, homologues, fragments and derivatives disclosed herein may be labeled with a revealing marker. The revealing marker may be any suitable marker that enables detection of the polypeptide, etc. Suitable markers include radioactive isotopes, such as 125 I, enzymes, antibodies, polynucleotides and linkers, such as biotin. Labeled polypeptides can be used in diagnostic procedures (such as immunoassays) to detect the amount of polypeptides in a sample. Polypeptides or labeled polypeptides can also be used in serological or cell-mediated immunoassays to detect the immunoreactivity of animals and humans to the polypeptides using standard protocols.

[0208] The anti-GPC-3 antibody polypeptides, variants, homologues, fragments and derivatives disclosed herein (optionally labeled) can also be immobilized to a solid phase, such as the surface of an immunoassay well or test strip. Such labeled and / or immobilized polypeptides can be packaged into a kit in a suitable container together with appropriate reagents, controls, instructions, etc. Such polypeptides and kits can be used in methods for detecting antibodies against a polypeptide or an allelic variant or species variant thereof by immunoassay.

[0209] Immunoassay methods are well known in the art and generally comprise: (a) providing a polypeptide comprising an epitope that can be bound by an antibody directed against the protein; (b) incubating a biological sample with the polypeptide under conditions that allow formation of an antibody-antigen complex; and (c) determining whether an antibody-antigen complex comprising the polypeptide is formed.

[0210] The anti-GPC-3 antibody polypeptides, variants, homologs, fragments and derivatives disclosed herein can be used in in vitro or in vivo cell culture systems to study the role of their corresponding genes and their homologs in cellular function, including their function in disease. For example, truncated or modified polypeptides can be introduced into cells to disrupt normal functions occurring in cells. The polypeptides can be introduced into cells by in situ expression of the polypeptides from recombinant expression vectors (see below). The expression vectors optionally carry an inducible promoter to control the expression of the polypeptides.

[0211] It is desirable to use appropriate host cells (e.g., insect cells or mammalian cells) to provide the post-translational modifications (e.g., myristoylation, glycosylation, truncation, lapidation, and phosphorylation of tyrosine, serine, or threonine) required to confer optimal biological activity on the recombinant expression product. Such cell culture systems expressing the anti-GPC-3 antibody polypeptides, variants, homologs, fragments, and derivatives disclosed herein can be used in assay systems to identify candidate substances that interfere with or enhance the function of the polypeptide in cells.

[0212] Polynucleotide sequence

[0213] The variable regions, monoclonal antibody sequences and humanized antibody sequences may comprise polynucleotides. These polynucleotides may comprise DNA or RNA.

[0214] They can be single-stranded or double-stranded. They can also be polynucleotides, including synthetic or modified nucleotides. A variety of different types of modifications to oligonucleotides are known in the art. These modifications include methylphosphonate backbones and phosphorothioate backbones, with acridine or polylysine chains added to the 3' end and / or 5' end of the molecule. For purposes of this article, it will be appreciated that the polynucleotides described herein can be modified by any method available in the art. Such modifications can be performed to enhance the in vivo activity or lifespan of the polynucleotides.

[0215] When the polynucleotide is double-stranded, both strands of the duplex, whether alone or in combination, are included in the methods and compositions described herein. When the polynucleotide is single-stranded, it is understood that the complementary sequence of the polynucleotide is also included.

[0216] Variants, derivatives and homologues

[0217] The terms "variant", "homologue" or "derivative" related to the nucleotide sequences described herein include any substitution, variation, modification, replacement, deletion or addition of one (or more) nucleotides in the sequence. The resulting sequence can encode a polypeptide having GPC-3 binding activity as described elsewhere herein.

[0218] As noted above, with respect to sequence identity, a "homolog" has, for example, at least 5% identity, at least 10% identity, at least 15% identity, at least 20% identity, at least 25% identity, at least 30% identity, at least 35% identity, at least 40% identity, at least 45% identity, at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to a related sequence.

[0219] The homology of nucleotides can be compared. For example, the homology of nucleotides can be compared. For example, the homology of nucleotides can be compared. For example, the homology of nucleotides can be compared. For example, the homology of nucleotides can be compared. For example, the homology of nucleotides can be compared. For example, the homology of nucleotides can be compared. For example, the homology of nucleotides can be compared. For example, the homology of nucleotides can be compared. For example, the homology of nucleotides can be compared. For example, the homology of nucleotides can be compared. For example, the homology of nucleotides can be compared. For example, the homology of nucleotides can be compared. For example, the homology of nucleotides can be compared. For example, the homology of nucleotides can be compared. For example, the homology of nucleotides can be compared.

[0220] Hybridization

[0221] We also describe nucleotide sequences that can selectively hybridize to any of the sequences described herein, such as 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1 variable regions, antibodies and humanized antibodies or any variant, fragment or derivative thereof, or the complement of any of the foregoing. The nucleotide sequence can be at least 15 nucleotides in length, such as at least 20, 30, 40 or 50 nucleotides in length.

[0222] As used herein, the term "hybridization" shall include "the process by which a nucleic acid strand joins with a complementary strand through base pairing" as well as the process of amplification as performed in polymerase chain reaction techniques.

[0223] A polynucleotide capable of selectively hybridizing to a nucleotide sequence described herein or its complement is typically at least 70% (e.g., at least 80% or 90%, such as at least 95% or 98%) homologous to the corresponding nucleotide sequence described herein over a region of at least 20 (e.g., at least 25 or 30, such as at least 40, 60 or 100 or more) consecutive nucleotides.

[0224] The term "selectively hybridizable" means that a polynucleotide used as a probe is used under conditions in which the target polynucleotide is found to hybridize to the probe at a level significantly above background. Background hybridization may occur because of the presence of other polynucleotides, for example, in the cDNA or genomic DNA library being screened. In this case, background means that the interaction between the probe and nonspecific DNA members of the library produces a signal level that is less than 10 times, for example, less than 100 times, stronger than the observed specific interaction with the target DNA. The strength of the interaction can be measured, for example, by radiolabeling the probe (e.g., with 32 P) to measure.

[0225] Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and are given a defined "stringency" as explained below.

[0226] Maximum stringency generally occurs at about Tm-5°C (5°C lower than the Tm of the probe); high stringency occurs at about 5°C to 10°C below Tm; medium stringency occurs at about 10°C to 20°C below Tm; and low stringency occurs at about 20°C to 25°C below Tm. As will be appreciated by those skilled in the art, maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences, while medium (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.

[0227] We disclose that the ability to produce a molecule under stringent conditions (e.g., 65°C and 0.1xSSC {1xSSC = 0.15 M NaCl, 0.015 M Na 3 Citrate pH 7.0}) to a nucleotide sequence that hybridizes to a nucleic acid or a fragment, homolog, variant or derivative thereof.

[0228] When the polynucleotide is double-stranded, both strands of the duplex, whether alone or in combination, are included in the present disclosure. When the polynucleotide is single-stranded, it is understood that the complementary sequence of the polynucleotide is also included and disclosed.

[0229] Polynucleotides that are not 100% homologous to sequences disclosed herein but belong to the present disclosure can be obtained in a variety of ways. Other variants of sequences described herein can be obtained by, for example, probing DNA libraries prepared from a series of individuals (e.g., individuals from different populations). In addition, other viral homologs / bacterial homologs or cell homologs can be obtained, particularly cell homologs found in mammalian cells (e.g., rats, mice, cattle, and primate cells), and such homologs and fragments thereof can usually be selectively hybridized with the sequences shown in the sequence table herein. Such sequences can be obtained by probing cDNA libraries made from other animal species or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of the disclosed sequences under medium to high stringency conditions.

[0230] The polynucleotides described herein can be used to generate primers (e.g., PCR primers, primers for alternative amplification reactions), probes (e.g., labeled with a revealing marker by conventional methods using radioactive or non-radioactive markers), or the polynucleotides can be cloned into a vector. Such primers, probes, and other fragments are at least 15 nucleotides in length, such as at least 20 nucleotides, such as at least 25, 30, or 40 nucleotides, and are also included in the term polynucleotides used herein. The fragment length may be less than 500, 200, 100, 50, or 20 nucleotides.

[0231] Polynucleotides such as DNA polynucleotides and probes can be produced recombinantly, synthetically or by any means available to those skilled in the art. They can also be cloned by standard techniques.

[0232] Typically, primers are produced synthetically, involving the stepwise preparation of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are available in the art.

[0233] Longer polynucleotides are usually produced using recombinant methods, such as using PCR (polymerase chain reaction) cloning technology. This involves preparing a pair of primers (e.g., about 15 to 30 nucleotides) flanking the sequence region of the desired clone, contacting the primers with mRNA or cDNA obtained from animal cells or human cells, performing a polymerase chain reaction under conditions that cause amplification of the desired region, separating the amplified fragments (e.g., by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers can be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.

[0234] Glypican-3

[0235] Glypican-3 is also known as the rat homolog of GPC3 and OCI-5. Its cytogenetic location is Xq26.2, and its genomic coordinates (GRCh38) are X:133,535,744-133,985,615.

[0236] Members of the glypican family, including GPC3, are heparan sulfate proteoglycans that are bound to the extracytoplasmic surface of the plasma membrane via covalent glycosylphosphatidylinositol (GPI) linkages. The main function of membrane-attached glypicans is to regulate the signaling of WNT, Hedgehog, fibroblast growth factor, and bone morphogenetic protein (Filmus et al., 2008).

[0237] To identify the molecular basis of Simpson-Golabi-Behmel syndrome (SGBS; see 312870), also known as Simpson dysmorphic syndrome (SDYS), Pilia et al. (1996) adopted a positional cloning approach using an X / autosomal translocation. They used the cell line GM0097, which was deposited in 1974 at the NIGMS collection. This cell line was derived from a female diagnosed with Beckwith-Wiedemann syndrome (BWS; 130650) and showed a karyotype with a de novo X;1 translocation. This karyotype indicated that she was affected by X-linked SGBS rather than BWS, which is determined by mutations on 11p. They mapped the breakpoints in the existing contigs assembled across Xq26 and found a gene, which they called GPC3, that was interrupted by this translocation. This gene was interrupted in another female patient with overgrowth and X;16 translocation and was deleted in 3 different SGBS families. The 2,130-bp cDNA encodes a deduced protein of 580 amino acids starting from the first 151 bp of the sequence. GPC3 shares many common features with the GPC1 gene (600395).

[0238] Filmus et al. in 1988 isolated rat Gpc3 as a developmentally regulated transcript in the intestine, and Filmus et al. in 1995 showed that Gpc3, which they called Oci-5, is a GPI-linked heparan sulfate proteoglycan.

[0239] Pilia et al. (1996) determined that the GPC3 gene contains 8 exons and encompasses approximately 500 kb.

[0240] By fluorescence in situ hybridization, Shen et al. (1997) located the GPC3 gene at Xq26 in humans and Xq36 in rats.

[0241] Sood et al. (2006) used DNA microarrays to compare gene expression patterns in normal human placenta with those in other tissues and found that several genes involved in growth and tissue remodeling were expressed at relatively high levels in villous sections of the placenta compared with other tissues. These genes included GPC3, CDKN1C (600856), and IGF2 (147470). Mutations in the GPC3 and CDKN1C genes occur in patients with Simpson-Golabi-Behmel syndrome and Beckwith-Wiedemann syndrome (130650), respectively, both of which are feto-placental overgrowth syndromes. In contrast, the absence of IGF2 is associated with fetal growth restriction in mice. The relatively high expression of genes that promote and inhibit growth suggested to Sood et al. (2006) that pathways controlling placental development are tightly and locally regulated.

[0242] Capurro et al. (2008) found that GPC3 inhibited soluble hedgehog activity in culture medium of SHH (600725)-expressing mouse embryonic fibroblasts and IHH (600726)-expressing human embryonic kidney cells. GPC3 interacted with SHH but not with the SHH receptor Patched (ptch 1; 601309), and it competed with Patched for binding to SHH. In addition, GPC3 induced SHH endocytosis and degradation. The heparan sulfate chains of GPC3 were not required for interaction with SHH, but membrane attachment via the GPI anchor was required.

[0243] Maurel et al. (2013) observed that the expression of microRNA-1291 (MIR1291; 615487) and GPC3 are both upregulated in hepatocellular carcinoma. They found that MIR1291 does not bind directly to GPC3 mRNA, but rather enhances the stability of GPC3 mRNA by binding to IRE1A (ERN1; 604033), an endoribonuclease that functions in the unfolded protein response of the endoplasmic reticulum, and directing its degradation. In the absence of MIR1291, IRE1A binds to a canonical site in the 3-prime UTR of GPC3 and cleaves the mRNA, directing its degradation through the unfolded protein response. Unlike most miRNAs, which typically bind to complementary sequences in the 3-prime UTR of the target mRNA, MIR1291 binds to a complementary site in the 5-prime UTR of IRE1A to direct its degradation.

[0244] In a preliminary study by Pilia et al. (1996), six of the eight exons of the GPC3 gene were examined and deletions were found in three of six patients with SGBS. This suggests that large deletions may be responsible for a significant proportion of cases of Simpson-Golabi-Behmel syndrome. This is probably not surprising, given the large region of genomic DNA covered by the GPC3 gene (approximately 500 kb) and the high proportion of deletions found in other diseases involving large genes, such as the dystrophin gene (300377) in patients with Duchenne muscular dystrophy (310200). Lindsay et al. (1997) conducted a study to determine the proportion and type of GPC3 gene deletions in 18 families with SGBS, representing approximately half of the reported cases. Deletions were detected in only five families (one of which had been previously reported). PCR analysis using primer pairs amplified fragments of each of the eight exons of the GPC3 gene and deletions were found in all exons of the gene except exon 3. The results suggest that large deletions may be less common in SGBS than initially thought. One patient with a deletion of exons 4 and 5 lacked characteristic facial dysmorphic features. This raises the possibility that defects in the GPC3 gene are involved in a more widespread overgrowth disorder.

[0245] Simpson-Golabi-Behmel syndrome, type 1

[0246] Veugelers et al. (2000) identified one SGBS patient with a GPC3 exon 7 deletion (300037.0002). Six SGBS patients showed point mutations in GPC3: one frameshift mutation, three nonsense mutations, and one splice mutation (300037.0004) predicted loss of function of the Glypican-3 protein. One missense mutation, W296R (300037.0003), altered a conserved amino acid found in all Glypican proteins identified at that time. GPC3 proteins that reproduced this mutation were poorly processed and failed to increase cell surface expression of heparan sulfate, indicating that this missense mutation is also a loss-of-function mutation.

[0247] Sakazume et al (2007) identified mutations in the GPC3 gene in seven Japanese boys with SGBS1. One of the boys had an affected brother. All mutations were predicted to result in complete loss of function. Only one patient had a large deletion, as well as five nonsense mutations and one frameshift mutation. No genotype / phenotype correlation was apparent.

[0248] Wilms tumor, somatic

[0249] White et al. (2002) identified only two non-conservative single base changes (300037.0006-300037.0007) in the GPC3 gene in Wilms tumor (194070) tissues, suggesting that GPC3 may play a role in the pathogenesis of Wilms tumor. They pointed out that Wilms tumor was found in many patients with Simpson-Golabi-Behmel syndrome (Hughes-Benzie et al., 1996; Xuan et al., 1999).

[0250] Capurro et al. (2008) showed that Gpc3-null mouse embryos exhibited significant overgrowth by embryonic day 12.5. They found that embryos between days 10.5 and 13.5 showed increased hedgehog signaling as measured by elevated Patched and Gli1(165220) mRNA levels.

[0251] The above text is adapted from the OMIM entry for glypican-3 (https: / / www.omim.org / entry / 300037), edited by Patricia A. Hartz, Cassandra l. nifta fin, Victor A. McKusick, Anne M. Stumpf, Ada Hamosh, George E. Tiller, and Michael J. Wright.

[0252] GPC-3 polypeptide and nucleic acid

[0253] The definitions of GPC-3 polypeptide homologues, variants, derivatives and fragments may be similar to those described in the previous paragraphs.

[0254] Reference to a GPC-3 polypeptide shall include reference to a homologue, variant, derivative or fragment of the GPC-3 polypeptide, where the context permits. Similarly, reference to a GPC-3 nucleic acid shall include reference to a homologue, variant, derivative or fragment of the GPC-3 nucleic acid, where the context permits.

[0255] An example of a Glypican-3 nucleic acid is the sequence with GenBank Accession No. NM_004484.4.

[0256] An example of a Glypican-3 polypeptide is the sequence having GenBank Accession No. NP_004475.1.

[0257] Generation of anti-GPC-3 antibodies

[0258] Anti-GPC-3 antibodies can be produced by recombinant DNA methods or synthetic peptide chemistry methods well known to those of ordinary skill in the art.

[0259] For example, anti-GPC-3 antibodies can be synthesized by techniques well known in the art, such as "Solid Phase Peptide Synthesis: A Practical Approach", E. Atherton and RC Sheppard, IRL Press, Oxford England. Similarly, multiple fragments can be synthesized and then linked together to form larger fragments. These synthetic peptide fragments can also be prepared by making amino acid substitutions at specific positions to test in vitro and in vivo activities.

[0260] Anti-GPC-3 antibodies can be synthesized in standard microchemical equipment and checked for purity using HPLC and mass spectrometry.Methods of peptide synthesis, HPLC purification and mass spectrometry are well known to those skilled in the art.

[0261] Anti-GPC-3 antibodies can also be expressed under in vitro and in vivo conditions in transformed host cells that have incorporated the DNA sequences described herein (such as variable sequences) or allelic variants thereof, and can be used to prevent and / or treat cancer, such as hepatocellular carcinoma.

[0262] The term "vector" includes expression vectors and transformation vectors. The term "expression vector" refers to a construct capable of in vivo or in vitro expression. The term "transformation vector" refers to a construct capable of being transferred from one species to another.

[0263] Vectors that can be used for expression include recombinant viral vectors, particularly recombinant retroviral vectors (RRV), such as lentiviral vectors and adenoviral vectors including combinations of retroviral vectors.

[0264] The term "recombinant retroviral vector" (RRV) refers to a vector with sufficient retroviral genetic information to allow the RNA genome to be packaged into viral particles capable of infecting target cells in the presence of packaging components. Infection of target cells includes reverse transcription and integration into the target cell genome. RRV carries non-viral coding sequences that will be delivered to target cells by the vector. RRV viruses cannot replicate independently to produce infectious retroviral particles in the final target cells. Typically, RRV lacks functional gag pol and / or env genes and / or other genes essential for replication. Vectors that can be used include recombinant poxvirus vectors, such as fowlpox virus (FPV), insect poxvirus, vaccinia virus (e.g., NYVAC), canarypox virus, MVA, or other non-replicating viral vector systems, such as those described in WO9530018.

[0265] Poxviruses can be engineered for recombinant gene expression and used as recombinant live vaccines in dual immunotherapy approaches. The primary rationale for using live attenuated viruses (e.g., viruses) as delivery vehicles and / or vector-based vaccine candidates stems from their ability to elicit a cell-mediated immune response. As described above, viral vectors can be used as delivery vehicles and vector-based vaccine candidates due to the immunogenicity of their structural proteins, which act as adjuvants to enhance the immune response, thereby making the nucleotide sequence of interest (NOI), such as a nucleotide sequence encoding an anti-GPC-3 antibody, more immunogenic.

[0266] Poxvirus vaccination strategies use recombinant technology to introduce NOIs into the poxvirus genome. If the NOI is integrated into the viral DNA at a site that is not essential for the viral life cycle, the newly generated recombinant poxvirus may be infectious, i.e., infect foreign cells and thereby express the integrated NOI. Recombinant poxviruses prepared in this way can be used as live vaccines for the prevention and / or treatment of disease.

[0267] Other requirements of poxvirus vector delivery systems include good immunogenicity and safety. MVA is a replication-impaired vaccinia virus strain with a good safety record. MVA does not replicate in most cell types and normal human tissues. Limited replication of MVA was observed in a few transformed cell types such as BHK21 cells. Carroll et al. (1997vacine15:387-394) have shown that recombinant MVA is as good as traditional recombinant vaccinia virus vectors in generating protective CD8+T cell responses, and is an effective alternative to more commonly used replicable vaccinia viruses. Vaccinia virus strains derived from MVA, or independently developed virus strains with MVA characteristics, make MVA particularly suitable for use in vaccines and are also suitable for use as delivery vectors.

[0268] The nucleotide sequence of interest and needing to be expressed can be operably connected to the transcription unit. The term "transcription unit" as described herein is a nucleic acid region containing a coding sequence and a signal for realizing the expression of these coding sequences independent of any other coding sequence. Therefore, each transcription unit usually comprises at least a promoter, an optional enhancer and a polyadenylation signal. The term "promoter" is used in the normal sense of this area, such as an RNA polymerase binding site. The promoter may include an enhancer element. The term "enhancer" includes a DNA sequence that is combined with other protein components of the transcription initiation complex and therefore promotes the transcription initiation directed by its associated promoter. The term "cell" includes any suitable organism. The cell may include a mammalian cell, such as a human cell.

[0269] The term "transformed cell" refers to a cell with a modified genetic structure. For example, as described herein, when a vector (such as an expression vector) is introduced into a cell, the cell has a modified genetic structure. The term "organism" includes any suitable organism. The organism can include mammals, such as humans.

[0270] As used herein, the term "transgenic organism" refers to an organism that contains a modified genetic structure. For example, an organism may have a modified genetic structure if a vector (such as an expression vector) is introduced into the organism.

[0271] Antibody expression

[0272] We also describe a method comprising transforming a host cell with a nucleotide sequence or with a nucleotide sequence described herein, wherein the nucleotide sequence includes: for example, the variable region of 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1, an antibody sequence or a humanized antibody sequence.

[0273] We also provide a method comprising culturing a transformed host cell - said cell having been transformed with a nucleotide sequence or said nucleotide sequence - under conditions suitable for expressing the anti-GPC-3 antibody encoded by said nucleotide sequence.

[0274] We also provide a method comprising culturing a transformed host cell - said cell having been transformed with a nucleotide sequence or said nucleotide sequence - under conditions suitable for expressing the anti-GPC-3 antibody encoded by said nucleotide sequence; and then recovering said anti-GPC-3 antibody from the transformed host cell culture.

[0275] Therefore, the nucleotide sequences encoding the anti-GPC-3 antibodies, fusion proteins or their functional equivalents can be used to produce recombinant DNA molecules that direct the expression of the anti-GPC-3 antibodies in appropriate host cells.

[0276] For example, anti-GPC-3 antibodies can be produced in recombinant E. coli, yeast, or mammalian expression systems and purified by column chromatography.

[0277] In some cases, it is advantageous to use antibody fragments rather than whole antibodies. The smaller size of the fragments allows for rapid clearance and can result in improved neutralization of viral activity, infection, progression, etc. Fab, Fv, and ScFv antibody fragments can all be expressed and secreted in E. coli, thus enabling the production of such fragments in large quantities.

[0278] The nucleotide sequence encoding the anti-GPC-3 antibody may be operably linked to a promoter sequence capable of directing the expression of the nucleotide sequence encoding the anti-GPC-3 antibody in a suitable host cell. When inserted into a host cell, the transformed host cell may be cultured under appropriate conditions until a sufficient level of the anti-GPC-3 antibody is obtained, and then the cells may be lysed and the anti-GPC-3 antibody isolated.

[0279] Host cells transformed with the nucleotide sequence encoding the anti-GPC-3 antibody can be cultured under conditions suitable for expressing and recovering the anti-GPC-3 antibody from the cell culture. The protein produced by the recombinant cells can be secreted or can be contained within the cell, depending on the sequence and / or vector used. As will be appreciated by those skilled in the art, the expression vector containing the nucleotide sequence encoding the anti-GPC-3 antibody can be designed with a signal sequence that directs the nucleotide sequence encoding the anti-GPC-3 antibody to be secreted through a specific prokaryotic or eukaryotic cell membrane. Other recombinant constructs can combine the nucleotide sequence encoding the anti-GPC-3 antibody with a nucleotide sequence encoding a polypeptide domain, which facilitates the purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12: 441-5 3', see also the discussion below on vectors containing fusion proteins).

[0280] The anti-GPC-3 antibody can also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Such purification-facilitating domains include, but are not limited to, metal chelating peptides, such as histidine-tryptophan modules that allow purification on immobilized metals (Porath J (1992) Protein Expr Purif 3-26328 1), protein A domains that allow purification on immobilized immunoglobulins, and domains used in the FLAGS extension / affinity purification system (Immunex Corp, Seattle, WA). The inclusion of a cleavable linker sequence, such as Factor XA or enterokinase (Invitrogen, San Diego, CA), between the purification domain and the anti-GPC-3 antibody facilitates purification.

[0281] The nucleotide sequences described herein can be engineered to alter the anti-GPC-3 antibody coding sequence for a variety of reasons, including but not limited to alterations in cloning, processing and / or expression of modified gene products. For example, mutations can be introduced using techniques well known in the art, such as site-directed mutagenesis to insert new restriction sites, alter glycosylation patterns, or change codon preferences.

[0282] In another embodiment, a nucleotide sequence encoding a native, modified or recombinant anti-GPC-3 antibody or the nucleotide sequence can be linked to a heterologous sequence to encode a fusion protein. For example, a fusion protein comprising an anti-GPC-3 antibody or an enzymatically active fragment or derivative thereof is linked to an affinity tag such as glutathione-S-transferase (GST), biotin, His6, ac-myc tag (see Emrich et al 1993 Biocem Biophys Res Commun 197(1):21220), hemagglutinin (HA) (as described in Wilson et al 1984 Cell 37 767) or FLAG epitope (Ford et al 1991 Protein Expr Purif Apr;2(2):95-107).

[0283] The fused recombinant protein may comprise an antigenic coprotein, such as GST, β-galactosidase, or lipoprotein D from Haemophillls influenzae, which is a relatively large coprotein that can be solubilized and facilitates its production and purification. Alternatively, the fusion protein may comprise a carrier protein, such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). In certain embodiments, the marker sequence may comprise a 6-histidine peptide, such as provided in the pQE vector (Qiagen Inc) and described in Gentz ​​et al. (1989 PNAS 86:821-824). Such fusion proteins are easily expressed in yeast culture (such as described in Mitchell et al 1993 Yeast 5:715-723) and are easily purified by affinity chromatography. The fusion protein may also be engineered to comprise a cleavage site between the nucleotide sequence encoding the anti-GPC-3 antibody and the heterologous protein sequence, so that the anti-GPC-3 antibody can be cleaved and purified from the heterologous portion. In another embodiment, the entire combined fusion protein can be used for the determination of the target protein. Alternatively, the co-protein can act as an adjuvant in the sense of providing a general stimulation of the immune system. The co-protein can be attached to the amino terminus or the carboxyl terminus of the first protein.

[0284] Although the presence / absence of marker gene expression indicates that the anti-GPC-3 antibody nucleotide sequence is also present, its presence and expression should be confirmed. For example, if the anti-GPC-3 antibody encoding nucleotide sequence is inserted into the marker gene sequence, recombinant cells containing the anti-GPC-3 antibody coding region can be identified by the lack of marker gene function. Alternatively, the marker gene can be placed in tandem with the anti-GPC-3 antibody encoding nucleotide sequence under the control of a single promoter.

[0285] Expression of the marker gene in response to induction or selection also typically indicates expression of anti-GPC-3 antibodies.

[0286] Other methods for quantifying expression of specific molecules include radiolabeling (Melby PC et al 1993 J Immunol Methods 159:235-44) or biotinylating (Duplaa C et al 1993 Anal Biochem 229-36) nucleotides, co-amplification of control nucleic acids, and standard curve methods onto which the experimental results are interpolated.

[0287] Quantitation of multiple samples can be accelerated by performing the assay in an ELISA format, where the target anti-GPC-3 antibody is present at multiple dilutions, and the spectrophotometric or calorimetric response gives rapid quantitation.

[0288] Altered anti-GPC-3 antibody nucleotide sequences may be prepared or used, including deletions, insertions or substitutions of various nucleotide residues, resulting in a nucleotide sequence encoding the same or a functionally equivalent anti-GPC-3 antibody. For example, the expressed anti-GPC-3 antibody may also have deletions, insertions or substitutions of amino acid residues that produce silent changes to produce a functionally equivalent anti-GPC-3 antibody. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues, as long as the binding affinity of the anti-GPC-3 antibody is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0289] Bispecific Antibodies

[0290] We disclose bispecific antibodies or bispecific antigen-binding fragments capable of binding GPC-3 and another antigen.

[0291] The second antigen may include a T cell surface antigen. An example of a T cell surface antigen is a CD protein, such as CD3 (GenBank Accession No.: NM_000733.4).

[0292] In particular, we disclose an anti-GPC-3 bispecific T cell engaging antibody that is capable of specifically recognizing and binding to GPC-3 and human CD3 molecules on the cell surface.

[0293] Such bispecific antibodies can be constructed by replacing the Fab region of one arm of the antibody with a sequence capable of binding to a T cell surface antigen (such as CD3). Such T cell surface antigen binding sequences can, for example, comprise the V region of a known antibody against a T cell surface antigen. H and V L For example, they can be provided in the form of anti-CD3 scFv fragments.

[0294] Detailed protocols for constructing bispecific antibodies capable of binding to CD3 are listed in the Examples.

[0295] The bispecific antibodies or bispecific antigen-binding fragments may be able to activate naive T cells, induce the release of cytokines (such as interferon-γ and IL-2) and / or induce the death of GPC-3 expressing cells.

[0296] Chimeric Antigen Receptor (CAR)

[0297] Chimeric antigen receptors (CARs), also known as chimeric T-cell receptors, artificial T-cell receptors, and chimeric immunoreceptors, are engineered receptors that have arbitrary specificity grafted onto immune effector cells.

[0298] In a typical CAR, the specificity of a monoclonal antibody is transplanted onto a T cell. The nucleic acid encoding the CAR can be transferred to the T cell using, for example, a retroviral vector. This allows the generation of large numbers of cancer-specific T cells for adoptive cell transfer. Phase I clinical studies of this approach have shown efficacy.

[0299] The target antigen binding domain of CAR is usually fused to the signal transduction intracellular domain (endodomain) through the spacer domain and the transmembrane domain. When CAR binds to the target antigen, it will cause the T cell expressing the CAR to transmit an activation signal.

[0300] We disclose a CAR comprising a Glypican-3 binding domain based on any of antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, or 1E1.

[0301] The CAR may comprise a Glypican-3 binding domain comprising

[0302] (a) Heavy chain variable region (V H ) having the complementarity determining regions (CDRs) of any of the above antibodies as described herein;

[0303] (b) Light chain variable region (V L) having the CDRs of any of the above antibodies as described herein.

[0304] One or more mutations (substitutions, additions or deletions) may be introduced into the or each CDR without negatively affecting the Glypican-3 binding activity. For example, each CDR may have one, two or three amino acid mutations.

[0305] CAR can contain the following V H Sequence one: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47.

[0306] CAR can contain the following V L Sequence one: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48.

[0307] The CAR may comprise a variant of any of these sequences having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity, provided that the variant sequence retains the ability to bind Glypican-3 when coupled to a complementary V L Domain or V Hdomain binding, if appropriate).

[0308] The percent identity between two polypeptide sequences can be readily determined using programs such as BLAST, which is freely available at http: / / blast.ncbi.nlm.nih.gov.

[0309] Transmembrane domain

[0310] CAR can also include a transmembrane domain that spans the membrane. It can include a hydrophobic alpha helix. The transmembrane domain can be derived from CD28, which provides good receptor stability.

[0311] Intracellular T cell signaling domain (intracellular domain)

[0312] The intracellular domain is the signaling portion of the CAR. After recognizing the antigen, the receptor aggregates and transmits the signal to the cell. The most commonly used intracellular domain component is CD3-ζ, which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen binding. CD3-ζ may not provide a fully effective activation signal, and additional co-stimulatory signals may be required. For example, chimeric CD28 and OX40 can be used together with CD3-ζ to transmit proliferation / survival signals, or all three can be used together.

[0313] The intracellular domain of CAR may include the CD28 intracellular domain as well as the OX40 and CD3-ζ intracellular domains.

[0314] The variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with such intracellular domain, provided that the sequence provides an effective transmembrane domain / intracellular T cell signaling domain.

[0315] Signal peptide

[0316] The CAR may comprise a signal peptide such that when the CAR is expressed in a cell (such as a T cell), the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface where it is expressed.

[0317] The core of the signal peptide may contain a long stretch of hydrophobic amino acids that have a tendency to form a single alpha-helix. The signal peptide may begin with a short stretch of positively charged amino acids, which helps enforce the correct topology of the polypeptide during the translocation process. At the end of the signal peptide, there is usually a stretch of amino acids that are recognized and cleaved by a signal peptidase. The signal peptidase can cleave during or after the translocation process is completed to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease.

[0318] The signal peptide may be located at the amino terminus of the molecule.

[0319] CAR may have the following general formula:

[0320] Signal peptide-phosphatidylinositol proteoglycan-3 binding domain-spacer domain-transmembrane domain-intracellular T cell signaling domain.

[0321] Spacer

[0322] The CAR may comprise a spacer sequence to connect the Glypican-3 binding domain and the transmembrane domain and to spatially separate the Glypican-3 binding domain from the intracellular domain. The flexible spacer enables the Glypican-3 binding domain to be oriented in different directions to achieve Glypican-3 binding.

[0323] The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge or a CD8 handle, or a combination thereof.The spacer may alternatively comprise an alternative sequence having similar length and / or domain spacing properties as an IgG1 Fc region, an IgG1 hinge or a CD8 handle.

[0324] The human IgG1 spacer can be altered to remove the Fc binding motif.

[0325] Examples of amino acid sequences of these spacers are given below: Hinge-CH2CH3 of human IgG1, human CD8 handle, human IgG1 hinge, IgG1 hinge-Fc, and IgG1 hinge-Fc modified to remove the Fc receptor recognition motif.

[0326] Diagnostic kits

[0327] We also provide diagnostic kits for detecting cancer (including hepatocellular carcinoma) in an individual or susceptibility to cancer in an individual.

[0328] The diagnostic kit may include means for detecting the expression, amount or activity of GPC-3 in an individual by any of the methods described herein. Thus, the diagnostic kit may include any one or more of the following: an anti-GPC-3 antibody, an antibody capable of binding to the same epitope as monoclonal antibody 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 or 1E1, a monoclonal antibody 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 or 1E1. 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, or 1E1, 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, Fab from 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, or 1E1, scFv comprising antibody 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, or 1E1 C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 or 1E1, or humanized monoclonal antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 or 1E1, etc.

[0329] The diagnostic kit may include instructions for use or other labels. The diagnostic kit may also include tools for treating or preventing cancer (e.g., hepatocellular carcinoma), such as any composition described herein, or any tool known in the art for treating cancer (e.g., hepatocellular carcinoma). In particular, the diagnostic kit may include, for example, the anti-GPC-3 antibody obtained by screening.

[0330] Hepatocellular Carcinoma / Liver Cancer

[0331] The anti-GPC-3 antibodies and binding agents disclosed herein can be used to treat, prevent, or ameliorate cancer, such as liver cancer.

[0332] Liver cancer (hepatocellular carcinoma, HCC) is the fifth most common malignant tumor in the world and the third leading cause of cancer mortality worldwide (Ferley J, Bray F. Pisani P, Parkin DM. GLOBOCAN 2002: Cancer Incidence, Mortality and Prevalence Worldwide. IARC Cancer Base No5 version 20. IARC Press, Lyon, France; 2004).

[0333] Hepatocellular carcinoma (HCC) has replaced gastric cancer as the third most common cancer among Singaporean men, accounting for 8.1% of all cancers diagnosed from 1998 to 2002 (Seow A, KW, Chia KS, Shi LM, Lee HP, Shanmugaratnam K (2004). “Trends in Cancer Incidence in Singapore 1968-2002.” Singapore Cancer Registry (Report no.6)). In Asia, about 250,000 new cases are reported each year in China alone, and HCC is endemic. Globally, it is the leading cause of cancer death (Bosch, FX, J. Ribes, et al. (2005). "Epidemiology of hepatocellular carcinoma." Clin Liver Dis 9 (2): 191-211, v.). Even in countries such as the United States and Europe where the prevalence of HCC is relatively low, the incidence of HCC is increasing (El-Serag, HB and A.C. Mason (1999). "Rising incidence of hepatocellular carcinoma in the United States." NEngl J Med 340(10):745-50).

[0334] More than 90% of HCCs are diagnosed at an advanced stage and are often associated with liver cirrhosis. HCCs often exhibit highly aggressive clinical behavior, with most patients dying within 12 months of diagnosis (El-Serag HB. Hepatocellular carcinoma: an epidemiologic view. J Clin Gastroenterol 2002; 35(5 Suppl 2): ​​S72-8).

[0335] Patients are usually in an advanced stage of the disease where surgery is not an option; in cases where surgical resection is performed, the two-year recurrence rate is still as high as 50% (Nagasue N, Kohno H, Chang YC, Taniura H, Yamanoi A, Uchida M, et al. Liver resection for hepatocellular carcinoma. Results of 229 consecutive patients during 11 years. Ann Surg 1993; 217: 375-84; Yamamoto J, Kosuge T, Takayama T, Shimada K, Yamasaki S, Ozaki H, et al. Recurrence of hepatocellular carcinoma after surgery. Br J Surg 1996; 83: 1219-22).

[0336] HCC is a cancer that is relatively refractory to chemotherapy. There is no particularly effective single-drug chemotherapy or multi-drug chemotherapy. Doxorubicin is the most commonly used chemotherapy drug for metastatic HCC, with a response rate of less than 20% (Johnson, PJ, R. Williams, et al. (1978). "Induction of remission in hepatocellular carcinoma with doxorubicin." Lancet 1(8072):1006-9) and there was no statistically significant survival advantage. Recent results from a trial using a 3-drug combination chemotherapy and interferon showed a response rate of 20.9% and a median survival of 8.67 months (Yeo, W., TS Mok, et al. (2005), "Arandomized phase III study of doxorubicin versus cisplatin / interferon alpha-2b / doxorubicin / fluorouracil (PIAF) combination chemotherapy for unresectable hepatocellular carcinoma." JNatl Cancer Inst 97(20):1532-8). This regimen did not show improved survival compared with doxorubicin alone and was associated with more toxicity.

[0337] Recent results of randomized clinical trials show that standard chemotherapy regimens have little effect in prolonging the survival of HCC patients (Yeo W, Mok TS, Zee B, Leung TW, Lai PB, Lau WY, et al. Arandomized phase III study of doxorubicin versus cisplatin / interferon alpha-2b / doxorubicin / fluorouracil (PIAF) combination chemotherapy for unresectable hepatocellular carcinoma. J Natl Cancer Inst 2005; 97: 1532-38).

[0338] In addition to tumor recurrence and metastasis, peritoneal ascites is another important cause of morbidity in patients with advanced HCC, which is usually caused by impaired liver function, portal vein obstruction, and increased endothelial cell permeability.

[0339] Prevention and treatment methods

[0340] Monoclonal antibodies 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1 can be used to treat disease in humans or other animals.

[0341] We show in the examples that this anti-GPC-3 antibody has anti-tumor activity. Specifically, the examples show that the anti-GPC-3 antibody can be used to treat and prevent hepatocellular carcinoma.

[0342] We disclose methods of treating cancer, including hepatocellular carcinoma. Methods of preventing (ie, prophylaxis) cancer, such as hepatocellular carcinoma, are also amenable to the same or similar methods.

[0343] Therefore, we provide the use of anti-GPC-3 antibodies in the treatment or prevention of proliferative diseases. Proliferative diseases may include cancer. Anti-GPC-3 antibodies can be used as drugs or therapies to treat cancer (such as hepatocellular carcinoma). They can be used to prevent the progression of such infections or diseases.

[0344] In general, our methods involve manipulating cells by modulating (e.g., downregulating) the expression, amount, or activity of GPC-3. Treatment may generally include contacting a tumor cell or a cell suspected of being a tumor cell with an anti-GPC-3 antibody. The methods may involve exposing a patient to an anti-GPC-3 antibody or variant thereof described herein.

[0345] The cells may be exposed to or additionally exposed to an anti-cancer agent, such as an antibody or other molecule known to be effective in preventing or treating cancer (e.g., liver cancer). In this case, the cells may be exposed to both the antibody and the anti-cancer agent, or sequentially to the antibody and the anti-cancer agent alone. The exposure may be repeated multiple times. Any combination of any amount or relative amount of anti-GPC-3 antibody and other agent may be used at any exposure time.

[0346] Therefore, we provide the use of a combination of an anti-GPC-3 antibody and an anti-cancer agent as described above in the treatment of cancer, such as liver cancer.

[0347] The cell may be a single cell, or may be a cluster of cells. The cell may be in vivo in an organism. The organism may be an organism known to have cancer, or may be an organism suspected of having cancer, or may be an organism susceptible to cancer. The treatment may comprise administering the one or more antibodies to the organism. As described above, a single antibody may be administered, or a combination of an anti-GPC-3 antibody and an anti-cancer agent may be administered. As described above, administration may be simultaneous administration or sequential administration. Thus, the treatment may comprise administering the anti-GPC-3 antibody and the anti-cancer agent to the individual simultaneously or sequentially.

[0348] To this end, a number of criteria reflecting treatment or prevention or patient health progress may be specified. Useful criteria for cancer cases may include TNM staging, Okuda system, CLIP score, BCLC staging known in the art. For example, measurement of tumor size and severity of cirrhosis, such as by measuring ascites volume, serum albumin and bilirubin levels, may be used to assess treatment.

[0349] The treatment of liver cancer is described in detail in Liu et al (2015), Cold Spring Harb Perspect Med. 2015 Sep; 5 (9).

[0350] Thus, as an example, a treated individual may show a reduction in such symptoms as measured by a suitable assay or test. A treated individual may show, for example, a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more reduction in one or more symptoms compared to an untreated individual.

[0351] For example, if the symptoms associated with the disease are significantly inhibited (i.e., 50% or more) relative to the control, the patient's disease can be defined as being "treated". The inhibition can be at least 75% relative to the control, such as 90%, 95% or 100% relative to the control. The term "treatment" is also meant to include the prevention or alleviation of cancer, such as liver cancer.

[0352] Antibody treatment methods involving the use of anti-GPC-3 antibodies can be combined with other methods for treating such diseases, including conventional drug-based methods.

[0353] treat

[0354] The mainstay of treatment is supportive care. Patients are encouraged to continue taking oral fluids, especially oral fluids. If patients are unable to maintain oral intake, they may need additional intravenous fluids to prevent dehydration and significant hemoconcentration. If platelet levels drop significantly, platelet transfusions are indicated.

[0355] Pharmaceutical composition

[0356] As disclosed herein, anti-GPC-3 antibodies can be used to treat or prevent cancer, such as liver cancer.

[0357] Anti-GPC-3 antibodies can be administered by a variety of routes, including enteral, parenteral and topical administration routes. For example, suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophoretic, intravenous, intramuscular, intraperitoneal, intranasal, subdural, rectal, etc.

[0358] According to other embodiments, a composition comprising an anti-GPC-3 antibody and a pharmaceutically acceptable carrier or excipient is provided for treating or preventing cancer, such as liver cancer.

[0359] Suitable pharmaceutically acceptable excipients include processing agents and drug delivery modifiers and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, glucose, hydroxypropyl-p-cyclodextrin, polyvinylpyrrolidone, low melting point wax, ion exchange resins, etc., and any combination of two or more thereof. Other suitable pharmaceutically acceptable excipients are described in "Remington's Pharmaceutical Sciences," Mack Pub. Co., New Jersey (1991), which is incorporated herein by reference.

[0360] The pharmaceutical composition containing the anti-GPC-3 antibody can be in any form suitable for the intended method of administration, including, for example, a solution, suspension or emulsion. Liquid carriers are generally used to prepare solutions, suspensions and emulsions. Liquid carriers expected to be used in practice include, for example, water, saline, a pharmaceutically acceptable organic solvent or multiple organic solvents, pharmaceutically acceptable oils or fats, and the like, and mixtures of two or more thereof. The liquid carrier may contain other suitable pharmaceutically acceptable additives, such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickeners, viscosity modifiers, stabilizers, and the like. Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as ethylene glycol.

[0361] Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, etc. For parenteral administration, the carrier may also be an oily ester, such as ethyl oleate, isopropyl myristate, etc. The composition may also be in the form of microparticles, microcapsules, liposomes, etc., and combinations of any two or more thereof.

[0362] The anti-GPC-3 antibody can be administered orally, parenterally, sublingually, by inhalation spray, rectally or topically in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles as required. Topical administration may also involve the use of transdermal administration, such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

[0363] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be prepared according to known techniques using suitable dispersants or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as 1,3-propylene glycol solutions. Acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, it has been found that fatty acids (such as oleic acid) can be used to prepare injections.

[0364] Suppositories for rectal administration of drugs can be prepared by mixing the drug with a suitable non-irritating excipient such as cocoa butter and polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.

[0365] Orally administered solid dosage forms can include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound can be mixed with at least one inert diluent (such as sucrose, lactose or starch). According to routine, this dosage form can also include other substances in addition to the inert diluent, such as lubricants such as magnesium stearate. In the case of capsules, tablets and pills, the dosage form can also include a buffer. Tablets and pills can also be prepared with enteric coatings.

[0366] Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art (such as water). Such compositions may also include adjuvants such as wetting agents, emulsifying agents and suspending agents, cyclodextrins, and sweeteners, flavoring agents and fragrances.

[0367] According to other embodiments, we provide methods of inhibiting any activity of GPC-3 in a human or animal subject, the method comprising administering to the subject an anti-GPC-3 antibody (or a composition comprising such a compound) in an amount effective to inhibit the relevant activity in the subject. Other embodiments provide methods of treating cancer, such as liver cancer, in a human or animal subject, comprising administering to the cell or to the human or animal subject a compound or composition as described herein in an amount effective to inhibit the activity of GPC-3 in the cell or subject. The subject may be a human or non-human animal subject. Inhibition of protein activity includes detectable suppression of the activity of the relevant protein compared to a control or compared to the expected protein activity.

[0368] An effective amount of an anti-GPC-3 antibody generally includes any amount sufficient to inhibit the activity of the relevant protein detectably by any assay described herein, by other assays known to those of ordinary skill in the art, or by detecting amelioration of symptoms in a subject with cancer, such as liver cancer.

[0369] Successful treatment of a subject may result in alleviation or relief of symptoms in a subject suffering from a medical or biological disease, such as arresting further development of the disease or preventing the disease. Thus, for example, treatment of cancer such as liver cancer may result in alleviation of symptoms as described above.

[0370] The amount of active ingredient that can be combined with carrier materials to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. However, it should be understood that the specific dosage level for any particular patient will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, route of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The therapeutically effective amount in a given situation can be easily determined by routine experimentation and is within the skill and judgment of an ordinary clinician.

[0371] The therapeutically effective dose is generally about 10 μg / kg / day to 100 mg / kg / day of anti-GPC-3 antibody, such as about 25 μg / kg / day to about 20 mg / kg / day or about 50 μg / kg / day to about 2 mg / kg / day of anti-GPC-3 antibody, which can be administered in one or more doses.

[0372] Anti-GPC-3 antibodies can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by monolayer or multilayer hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. In addition to the compound, the composition of the present invention in liposome form may contain stabilizers, preservatives, excipients, etc. Lipids that can be used include natural and synthetic phospholipids and phosphatidylcholines (lecithins). Methods for forming liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, NW, p. 33 et seq (1976).

[0373] Although anti-GPC-3 antibodies can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other agents for treating a disease. Representative agents used in combination with anti-GPC-3 antibodies for treating cancers such as liver cancer include, for example, Sorafenib, Lenvatinib, and Regorafenib.

[0374] When an additional active agent is used in combination with an anti-GPC-3 antibody, the additional active agent can generally be used in a therapeutic amount as set forth in PHYSICIANS' DESK REFERENCE (PDR) 53rd Edition (1999), which is incorporated herein by reference, or in a therapeutically effective amount known to those of ordinary skill in the art.

[0375] The anti-GPC-3 antibody and other therapeutically active agent may be administered at the recommended maximum clinical dose or at a lower dose. The dosage level of the active anti-GPC-3 antibody in the composition may be varied to obtain the desired therapeutic response depending on the route of administration, severity of the disease, and patient response. The combination may be administered as separate compositions or as a single dosage form containing both agents. When administered as a combination, the therapeutic agents may be formulated as separate compositions administered at the same time or at different times, or the therapeutic agents may be administered as a single composition.

[0376] Bioavailability

[0377] In some embodiments, the compounds disclosed herein (and combinations) are orally bioavailable. Oral bioavailability refers to the proportion of an oral drug that reaches the systemic circulation. Factors that determine the oral bioavailability of a drug are dissolution, membrane permeability, and metabolic stability. Typically, oral bioavailability is determined first using a screening cascade of in vitro techniques and then using in vivo techniques.

[0378] Dissolution, in vitro solubility experiments can be performed at an appropriate pH to simulate the GIT to predict the dissolution of the drug in the aqueous contents of the gastrointestinal tract (GIT). In some embodiments, the anti-GPC-3 antibody can have a minimum solubility of 50 mg / ml. Solubility can be determined by standard procedures known in the art (such as described in Adv. Drug Deliv. Rev. 23, 3-25, 1997).

[0379] Membrane permeability refers to the passage of the compound through the cells of the GIT. Lipophilicity is a key property that predicts membrane permeability and is determined by in vitro Log D 7.4 The LogD of the anti-GPC-3 antibody was determined by measurement. 7.4 It may range from -2 to +4 or from -1 to +2. The log D may be determined by standard procedures known in the art, such as described in J. Pharm. Pharmacol. 1990, 42:144.

[0380] In the presence of efflux transporters such as p-glycoprotein, cell monolayer assays such as CaCO 2 ) greatly increases the prediction of favorable membrane permeability, the so-called caco-2 flux. The caco-2 flux of anti-GPC-3 antibodies can be greater than 2×10 -6 cms -1 , for example, greater than 5×10 -6 cms -1 The caco flux value can be determined by standard procedures known in the art (eg, as described in J. Pharm. Sci, 1990, 79, 595-600).

[0381] Metabolic stability refers to the ability of the GIT or liver to metabolize a compound during absorption: first pass effect. Detection systems such as microsomes and hepatocytes predict metabolic potential. In some embodiments, the compounds of the examples may show a metabolic stability commensurate with a liver extract of less than 0.5 in the detection system. Examples of detection systems and data processing are described in Curr. Opin. Drug Disc. Devel., 201, 4, 36-44, Drug Met. Disp., 2000, 28, 1518-1523.

[0382] Due to the interaction of the above processes, further support for the oral bioavailability of drugs in humans can be obtained through in vivo experiments in animals. In these studies, the absolute bioavailability is determined by administering the compounds alone or in admixture by the oral route. The intravenous route is also used for absolute determination (% absorption). Examples of oral bioavailability assessments in animals can be found in Drug Met. Disp., 2001, 29, 82-87; J. Med Chem., 1997, 40, 827-829, Drug Met. Disp., 1999, 27, 221-226.

[0383] The term "pharmaceutically acceptable carrier" as used herein generally refers to an organic or inorganic material that cannot react with the active ingredient. Carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; stearic acid; magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; agar; alginic acid; pyrogen-free water; isotonic saline; and phosphate buffer; skim milk powder; and other non-toxic compatible substances used in pharmaceutical preparations. Wetting agents and lubricants such as sodium lauryl sulfate, and colorants, flavoring agents, lubricants, excipients, tableting agents, stabilizers, antioxidants and preservatives may also be present.

[0384] The term "therapeutically effective amount" as used herein generally refers to the amount of an agent sufficient to achieve the treatment defined herein when applied to a subject in need of such treatment, such as the amount of a compound as an active ingredient. The therapeutically effective amount of the compound, salt, derivative, isomer or enantiomer of the present invention will depend on a variety of factors, including, for example, the age and weight of the subject, the exact condition to be treated and its severity, the nature of the formulation and the route of administration, which will ultimately be determined by the attending physician or veterinarian. However, the effective amount of the compound of the present invention for treating diseases associated with bacterial or viral infections, particularly bacterial meningitis, is generally in the range of about 10 mg / kg to about 40 mg / kg of subject (mammal) body weight per day, more generally about 40 mg / kg body weight per day. Therefore, for an adult subject of 70 kg, the actual amount per day is generally about 2,800 mg, and the amount can be administered in a single dose per day or more generally in multiple (e.g., two, three, four, five or six) sub-doses per day, so that the total daily dose is the same. The effective amount of the salt of the present invention can be determined as the ratio of the effective amount of the compound itself.

[0385] The term "treatment" as used herein refers to any treatment of a condition or disease in animals, particularly mammals, and more particularly humans, and includes: preventing the condition or disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; inhibiting the disease or condition, i.e., arresting its development; alleviating the disease or condition, i.e., causing regression of the condition; or alleviating a condition caused by the disease, i.e., a symptom of the disease.

[0386] Chemical derivatives

[0387] As used herein, the term "derivative" or "derivative" includes chemical modifications of a compound. Examples of such chemical modifications are replacement of hydrogen with a halogen group, an alkyl group, an acyl group, or an amino group.

[0388] Chemical modification

[0389] In one embodiment, the compound may be a chemically modified compound.

[0390] Chemical modification of compounds can enhance or weaken hydrogen bonding interactions, charge interactions, hydrophobic interactions, van der Waals interactions, or dipole interactions between the compound and the target.

[0391] In one aspect, the identified compounds can serve as models (eg, templates) for the development of other compounds.

[0392] individual

[0393] Delivering a compound to a subject. As used herein, the term "subject" refers to a vertebrate, particularly a member of a mammalian species. The term includes, but is not limited to, livestock, sports animals, primates, and humans.

[0394] Example

[0395] Example 1. Discovery

[0396] Using phage display technology, recombinant human GPC-3 protein was used to isolate binders from the Fab sequence library constructed by SIgN.

[0397] Of the 570 clones screened, 24 clones that showed the highest binding to GPC-3 in ELISA were isolated and cloned as IgG 1 Type for further characterization: 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1.

[0398] Example 2. Binding affinity to GPC-3

[0399] Twenty-four antibody clones were tested in 3 batches against recombinant human GPC-3 protein in ELISA to evaluate binding affinity to the target, using humanized GC33 clone (Chugai pharmaceutical, Roche) as a reference control antibody ( Figure 1 A. Figure 1 B and Figure 1 C).

[0400] Except for clones 1E1 and 3D12, all other 22 clones showed medium to high affinity to human GPC-3 ( Figure 1 D).

[0401] Example 3. Specific binding of anti-GPC-3 antibodies to GPC-3 expressing cells

[0402] Using HepG2(GPC-3 高 )、Hep3B(GPC-3 中 ) and SK-Hep1(GPC-3 阴性 ) cells to measure the ability of anti-GPC-3 antibodies to specifically recognize and bind to cell surface GPC-3. HepG2 and Hep3B cells consistently express GPC-3, while SK-Hep1 cells do not express GPC-3.

[0403] Briefly, HepG2(GPC-3 高 )、Hep3B(GPC-3 中 ) and SK-Hep1(GPC-3 阴性 ) cells were seeded in a 96-well round-bottom plate at 50,000 cells per well in a cell suspension and then incubated with different concentrations of anti-GPC-3 antibodies. Binding was measured by flow cytometry using labeled secondary antibodies.

[0404] In this assay, most anti-GPC-3 antibodies strongly bound to HepG2 and Hep3B cells expressing GPC-3, but did not bind or bound weakly to GPC-3 negative SK-Hep1 cells ( Figure 2 ). Clones 1C4, 1D2, 1D3, 1D8, 1F4, 1H1, 1H10, 2B5, 2F1, 3A9, 3D12, 4A5, 4H1, and 5C4 showed highly specific binding to GPC-3, as they did not bind to SK-Hep1 cells even at the highest antibody concentration (20 nM). This suggests that these antibodies bind to the cell surface in a highly specific manner that is dependent on GPC-3 expression.

[0405] Example 4. Cross-species reactivity of anti-GPC-3 antibodies against cynomolgus monkey and mouse GPC-3 proteins Since Glypican-3 is a highly conserved gene, we performed a cross-reactivity ELISA to evaluate the cross-species reactivity of our anti-GPC-3 antibodies, in which recombinant human, cynomolgus monkey, and mouse GPC-3 proteins were all biotinylated and used in binding ELISA to test their binding to each anti-GPC-3 IgG 1 Clonal combination.

[0406] Except for clone 1D8 and humanized GC33, all other anti-GPC-3 IgG 1 All clones showed cross-reactivity to cynomolgus monkey GPC-3 and mouse GPC-3 ( Figure 3 ), indicating that these antibodies recognize a common epitope of GPC-3 protein spanning human antigen, cynomolgus monkey antigen, and mouse antigen.

[0407] Subsequently, the ability of anti-GPC-3 antibodies to specifically recognize and bind to mouse GPC-3 on the cell surface was measured using mouse GPC-3 stably transduced Hepa1-6 cells (designated "Hepa1-6-mGPC-3") and parental Hepa1-6 cells.

[0408] Most anti-GPC-3 antibodies strongly bound to Hepa1-6-mGPC-3 cells at 20 nM staining concentration, but not to parental Hepa1-6 cells ( Figure 4 A).

[0409] Clones 1C4, 1D2, 1D3, 1F1, 1H1, 1H10, 2B5, 2F1, 3A9, 3C6, 3G12, 4A12, 4G11, 4H1, and 5C4 showed highly specific binding to mouse GPC-3, although their cell binding ratio to parental Hepa1-6 cells was not high ( Figure 4 B).

[0410] Interestingly, although clones 1F4, 3D12, and 4F9 showed positive binding signals in the cross-reactive binding ELISA ( Figure 3 ), but they were unable to effectively stain the mouse GPC-3 antigen expressed on the cell surface ( Figure 4 ).

[0411] Example 5. In vitro activity: Induction of antibody-dependent cell-mediated cytotoxicity (ADCC) To evaluate the ability of anti-GPC-3 antibodies to induce ADCC, the cells were cultured in the presence or absence of each of the different anti-GPC-3 IgGs. 1In the case of clones (2.5 μg / ml), GPC-3 positive HepG2 cells were mixed with naive natural killer (NK) cells at a ratio of 1:2.5 (E:T ratio = 2.5:1).

[0412] The cell index values ​​were then measured continuously at 15-minute intervals for 44 hours using the xCellingence RTCA system. In this assay, the cell index values ​​of tumor cells are measured by the impedance of the current through the transistor plate caused by tumor cell adhesion. Endpoint cytotoxicity was calculated and is shown in Figure 5 middle.

[0413] Compared with NK cell-induced cytotoxicity alone, a significant increase in killing was observed in more than half of the anti-GPC-3 antibody clones, with clones 1C4, 1D2, and 4H1 showing the highest antibody-dependent cell-mediated cytotoxicity.

[0414] Example 6. Use in chimeric antigen receptor (CAR) T cell constructs

[0415] The antibody sequences can also be used to construct anti-GPC-3 CAR T cells.

[0416] Fifteen clones were selected to express as CAR in the second generation CAR format (CD3ζ+4-1BB intracellular domain).

[0417] To evaluate the function of these anti-GPC-3CAR T cells, the cells were transfected with IL-2 (50 units / ml) by TransAct TM T cells from healthy donors were activated with (CD3 / CD28 agonists) for 24 hours and transduced with the CAR gene via a lentiviral-based transduction process. 72 hours after transduction, the efficacy of these anti-GPC-3CAR T cells was tested for T cell-mediated cytotoxicity using the xCELLigence RTCA system.

[0418] CAR T cells have been shown to be highly effective in killing tumor cells.

[0419] While mock-transduced T cells showed cytolysis of 10% to 15% of GPC-3-expressing HepG2 cells, anti-GPC-3CAR T cells were able to kill 30% to 100% of HepG2 cells ( Figure 6 Within 48 hours of co-culture, anti-GPC-3 CAR T cells based on clones 1C4, 1D3, 1F1, 2F1, 3C6, 1D2, 1H1, 2B5, 1D8, 4A5, 4H1, and 5C4 could lyse 90% to 100% of GPC-3 高HepG2 cells. Killing by these CAR T cells was highly specific for antigen presentation, as the same CAR T cells only showed expression of GPC-3 阴性 Very slight background killing of SK-Hepl cells (10% to 30%).

[0420] In addition, in the presence of anti-GPC-3CAR T cells and GPC-3 高 HepG2 cells or GPC-3 阴性 After 24 and 48 h of co-culture with SK-Hep1 cells, cell culture supernatants were collected and subjected to ELISA to evaluate cytokine (interferon-γ and IL-2) release by anti-GPC-3CAR T cells. 高 When co-cultured with HepG2 cells, anti-GPC-3 CAR T cells based on clones 1C4, 1D3, 1F1, 2F1, 3C6, 1D2, 1H1, 2B5, 1D8, 4A5, 4H1, and 5C4 secreted large amounts of interferon-γ and IL-2. 阴性 When SK-Hep1 cells were injected, the secretion levels of interferon-γ and IL-2 were extremely low, indicating that these anti-GPC-3CAR T cells had higher specificity in responding to and killing GPC-3 expressing cells ( Figure 7 ).

[0421] Interestingly, Figure 6 and Figure 7 All showed moderate nonspecific off-target reactivity of anti-GPC-3CAR T cells expressing mouse GC33 clone.

[0422] Example 7. Nucleotide and amino acid sequences of antibodies

[0423] The amino acid sequences and nucleotide sequences of 24 fully human monoclonal antibodies and their derivatives that can specifically bind to Glypican-3 and trigger killing of antigen-expressing cells are as follows.

[0424] The antibodies are 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4 and 1E1.

[0425] Sequence

[0426] Amino acid sequence

[0427] CDRs are shown in bold and in bold (CDR1), italic (CDR2), orUnderline (CDR3) is highlighted.

[0428]

[0429]

[0430] Table D1. Heavy chain and light chain amino acid sequence ID number (sequence as follows) Heavy chain variable region of human IGG1 (V H )Amino acid sequence

[0431] SEQ ID NO: 1: amino acid sequence of the heavy chain variable region of 5C4

[0432] SEQ ID NO: 3: amino acid sequence of the heavy chain variable region of 4H1

[0433]

[0434] SEQ ID NO: 5: amino acid sequence of the heavy chain variable region of 1D2

[0435]

[0436] SEQ ID NO:7:1C4 heavy chain variable region amino acid sequence

[0437]

[0438] SEQ ID NO:9:2B5 heavy chain variable region amino acid sequence

[0439]

[0440] SEQ ID NO: 11: amino acid sequence of the heavy chain variable region of 1F1

[0441]

[0442] SEQ ID NO: 13: amino acid sequence of the heavy chain variable region of 1H1

[0443]

[0444] SEQ ID NO: 15: amino acid sequence of the heavy chain variable region of 4A5

[0445]

[0446] SEQ ID NO: 17: amino acid sequence of the heavy chain variable region of 1D8

[0447]

[0448] SEQ ID NO: 19: amino acid sequence of the heavy chain variable region of 1D3

[0449]

[0450] SEQ ID NO: 21: amino acid sequence of the heavy chain variable region of 2F1

[0451]

[0452] SEQ ID NO: 23: amino acid sequence of the heavy chain variable region of 3C6

[0453]

[0454] SEQ ID NO: 25: amino acid sequence of the heavy chain variable region of 3D12

[0455]

[0456] SEQ ID NO: 27: amino acid sequence of the heavy chain variable region of 3A9

[0457]

[0458] SEQ ID NO: 29: amino acid sequence of the heavy chain variable region of 1F4

[0459] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTVADTAVYYCAR LYKRRPFDY QUR

[0460] SEQ ID NO:31: amino acid sequence of the heavy chain variable region of 1H10

[0461] QLQLQESGGGLVQPGGSLRLSCAASGFTFSSYVMSWVRQAPGKGLEWVSTISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCVM GWYFDL WGRGTL

[0462] SEQ ID NO: 33: amino acid sequence of the heavy chain variable region of 3C12

[0463] QVTLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLALIYWNDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCAH SRIAARRVLDY QUR

[0464] SEQ ID NO: 35: amino acid sequence of the heavy chain variable region of 4G11

[0465] QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCAR DTSGVAFDY QUR

[0466] SEQ ID NO: 37: amino acid sequence of the heavy chain variable region of 4A12

[0467] DVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAE GRIQLDY QUR

[0468] SEQ ID NO:39: amino acid sequence of the heavy chain variable region of 1A12

[0469] QVQLQQWGAGLLKPSETLSLTCAAYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCAR LYKRRPFDY QUR

[0470] SEQ ID NO: 41: amino acid sequence of the heavy chain variable region of 3G12

[0471]

[0472] SEQ ID NO: 43: amino acid sequence of the heavy chain variable region of 4F9

[0473]

[0474] SEQ ID NO: 45: amino acid sequence of the heavy chain variable region of 4G4

[0475]

[0476] SEQ ID NO: 47: amino acid sequence of the heavy chain variable region of 1E1

[0477] The light chain variable region of human IgG1 (V L )Amino acid sequence

[0478] SEQ ID NO: 2:5C4 light chain variable region amino acid sequence

[0479]

[0480] SEQ ID NO: 4: Light chain variable region amino acid sequence of 4H1

[0481]

[0482] SEQ ID NO:6:1D2 light chain variable region amino acid sequence

[0483]

[0484] SEQ ID NO: 8:1C4 light chain variable region amino acid sequence

[0485]

[0486] SEQ ID NO: 10: Light chain variable region amino acid sequence of 2B5

[0487]

[0488] SEQ ID NO: 12: Light chain variable region amino acid sequence of 1F1

[0489]

[0490] SEQ ID NO: 14: Light chain variable region amino acid sequence of 1H1

[0491]

[0492] SEQ ID NO: 16: Light chain variable region amino acid sequence of 4A5

[0493]

[0494] SEQ ID NO: 18: Light chain variable region amino acid sequence of 1D8

[0495]

[0496] SEQ ID NO: 20:1D3 light chain variable region amino acid sequence

[0497]

[0498] SEQ ID NO: 22: Light chain variable region amino acid sequence of 2F1

[0499]

[0500] SEQ ID NO: 24: Light chain variable region amino acid sequence of 3C6

[0501]

[0502] SEQ ID NO: 26: Light chain variable region amino acid sequence of 3D12

[0503]

[0504] SEQ ID NO: 28: Light chain variable region amino acid sequence of 3A9

[0505]

[0506] SEQ ID NO:30: Light chain variable region amino acid sequence of 1F4

[0507]

[0508] SEQ ID NO:32: Light chain variable region amino acid sequence of 1H10

[0509]

[0510] SEQ ID NO: 34: Light chain variable region amino acid sequence of 3C12

[0511]

[0512] SEQ ID NO: 36: Light chain variable region amino acid sequence of 4G11

[0513]

[0514] SEQ ID NO:38: Light chain variable region amino acid sequence of 4A12

[0515]

[0516] SEQ ID NO:40: Light chain variable region amino acid sequence of 1A12

[0517]

[0518] SEQ ID NO: 42: Light chain variable region amino acid sequence of 3G12

[0519]

[0520] SEQ ID NO: 44: Light chain variable region amino acid sequence of 4F9

[0521]

[0522] SEQ ID NO: 46:4G4 light chain variable region amino acid sequence

[0523]

[0524] SEQ ID NO: 48: Light chain variable region amino acid sequence of 1E1

[0525] Nucleotide sequence

[0526] CDRs are shown in bold and in bold (CDR1), italic (CDR2), or Underline (CDR3) is highlighted.

[0527]

[0528]

[0529] Table D2. Heavy chain and light chain nucleotide sequence ID number (sequence as follows) Heavy chain variable region of human IGG1 (V H ) Nucleotide sequence

[0530] SEQ ID NO: 49: Heavy chain variable region nucleotide sequence of 5C4

[0531]

[0532] SEQ ID NO: 51: Heavy chain variable region nucleotide sequence of 4H1

[0533]

[0534] SEQ ID NO: 53: Heavy chain variable region nucleotide sequence of 1D2

[0535]

[0536] SEQ ID NO: 55: Heavy chain variable region nucleotide sequence of 1C4

[0537]

[0538] SEQ ID NO: 57: Heavy chain variable region nucleotide sequence of 2B5

[0539]

[0540]

[0541] SEQ ID NO: 59: Heavy chain variable region nucleotide sequence of 1F1

[0542]

[0543] SEQ ID NO:61: Heavy chain variable region nucleotide sequence of 1H1

[0544]

[0545] SEQ ID NO: 63: Heavy chain variable region nucleotide sequence of 4A5

[0546]

[0547]

[0548] SEQ ID NO: 65: Heavy chain variable region nucleotide sequence of 1D8

[0549]

[0550] SEQ ID NO: 67: Heavy chain variable region nucleotide sequence of 1D3

[0551]

[0552] SEQ ID NO: 69: Heavy chain variable region nucleotide sequence of 2F1

[0553]

[0554]

[0555] SEQ ID NO: 71: Heavy chain variable region nucleotide sequence of 3C6

[0556]

[0557] SEQ ID NO: 73: Heavy chain variable region nucleotide sequence of 3D12

[0558]

[0559] SEQ ID NO: 75: Heavy chain variable region nucleotide sequence of 3A9

[0560]

[0561]

[0562] SEQ ID NO: 77: Heavy chain variable region nucleotide sequence of 1F4

[0563]

[0564] SEQ ID NO: 79: Heavy chain variable region nucleotide sequence of 1H10

[0565]

[0566] SEQ ID NO:81: Heavy chain variable region nucleotide sequence of 3C12

[0567]

[0568] SEQ ID NO: 83: Nucleotide sequence of heavy chain variable region of 4G11

[0569]

[0570] SEQ ID NO:85: Heavy chain variable region nucleotide sequence of 4A12

[0571]

[0572] SEQ ID NO:87: Heavy chain variable region nucleotide sequence of 1A12

[0573]

[0574] SEQ ID NO: 89: Nucleotide sequence of heavy chain variable region of 3G12

[0575]

[0576] SEQ ID NO:91: 4F9 heavy chain variable region nucleotide sequence

[0577]

[0578] SEQ ID NO:93: 4G4 heavy chain variable region nucleotide sequence

[0579]

[0580] SEQ ID NO:95: Heavy chain variable region nucleotide sequence of 1E1

[0581] The light chain variable region of human IgG1 (V L ) Nucleotide sequence

[0582] SEQ ID NO: 50:5C4 light chain variable region nucleotide sequence

[0583]

[0584] SEQ ID NO: 52: 4H1 light chain variable region nucleotide sequence

[0585]

[0586] SEQ ID NO: 54: nucleotide sequence of the light chain variable region of 1D2

[0587]

[0588] SEQ ID NO: 56: nucleotide sequence of the light chain variable region of 1C4

[0589]

[0590] SEQ ID NO: 58: 2B5 light chain variable region nucleotide sequence

[0591]

[0592] SEQ ID NO:60: nucleotide sequence of the light chain variable region of 1F1

[0593]

[0594]

[0595] SEQ ID NO:62: nucleotide sequence of the light chain variable region of 1H1

[0596]

[0597] SEQ ID NO:64:4A5 light chain variable region nucleotide sequence

[0598]

[0599] SEQ ID NO: 66: nucleotide sequence of the light chain variable region of 1D8

[0600]

[0601]

[0602] SEQ ID NO: 68: nucleotide sequence of light chain variable region of 1D3

[0603]

[0604] SEQ ID NO: 70: nucleotide sequence of the light chain variable region of 2F1

[0605]

[0606] SEQ ID NO: 72: 3C6 light chain variable region nucleotide sequence

[0607]

[0608]

[0609] SEQ ID NO: 74: Nucleotide sequence of light chain variable region of 3D12

[0610]

[0611] SEQ ID NO: 76: 3A9 light chain variable region nucleotide sequence

[0612]

[0613] SEQ ID NO: 78: nucleotide sequence of light chain variable region of 1F4

[0614]

[0615] SEQ ID NO:80: Light chain variable region nucleotide sequence of 1H10

[0616]

[0617] SEQ ID NO:82: Nucleotide sequence of light chain variable region of 3C12

[0618]

[0619] SEQ ID NO: 84: 4G11 light chain variable region nucleotide sequence

[0620]

[0621] SEQ ID NO:86: 4A12 light chain variable region nucleotide sequence

[0622]

[0623]

[0624] SEQ ID NO:88:1A12 light chain variable region nucleotide sequence

[0625]

[0626] SEQ ID NO:90: ​​Nucleotide sequence of light chain variable region of 3G12

[0627]

[0628] SEQ ID NO:92: 4F9 light chain variable region nucleotide sequence

[0629]

[0630]

[0631] SEQ ID NO:94:4G4 light chain variable region nucleotide sequence

[0632]

[0633] SEQ ID NO:96: Light chain variable region nucleotide sequence of 1E1

[0634] Example 8. Use as anti-GPC-3 bispecific T cell engaging antibody

[0635] Anti-GPC-3 bispecific T cell engaging antibodies were constructed using anti-GPC-3 antibodies. In these constructs, the Fab region of one arm from each of the five anti-GPC-3 antibody clones (1C4, 1D2, 2B5, 4H1, 5C4) was replaced with an anti-CD3 scFv fragment as shown below.

[0636] Clone <![CDATA[Heavy chain (V H ) amino acid sequence]]> <![CDATA[Light chain (V L ) amino acid sequence]]> Anti-CD3 SEQ ID NO:97 SEQ ID NO:98

[0637] Table D3. Anti-CD3 heavy chain and light chain amino acid sequence ID number (sequence as follows)

[0638] Anti-CD3 heavy chain variable region (V H )Amino acid sequence

[0639] SEQ ID NO:97: Anti-CD3 heavy chain variable region amino acid sequence

[0640] DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSS

[0641] Variable region of anti-CD3 light chain (V L ) Amino acid sequence

[0642] SEQ ID NO:98: Amino acid sequence of variable region of anti-CD3 light chain

[0643] DIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIK

[0644] Clone <![CDATA[Heavy chain (V H ) nucleotide sequence]]> <![CDATA[Light chain (V L ) nucleotide sequence]]> Anti-CD3 SEQ ID NO:99 SEQ ID NO:100

[0645] Table D4. ID numbers of nucleotide sequences of anti-CD3 heavy chain and light chain (sequences are as follows)

[0646] Variable region of anti-CD3 heavy chain (V H ) Nucleotide sequence

[0647] SEQ ID NO:99: Nucleotide sequence of variable region of anti-CD3 heavy chain

[0648] gatgtgcagctggtgcagagcggggcagaggtgaaaaagcctggggcaagcgtcaaagtcagttgtaaagcctccggctacacattcactaggtatactatgcactgggtgcgccaggcccctggccaggggctggagtggatcggctacattaacccaagcagagggtacacaaattatgctgactccgtgaaaggcaggtttactatcaccaccgataagtccacctctacagcatacatggagctgagcagcctgcgaagcgaagacactgcaacctactattgcgcccggtactatgacgatcattactgtctggattattggggacagggcactaccgtgacagtctctagt

[0649] Variable region of anti-CD3 light chain (V L ) Nucleotide sequence

[0650] SEQ ID NO:100: Nucleotide sequence of variable region of anti-CD3 light chain

[0651] Gatattgtgctgacccagtctccagccacactgagtctgtcacccggcgaacgagccaccctgagctgccgggccagccagtccgtctcttacatgaactggtatcagcagaagcccggaaaagcccctaagcggtggatctacgacacaagcaaagtggcttccggcgtccccgcacgattcagtggctcagggagcggaactgactattctctgaccattaatagtctggaggctgaagatgccgctacctactattgtcagcagtggtcaagcaaccctctgacattcggggggggaactaaagtggaaatcaag

[0652] Anti-CD3 scFv amino acid sequence

[0653] SEQ ID NO:101: Anti-CD3 scFv amino acid sequence

[0654] DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIK

[0655] Anti-CD3 scFv nucleotide sequence

[0656] SEQ ID NO:102: Anti-CD3 scFv nucleotide sequence

[0657] gatgtgcagctggtgcagagcggggcagaggtgaaaaagcctggggcaagcgtcaaagtcagttgtaaagcctccggctacacattcactaggtatactatgcactgggtgcgccaggcccctggccaggggctggagtggatcggctacattaacccaagcagagggtacacaaattatgctgactccgtgaaaggcaggtttactatcaccaccgataagtccacctctacagcatacatggagctgagcagcctgcgaagcgaagacactgcaacctactattgcgcccggtactatgacgatcattactgtctggattattggggacagggcactaccgtgacagtctctagtggggagggaacatccactgggtctggagggagtggaggctcaggaggagcagacgatattgtgctgacccagtctccagccacactgagtctgtcacccggcgaacgagccaccctgagctgccgggccagccagtccgtctcttacatgaactggtatcagcagaagcccggaaaagcccctaagcggtggatctacgacacaagcaaagtggcttccggcgtccccgcacgattcagtggctcagggagcggaactgactattctctgaccattaatagtctggaggctgaagatgccgctacctactattgtcagcagtggtcaagcaaccctctgacattcggggggggaactaaagtggaaatcaag

[0658] Knob-in-hole mutations were introduced to promote correct heavy-chain pairing and LALA mutations were introduced to abrogate Fcγ receptor binding( Figure 8 A).

[0659] HepG2 (GPC-3 阳性 ), SK-Hep1 (GPC-3 阴性 ), and human T cells (CD3 阳性) These anti-GPC-3 bispecific T cell engaging antibodies were assayed for their ability to specifically recognize and bind to GPC-3 and human CD3 molecules on the cell surface. HepG2 cells and human T cells constitutively express high levels of GPC-3 and CD3, respectively, while SK-Hep1 cells do not express GPC-3 or CD3.

[0660] Briefly, HepG2 (GPC-3 阳性 ), SK-Hep1 (GPC-3 阴性 ), and human T cells (CD3 阳性 ) were seeded at 100,000 cells per well into the cell suspension of a 96-well round bottom plate and then incubated with different concentrations of the anti-GPC-3 bispecific T cell engaging antibodies. Binding was measured by flow cytometry using a labeled secondary antibody. In this assay, all 5 anti-GPC-3 bispecific T cell engaging antibodies strongly bound to HepG2 cells expressing GPC-3( Figure 8 B) as well as to human T cells expressing CD3( Figure 8 D). However, these antibodies did not bind to GPC-3 阴性 SK-Hep1 cells( Figure 8 C). This indicates that these anti-GPC-3 bispecific T cell engaging antibodies bind to the cell surface in a highly specific manner that is dependent on GPC-3 and CD3 expression.

[0661] To evaluate the function of these anti-GPC-3 bispecific T cell engaging antibodies, T cells were isolated from healthy donors and co-cultured with HepG2 (GPC-3 阳性 ) or SK-Hep1 (GPC-3 阴性 ) cells in the presence of the anti-GPC-3 bispecific T cell engaging antibodies at 3 different concentrations (2.5 nM, 312.5 pM, and 39 pM). After 24 hours, the T cells were harvested and the activation status of the T cells was analyzed by assessing the surface expression of the T cell activation markers CD25 and CD69.

[0662] The results showed that all five anti-GPC-3 bispecific T cell engaging antibodies could strongly activate human naive T cells( 阳性 A and Figure 9 C) when co-cultured with GPC-3 Figure 9 HepG2 cells, but did not activate human naive T cells( 阴性 B and Figure 9 D) when co-cultured with GPC-3 Figure 9 SK-Hep1 cells.

[0663] In addition, cell culture supernatants were collected and subjected to ELISA to evaluate the release of cytokines (interferon-γ and IL-2) from T cells treated with anti-GPC-3 bispecific T cell engaging antibodies.

[0664] In the presence of anti-GPC-3 bispecific T cell engaging antibodies (clones 1C4, 1D2, 2B5, 4H1, and 5C4), activated human T cells secreted significantly higher amounts of interferon-γ and IL-2 阳性 when co-cultured with GPC-3 Figure 9 E and Figure 9 G) HepG2 cells. However, when co-cultured with GPC-3 阴性 SK-Hep1 cells, the release of interferon-γ and IL-2 remained low Figure 9 F and Figure 9 H), indicating that these anti-GPC-3 bispecific T cell engaging antibodies have high specificity.

[0665] The efficacy of these anti-GPC-3 bispecific T cell engaging antibodies was further examined by antibody-dependent T cell-mediated cytotoxicity assay.

[0666] Briefly, GPC-3 阳性 HepG2 cells (T: target cells) were seeded onto xCelligence E-plates and cultured overnight, and the cell index values were measured. The next day, after adding various concentrations of anti-GPC-3 bispecific T cell engaging antibodies, primary T cells (E: effector cells) isolated from human PBMCs were added to the target cells at an effector-to-target cell ratio of 4:1 (E:T = 4:1). The xCellingence RTCA system was used to continue the assay for an additional 72 hours to measure the target cell index values.

[0667] All other anti-GPC-3 bispecific T cell engaging antibodies (clones 1C4, 1D2, 4H1, and 5C4) killed 50% to 70% of HepG2 cells within 72 hours at concentrations as low as 0.1 nM 阳性 except for clone 2B5 which induced deep cell lysis of target HepG2 (GPC-3 Figure 10 A and Figure 10 ) cells only at high concentrations of 10 nM and 1 nM.

[0668] In summary, the results of the above assays showed that our anti-GPC-3 antibodies in the form of bispecific T cell engaging antibodies have high specificity and efficacy in inducing the death of GPC-3 expressing cells.

[0669] Example 9. Use in Chimeric Antigen Receptor (CAR) T Cell Constructs

[0670] First, the in vivo activity of anti-GPC-3 CAR T cells was evaluated using immunocompromised NSG (NOD-scid IL2γ null ) mice through a prophylactic model ( Figure 11 ).

[0671] On day 0, 2 million Hep3B cells were subcutaneously injected into the right flanks of NSG mice (female only). Four days later, when no measurable tumors were observed, the mice were randomly grouped and injected with 10 million anti-GPC-3 CAR T cells (clone 5C4) or mock T cells. All mice in the "tumor only" group (not injected with T cells) and the "mock T" group developed large tumors and were sacrificed on day 41, while all mice in the "CAR T" group remained tumor-free. Eighty-four days after the initial tumor inoculation, 1 million Hep3B cells were subcutaneously injected into the right flank to challenge all tumor-free mice (4 mice) in the CAR T group and a group of naive mice (5 mice, "tumor only 2"), and their tumor growth and body weights were continuously monitored. All 5 mice in the "tumor only 2" group developed fairly large tumors, but 2 mice in the CAR T group remained tumor-free until day 165 (81 days after rechallenge) ( Figure 11 A).

[0672] These data indicate that 5C4 CAR T cells prevent the formation of GPC-3 阳性 HCC tumors in the xenograft model and provide long-term protection for NSG mice against the growth of GPC-3 阳性 HCC tumors upon tumor rechallenge. In addition, anti-GPC-3 CAR T cells (clone 5C4) did not cause severe toxicity in NSG mice, as there was no significant decrease in body weight after anti-GPC-3 CAR T treatment ( Figure 11 B). Six days after T cell injection, the upregulation of serum levels of T cell-related cytokines (perforin, granzyme B, sCD137, GM-CSF) from the "CAR T" group compared to the basal levels detected in the "mock T" group also reflected the efficacy of anti-GPC-3 CAR T cells ( Figure 11 C).

[0673] To test whether anti-GPC-3 CAR T cells could inhibit the growth of established tumors in vivo, we selected 3 different anti-GPC-3 antibody clones to generate anti-GPC-3 CAR constructs (clone 1D2, 4H1, and 5C4). Regardless of the different clones, 20% CAR expression could be obtained through lentiviral transduction ( Figure 12 A).

[0674] These anti-GPC-3 CAR T cells showed strong and specific cytotoxicity against target cells in vitro, as they killed 70% to 80% of GPC-3 阳性 Hep3B cells, but did not kill GPC-3 阴性 SK-Hep1 cells ( Figure 12 B). After subcutaneous inoculation of 2 million GPC-3 阳性 Hep3B cells into NSG mice (female only) for 26 days, the mice were grouped according to the measurable tumor size for CAR T cell injection. 10 million different anti-GPC-3 CAR T cells (4 mice per group) or mock T cells (3 mice) were intravenously injected into the tail veins of these mice.

[0675] On day 45 (19 days after CAR T cell injection), the tumors in the mice treated with 1D2 CAR T cells (average size of 1749 mm 3 ) began to shrink and reached an average of 643 mm on day 60 3 . The tumor growth in the mice treated with 4H1 CAR T cells or 5C4 CAR T cells was much slower than that in the mice treated with mock T cells, and a further decrease in tumor size was observed in the 5C4 CAR T group starting from day 53 ( Figure 12 C).

[0676] Interestingly, when the average tumor size exceeded 2000 mm 3 , the body weights of the mice in the mock T group significantly decreased 45 days after tumor inoculation, while the body weights of all the mice injected with CAR T remained stable ( Figure 12 D).

[0677] These data indicate that our anti-GPC-3 CAR T cells effectively inhibited the growth of GPC-3 阳性 HCC xenografts in vivo without causing severe toxicity.

[0678] Subsequently, we aimed to test whether the combined use of our anti-GPC-3 CAR T cell therapy with immune checkpoint blockade antibodies (such as anti-PD1 antibody) was effective.

[0679] Similarly, 2 million Hep3B cells were subcutaneously injected into the right flanks of NSG mice (female only). After 24 days, the mice were grouped according to the measurable tumor size and intravenously injected with anti-GPC-3 CAR T cells (clone 5C4) or mock T cells via the tail vein. 10 days after the injection of CAR T cells, when the average size of the tumors in both groups exceeded 1000 mm 3For tumors, anti-PD1 IgG was intraperitoneally injected into all mice 4 antibody, 5 times in total, at intervals of 3 to 5 days.

[0680] The tumors in the mock T group mice were unresponsive to the anti-PD1 antibody, so the tumors continued to grow. In contrast, within 5 days after the first injection of the anti-PD1 antibody, the tumor size of the CAR T group mice decreased sharply and continued to shrink until the average tumor size was less than 100 mm 3 , and it lasted for more than 20 days( Figure 13 A).

[0681] Although slight fluctuations were observed in the CAR T group, the body weights of both the mock T and CAR T group mice remained stable( Figure 13 B).

[0682] The result of effective tumor suppression by the combined use of anti-GPC-3 CAR T and anti-PD1 antibody was that 75% of the mice treated with anti-GPC-3 CAR T cells and then with anti-PD1 antibody survived for more than 70 days, while the median survival time of the mice treated with mock T and then with anti-PD1 antibody was only 45 days( Figure 13 C).

[0683] Interestingly, on the second day after the second injection of the anti-PD1 antibody (day 40, 16 days after the infusion of CAR T cells), the total number of human T cells in the CAR T group mice was significantly higher than that in the mice treated with mock T cells( Figure 13 D). In addition, the persistence of peripheral CAR+CD3+ T cells, peripheral CD4+CD3+ T cells, and peripheral CD8+CD3+ T cells was consistent with that of total T cells( Figure 13 E). In addition, the efficacy of the combined therapy of anti-GPC-3 CAR T cells and anti-PD1 antibody was also observed through the upregulation of the serum levels of T cell-related cytokines (perforin, granzyme B, sCD137, GM-CSF) in the anti-GPC-3 CAR T cell treatment group( Figure 13 F).

[0684] Since most anti-GPC-3 antibody clones can cross-react with the murine homolog of the GPC-3 protein, we injected CAR T cells derived from 8 different murine cross-reactive anti-GPC-3 clones (1C4, 1D2, 1F1, 1H1, 2B5, 4A5, 4H1, and 5C4) and 1 anti-GPC-3 clone (1D8) that does not cross-react with murine GPC-3 into NSG mice (6 mice per group, mixed gender). We detected the potential toxicity of these anti-GPC-3 CAR T cells to normal murine tissues in the absence of tumor cells expressing high levels of the GPC-3 protein by monitoring the body weight of each mouse as well as other abnormal physiological changes or behaviors. The results clearly showed that, similar to the "untreated" group ( Figure 14 A), mock T group ( Figure 14 B), and 1D8 CAR T group ( Figure 14 E), the 8 different anti-GPC-3 CAR T cells that can bind to murine GPC-3-expressing cells did not cause obvious toxicity in vivo until the 5-week time point ( Figure 14 C, Figure 14 D, Figure 14 F, Figure 14 G, Figure 14 H, Figure 14 I, Figure 14 J, and Figure 14 K), and GvHD may occur at any time after this time point.

[0685] This indicates that either murine GPC-3 is not expressed in normal murine tissues or the affinity between murine GPC-3 protein and the scFv fragments from 8 anti-GPC-3 clones is at an optimal level, such that the low expression of murine GPC-3 in normal murine tissues does not cause strong activation of these anti-GPC-3 CAR T cells.

[0686] Finally, to examine whether the use of CAR T cell therapy can be extended to the murine system, we first stained parental MC38 cells and MC38 cells stably transduced with murine GPC-3 (named "MC38-mGPC-3") with the representative anti-GPC-3 IgG clone 5C4, and then performed flow cytometry analysis ( Figure 15 A).

[0687] The murine CAR T construct based on clone 5C4 was designed by replacing the human T cell activation domain with its murine homolog counterpart and cloned into a retrovirus-based vector. Murine CAR T cells against GPC-3 were generated from murine T cells isolated from murine spleen cells by retroviral transduction method, and murine CAR expression was detected by flow cytometry analysis ( Figure 15B). The in vitro activity of these murine CAR T cells was verified by more than 80% cytotoxicity against MC38-mGPC-3 cells within 96 hours using xCelligence impedance detection ( Figure 15 C). To test the therapeutic effect of murine anti-GPC-3 CAR T cells in vivo, 800,000 MC38-mGPC-3 cells were subcutaneously injected into WT C57BL / 6 mice. When the tumors grew to a measurable size (about 100 mm 3 ), the mice were re-grouped and injected intravenously with anti-GPC-3 murine CAR T cells (clone 5C4) or mock T cells, or left untreated ("tumor only").

[0688] By day 33 (21 days after CAR T cell infusion), all mice in the mock T group and the tumor only group developed large-sized tumors, while mice from the anti-GPC-3 murine CAR T cell injection group developed very small tumors and 1 mouse remained tumor-free ( Figure 15 D). Monitoring of the mouse body weight showed that these murine CAR T cells did not cause significant toxicity whether in the presence of MC38-mGPC-3 tumor cells expressing mGPC-3 protein ( Figure 15 E) or in the absence of MC38-mGPC-3 tumor cells expressing mGPC-3 protein ( Figure 15 F).

[0689] These experiments not only demonstrated the efficacy of murine anti-GPC-3 CAR T cells in vitro and in vivo, but also elucidated the extended use of the anti-GPC-3 antibody sequence in the form of murine CAR T constructs for future studies of anti-GPC-3 CAR T therapy in homologous mouse models.

[0690] Example 10: Anti-GPC-3 IgG 1 Binding affinity of antibodies

[0691] Using a Biacore TM T200 system, the affinity of 24 anti-GPC-3 IgG 1 antibodies was measured by surface plasmon resonance (SPR) analysis and shown in Table E3 (as follows).

[0692]

[0693]

[0694] Table E3: Binding affinity (K 1 of 24 anti-GPC-3 IgG D) Association rate (Ka) and dissociation rate (Kd).

[0695] All 24 antibody clones showed nanomolar (nM) binding affinities for the recombinant GPC-3 protein, ranging from a lowest affinity of 44.17 nM (clone 4H1) to a highest affinity of 4.177 nM (clone 1D2).

[0696] References

[0697] 1. Capurro, M.I., Xu, P., Shi, W., Li, F., Jia, A., Filmus, J. Glypican-3 inhibits Hedgehog signaling during development by competing with Patched for Hedgehog binding. Dev. Cell 14: 700 - 711, 2008.

[0698] 2. Filmus, J., Capurro, M., Rast, J. Glypicans Genome Biol. 9: 224, 2008. Note: Electronic Article.

[0699] 3. Filmus, J., Church, J.G., Buick, R.N. Isolation of a cDNA corresponding to a developmentally regulated transcript in rat intestine. Molec. Cell Biol. 8: 4243 - 4249, 1988.

[0700] 4. Filmus, J., Shi, W., Wong, Z.M., Wong, M.J. Identification of a new membrane-bound heparan sulphate proteoglycan. Biochem. J. 311: 561 - 565, 1995.

[0701] 5. Hughes-Benzie, R.M., Pilia, G., Xuan, J.Y., Hunter, A.G.W., Chen, E., Golabi, M., Hurst, J.A., Kobori, J., Marymee, K., Pagon, R.A., Punnett, H.H., Schelley, S., Tolmie, J.L., Wohlferd, M.M., Grossman, T., Schlessinger, D., MacKenzie, A.E. Simpson-Golabi-Behmel syndrome: genotype / phenotype analysis of 18 affected males from 7 unrelated families. Am. J. Med. Genet. 66: 227-234, 1096.

[0702] 6. Lindsay, S., Ireland, M., O′Brien, O., Clayton-Smith, J., Hurst, J.A., Mann, J., Cole, T., Sampson, J., Slaney, S., Schlessinger, D., Burn, J., Pilia, G. Large scale deletions in the GPC3 gene may account for a minority of cases of Simpson-Golabi-Behmel syndrome. J. Med. Genet. 34: 480-483, 1997.

[0703] 7. Maurel, M., Dejeans, N., Taouji, S., Chevet, E., Grosset, C.F. MicroRNA-1291-mediated silencing of IRE1-alpha enhances glypican-3 expression. RNA 19: 778-788, 2013.

[0704] 8. Penisson - Besnier, I., Lebouvier, T., Moizard, M.-P., Ferre, M., Barth, M., Marc, G., Raynaud, M., Bonneau, D. Carotid artery dissection in an adult with the Simpson - Golabi - Behmel syndrome. Am. J. Med. Genet. 146A: 464 - 467, 2008.

[0705] 9. Pilia, G., Hughes - Benzie, R.M., MacKenzie, A., Baybayan, P., Chen, E.Y., Huber, R., Neri, G., Cao, A., Forabosco, A., Schlessinger, D. Mutations in GPC3, a glypican gene, cause the Simpson - Golabi - Behmel overgrowth syndrome. Nature Genet. 12: 241 - 247, 1996.

[0706] 10. Rodriguez - Criado, G., Magano, L., Segovia, M., Gurrieri, F., Neri, G., Gonzalez - Meneses, A., Gomez de Terreros, I., Valdez, R., Gracia, R., Lapunzina, P. Clinical and molecular studies on two further families with Simpson - Golabi - Behmel syndrome. Am. J. Med. Genet. 138A: 272 - 277, 2005.

[0707] 11. Romanelli, V., Arroyo, I., Rodriguez, J.I., Magano, L., Arias, P., Incera, I., Gracia - Bouthelier, R., Lapunzina, P. Germinal mosaicism in Simpson - Golabi - Behmel syndrome. (Letter) Clin. Genet. 72: 384 - 386, 2007.

[0708] 12.Sakazume,S.,Okamoto,N.,Yamamoto,T.,Kurosawa,K.,Numabe,H.,Ohashi,Y.,Kako,Y.,Nagai,T.,Ohashi,H.GPC3 mutations in seven patienis with Simpson-Golabi-Behmel syndrome.Am.J.Med.Genet.143A:1703-1707,2007.

[0709] 13.Shen,T.,Sonoda,G.,Hamid,J.,Li,M.,Filmus,J.,Buick,R.N.,Testa,J.R.Mapping of the Simpson-Golabi-Behmel overgrowth syndrome gene(GPC3)tochromosome X in human and rat by fluorescence in situ hybridization.MammalianGenome 8:72only,1997.

[0710] 14.Shi,W.,Filmus,J.A patient with the Simpson-Golabi-Behmel syndromedisplays a loss-of-function point mutation in GPC3 that inhibits theattachment of this proteoglycan to the cell surface.(Letter)Am.J.Med.Genet.149A:552-554,2009.

[0711] 15.Sood,R.,Zehnder,J.L.,Druzin,M.L.,Brown,P.O.Gene expression Pattemsin human placenta.Proc.Nat.Acad.Sci.103:5478-5483,2006.

[0712] 16. Veugelers, M., De Cat, B., Muyldermans, S.Y., Reekmans, G., Delande, N., Frints, S., Legius, E., Fryms, J.-P., Schrander-Stumpel, C., Weidle, B., Magdalena, N., David, G. Mutational analysis of the GPC3 / GPC4 glypican gene cluster on Xq26 in patients with Simpson-Golabi-Behmel syndrome: identification of loss-of-function mutations in the GPC3 gene. Hum. Molec. Genet. 9: 1321-1328, 2000.

[0713] 17. White, G.R.M., Kelsey, A.M., Varley, J.M., Birch, J.M. Somatic glypican 3 (GPC3) mutations in Wilms′ tumour. Brit. J. Cancer 86: 1920-1922, 2002.

[0714] 18. Xuan, J.Y., Hughes-Benzie, R.M., MacKenzie, A.E. A small interstitial deletion in the GPC3 gene causes Simpson-Golabi-Behmel syndrome in a Dutch-Canadian family. J. Med. Genet. 36: 57-58, 1999.

[0715] In this specification and its claims, the verb "comprise" and its inflected forms are used in its non-restrictive sense, to mean that items following the word are included, but items not specifically mentioned are not excluded. Further, the indefinite article "a" or "an" preceding an element does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. Thus, the indefinite article "a" generally means "at least one".

[0716] Each application and patent mentioned in this text, and each document cited or referenced in each of the above-mentioned applications and patents, including each document cited or referenced during the examination of each application and patent ("application cited documents"), and any manufacturer's instructions or catalogs of any product cited or mentioned in each application and patent and any application cited document, are hereby incorporated by reference into this text. In addition, all documents cited in this text, all documents cited in all documents cited in this text, and any manufacturer's instructions or catalogs of any product cited or mentioned in this text are hereby incorporated by reference into this text.

[0717] Various modifications and variations of the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art of molecular biology or related fields are intended to fall within the scope of the claims.

Claims

1. An antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (V H ) sequence and a light chain variable region (V L ) sequence selected from the following clones: 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1, and capable of specifically binding to glypican-3 (GPC-3) (Genbank accession number: NP_004475.1), or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

2. The antibody or antigen-binding fragment according to claim 1, wherein said V H and V L sequences are selected from: SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO:9 and SEQ ID NO:10; SEQ ID NO:11 and SEQ ID NO:12; SEQ ID NO:13 and SEQ ID NO:14; SEQ ID NO:15 and SEQ ID NO:16; SEQ ID NO:17 and SEQ ID NO:18; SEQ ID NO:19 and SEQ ID NO:20; SEQ ID NO:21 and SEQ ID NO:22; SEQ ID NO:23 and SEQ ID NO:24; SEQ ID NO:25 and SEQ ID NO:26; SEQ ID NO:27 and SEQ ID NO:28; SEQ ID NO:29 and SEQ ID NO:30; SEQ ID NO:31 and SEQ ID NO:32; SEQ ID NO:33 and SEQ ID NO:34; SEQ ID NO:35 and SEQ ID NO:36; SEQ ID NO:37 and SEQ ID NO:38; SEQ ID NO:39 and SEQ ID NO:40; SEQ ID NO:41 and SEQ ID NO:42; SEQ ID NO:43 and SEQ ID NO:44; SEQ ID NO:45 and SEQ ID NO:46; and SEQ ID NO:47 and SEQ ID NO:

48.

3. The antibody or antigen-binding fragment according to claim 1 or 2, which is capable of binding to an epitope of human glypican-3 (GPC-3) with an affinity of 55 nM or less, 50 nM or less, 45 nM or less, 40 nM or less, 35 nM or less, 30 nM or less, 25 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less, as measured by ELISA. 50 ​ 4. The antibody or antigen-binding fragment according to claim 1, 2 or 3, which is capable of binding to a cell line selected from the group consisting of HepG2 (GPC-3 高 ) and Hep3B (GPC-3 中 ).

5. An antibody or antigen-binding fragment according to any one of the preceding claims, which is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) of GPC-3-expressing cells in the presence of natural killer (NK) cells.

6. An antibody or antigen-binding fragment according to any one of the preceding claims, which is a humanized antibody comprising a human constant region.

7. An antibody or antigen-binding fragment according to any one of the preceding claims, which comprises a monoclonal antibody, a humanized monoclonal antibody, Fv, F(ab’), F(ab’) 2 or a single-chain Fv (scFv) fragment, preferably a single-chain Fv fragment.

8. An antibody or antigen-binding fragment according to any one of the preceding claims, which is a monoclonal antibody selected from the group consisting of: 5C4, 4H1, 1D2, 1C4, 2B5, 1F1, 1H1, 4A5, 1D8, 1D3, 2F1, 3C6, 3D12, 3A9, 1F4, 1H10, 3C12, 4G11, 4A12, 1A12, 3G12, 4F9, 4G4, and 1E1.

9. An antibody or antigen-binding fragment according to any one of the preceding claims, further comprising a heavy chain variable region (V H ) sequence and a light chain variable region (V L ) sequence that are capable of binding to CD3 (Genbank accession number: NM_000733.4).

10. The antibody or antigen-binding fragment according to claim 9, which is further capable of specifically binding to CD-3 or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

11. The antibody or antigen-binding fragment according to claim 9 or 10, wherein, The heavy chain variable region (V H ) sequence and the light chain variable region (V L ) sequence that can bind to CD3 comprise the sequences shown in SEQ ID NO:97 and SEQ ID NO:

98.

12. The antibody or antigen-binding fragment according to claim 9, 10, or 11, which is capable of activating T cells.

13. A chimeric antigen receptor (CAR) or a chimeric antigen receptor T cell (CAR T) expressing the chimeric antigen receptor, the chimeric antigen receptor (CAR) comprising: (a) an antigen-binding fragment according to any one of the preceding claims; (b) a transmembrane domain; and (c) a co-stimulatory intracellular signaling domain.

14. A nucleic acid capable of encoding an antibody or antigen-binding fragment according to any one of the preceding claims, said nucleic acid preferably comprising a sequence selected from the group consisting of: SEQ ID NO:49 and SEQ ID NO:50; SEQ ID NO:51 and SEQ ID NO:52; SEQ ID NO:53 and SEQ ID NO:54; SEQ ID NO:55 and SEQ ID NO:56; SEQ ID NO:57 and SEQ ID NO:58; SEQ ID NO:59 and SEQ ID NO:60; SEQ ID NO:61 and SEQ ID NO:62; SEQ ID NO:63 and SEQ ID NO:64; SEQ ID NO:65 and SEQ ID NO:66; SEQ ID NO:67 and SEQ ID NO:68; SEQ ID NO:69 and SEQ ID NO:70; SEQ ID NO:71 and SEQ ID NO:72; SEQ ID NO:73 and SEQ ID NO:74; SEQ ID NO:75 and SEQ ID NO:76; SEQ ID NO:77 and SEQ ID NO:78; SEQ ID NO:79 and SEQ ID NO:80; SEQ ID NO:81 and SEQ ID NO:82; SEQ ID NO:83 and SEQ ID NO:84; SEQ ID NO:85 and SEQ ID NO:86; SEQ ID NO:87 and SEQ ID NO:88; SEQ ID NO:89 and SEQ ID NO:90; SEQ ID NO:91 and SEQ ID NO:92; SEQ ID NO:93 and SEQ ID NO:94; and SEQ ID NO:95 and SEQ ID NO:96, such as a nucleic acid contained in an expression vector.

15. A host cell comprising the nucleic acid according to claim 14, wherein, the host cell preferably includes Chinese hamster ovary (CHO) or HEK293 cells.

16. A pharmaceutical composition comprising: an antibody or antigen-binding fragment, CAR, CAR T, nucleic acid or host cell according to any one of the preceding claims, and a pharmaceutically acceptable excipient, diluent or carrier.

17. A compound comprising an antibody or antigen-binding fragment according to any one of claims 1 to 12, said antibody or antigen-binding fragment being linked to a cytotoxic agent, for example linked to a cytotoxic agent with a cleavable linker.

18. The compound according to claim 17, wherein, the compound comprises an antibody-drug conjugate.

19. Use of an antibody, antigen-binding fragment, CAR, CAR T, nucleic acid, host cell, pharmaceutical composition, compound or antibody-drug conjugate according to any one of the preceding claims in a method for treating, preventing or alleviating cancer such as hepatocellular carcinoma (HCC).

20. A method for preparing an antibody or antigen-binding fragment, the method comprising expressing a nucleic acid according to claim 14 in a host cell, preferably in the host cell according to claim 15, and optionally isolating the expressed antibody or antigen-binding fragment.

21. Use of an antibody, antigen-binding fragment, CAR, CAR T, nucleic acid, pharmaceutical composition, compound or antibody-drug conjugate according to any one of claims 1 to 19 in the preparation of a medicament for treating, preventing or alleviating cancer such as hepatocellular carcinoma (HCC).

22. A method for detecting hepatocellular carcinoma (HCC) cells, the method comprising detecting the regulation of the expression, amount or activity of glypican-3 (GPC-3) in or of the cells with an antibody or antigen-binding fragment according to any one of claims 1 to 12.

23. A method for diagnosing hepatocellular carcinoma (HCC) in an individual, wherein the method comprises: detecting the expression level of regulated glypican-3 (GPC-3) in the cells by contacting the cells of the individual with an antibody or antigen-binding fragment according to any one of claims 1 to 12; comparing with the expression level of glypican-3 in the cells of an individual known not to have hepatocellular carcinoma (HCC); wherein an increase in the expression level of glypican-3 indicates that the individual has or may have hepatocellular carcinoma (HCC).

24. A diagnostic kit for hepatocellular carcinoma (HCC), the kit comprising an antibody or antigen-binding fragment according to claims 1 to 12 or a nucleic acid according to claim 14 and instructions for use.

25. An antibody, antigen-binding fragment, nucleic acid, pharmaceutical composition, compound, antibody-drug conjugate, use, method or diagnostic kit as described above with reference to FIGS. 1 to 15 of the drawings and as shown in FIGS. 1 to 15 of the drawings.

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