GPC3 antibody-drug conjugate and its use

Abstract

The present invention provides an antibody-drug conjugate having a structure represented by Formula I, and uses thereof. The antibody-drug conjugate of the present invention has good inhibitory activity against tumor cell growth both in vivo and in vitro, and has low cytotoxicity. JPEG2025531401000026.jpg5170 (wherein Ab is an anti-GPC3 antibody, L is a linker, and D is a cytotoxin.)

Description

[Technical Field]

[0001] The present invention relates to the field of medicinal chemistry, and in particular to a GPC3 antibody-drug conjugate and its use. [Background technology]

[0002] Liver cancer is the fifth most common cancer worldwide and the third leading cause of cancer-related deaths. Hepatocellular carcinoma (HCC) is the predominant form of liver cancer, accounting for 90% of all liver cancer cases. Currently, known treatments are multidisciplinary comprehensive treatments centered on surgical resection. Although short-term effects have improved, long-term effects remain insufficient. Therefore, exploring new, more effective strategies for liver cancer treatment has become a focus of research both domestically and internationally. Furthermore, the development of new liver cancer therapeutics remains an urgent task.

[0003] GPC3, collectively known as Glypican-3, is a member of the heparan sulfate proteoglycan family that binds to cell surfaces via glycosylphosphatidylinositol on the cell membrane. The GPC3 protein core consists of two subunits: the N-terminal subunit is approximately 40 kDa and the C-terminal subunit is approximately 30 kDa. Six Glypicans (GPC1-6) have been identified in mammals. GPC3 plays an important role in regulating cell proliferation, differentiation, adhesion, and migration. Studies have shown that GPC3 interacts with Wnt proteins and Frizzled receptors, forming complexes that induce downstream signaling. The GPC3 core protein can function as a Wnt co-receptor or receptor.

[0004] In humans, the GPC3 protein expressed by the GPC3 gene is found in extremely low amounts in adult normal tissues, and is low or absent in cancers such as gastric, breast, and ovarian cancers. However, it is frequently overexpressed (70%-80%) in hepatocellular carcinoma (HCC). GPC3 is a liver cancer-specific antigen, and research has shown that it can help identify the nature of HCC lesions early on, significantly improving diagnostic accuracy. Therefore, GPC3 is thought to have great potential in liver cancer immunotherapy.

[0005] GPC3-targeting therapy has attracted increasing attention in recent years. Currently, therapeutic strategies based on GPC3 targeting focus on antibody drugs, cell therapy, and vaccines. Developed therapeutic anti-GPC3 antibodies include GC33, YP7, and HN3, as well as bispecific antibodies (such as ERY974 and CM350, currently in clinical trials). Some of these antibodies inhibit Wnt signaling in liver cancer cells. Furthermore, chimeric antigen receptor (CAR) T cell immunotherapy for liver cancer treatment is currently under development. In mice bearing xenografted or in situ liver tumors, CAR-T cells can eliminate GPC3-positive cancer cells by inducing perforin- and granzyme-mediated cell death and reducing WNT signaling in tumor cells. Summary of the Invention

[0006] The inventors of the present application have prepared anti-GPC3 antibody-drug conjugates (ADCs) through extensive experiments and creative efforts, and have confirmed that they have good biological activity, thereby completing the present invention.

[0007] Thus, in a first aspect of the present invention, there is provided an antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein said antibody-drug conjugate has the structure shown in Formula I:

[0008] [ka]

[0009] where: The Ab is an anti-GPC3 antibody, the anti-GPC3 antibody comprising a heavy chain and a light chain, wherein CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and CDR1, CDR2, and CDR3 of the light chain variable region comprise the sequences shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; L is linker; D is a cytotoxin; p is any number between 1 and 8 (e.g., 1, 1.5, 2, 2.5, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 6.10, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 6.20, 6.21, 6.22, 6.23, 6.24, 6.25, 6.26, 6.27, 6.28, 6.29 ... 3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8, or 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5, or 7.5-8).

[0010] In Formula I, LD indicates that the linker and the cytotoxin are covalently linked to form the LD molecule, and Ab-(LD) p indicates that p LD molecules were covalently bound to Ab.

[0011] In the present invention, the drug-antibody ratio (DAR) refers to the number of drug molecules bound to an antibody (e.g., p in Formula I). ​​The number of drug molecules contained in the antibody-drug conjugate described herein may be an integer or a decimal. Whether it is an integer or a decimal, it refers to the average number of drug molecules bound to each antibody. "p is any number between 1 and 8" means that p may be any integer selected from 1 to 8 (inclusive) or any decimal selected from 1 to 8, such as 2.3, 3.9, 4.0, or 4.2. Those skilled in the art will also understand that even if the same manufacturing method is used, the DAR values ​​of antibody-drug conjugates produced in different batches will not necessarily be completely identical and may vary, for example, within a range of no more than 0.5.

[0012] The drug-to-antibody ratio (DAR) can be determined by conventional means such as mass spectrometry, ELISA assay, HIC, and HPLC. The quantitative distribution of ADCs with respect to p can also be measured. In some cases, separation, purification, and validation of homogeneous ADCs with a certain p value from ADCs with other drug loads can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0013] In some embodiments, the linker is 6-maleimidohexanoyl (MC), maleimidopropionyl (MP), N-succinimido 4-(2-pyridylthio)valerate (SPP), 4-(N-maleimidomethyl)-cyclohexane-1-formyl (MCC), N-succinimido(4-iodo-acetyl)aminobenzoate (SIAB), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), MA-PEG4-VC-PAB-DMEA,

[0014] [ka]

[0015] is selected from.

[0016] In some embodiments, the linker is 6-maleimidohexanoyl (MC), 6-maleimidohexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), MA-PEG4-VC-PAB-DMEA,

[0017] [ka]

[0018] is selected from.

[0019] Here, the structural formulae of MC, MC-vc-PAB, and MA-PEG4-VC-PAB-DMEA are as follows:

[0020] [Table 1]

[0021] In some embodiments, the cytotoxin is SN-38, gemcitabine, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), maytansinoids (e.g., Maytansine DM1, Maytansine DM4), calicheamicin, MGBA (e.g., duocarmycin), doxorubicin, ricin, diphtheria toxin, duocarmycin SA,

[0022] [ka]

[0023] The compound is selected from I131, interleukins, tumor necrosis factors, chemokines and nanoparticles.

[0024] In some embodiments, the cytotoxin is monomethylauristatin E (MMAE), duocarmycin SA, monomethylauristatin F (MMAF),

[0025] [ka]

[0026] is selected from.

[0027] Here, the structural formulae of MMAE, MMAF, and duocarmycin SA are as follows:

[0028] [Table 2]

[0029] In some embodiments, the LD is MC-vc-PAB-MMAE, MA-PEG4-VC-PAB-DMEA-Duocarmycin SA, MC-MMAF,

[0030] [ka]

[0031] is selected from.

[0032] Here, the structural formulae of MC-vc-PAB-MMAE, MA-PEG4-VC-PAB-DMEA-Duocarmycin SA, and MC-MMAF are as follows:

[0033] [Table 3]

[0034] When the above four LDs are covalently bound to Abs, the binding method and binding site are all the same, that is, they are all formed by binding between the succinimide at the end of the LD and the thiol group in the antibody. For example, when MC-vc-PAB-MMAE is covalently bound to Abs, the structural formula of the formed ADC is as follows:

[0035] [ka]

[0036] In the ADC formed above, the antibody Ab is bound to the carbon atom of the succinimide at the LD end via -S-. This -S- is not a separately added external thiol group, but a thiol group possessed by the antibody itself after the antibody Ab is reduced and the disulfide bond is opened.

[0037] In some embodiments, the sequences of the heavy chain variable region CDR1, CDR2, and CDR3 of the anti-GPC3 antibody are represented by SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the sequences of the light chain variable region CDR1, CDR2, and CDR3 of the anti-GPC3 antibody are represented by SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

[0038] In some embodiments, the sequences of the heavy chain variable region FR1, FR2, FR3, and FR4 of the anti-GPC3 antibody are set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

[0039] In some embodiments, the sequences of the light chain variable regions FR1, FR2, FR3, and FR4 of the anti-GPC3 antibody are represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; or the sequences of the light chain variable regions FR1, FR2, FR3, and FR4 of the anti-GPC3 antibody are represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, and SEQ ID NO: 15, respectively.

[0040] In some embodiments, the sequence of the heavy chain variable region of the anti-GPC3 antibody is set forth in SEQ ID NO:8.

[0041] In some embodiments, the sequence of the light chain variable region of the anti-GPC3 antibody is set forth in SEQ ID NO:16 or SEQ ID NO:18.

[0042] In some embodiments, the sequence of the heavy chain variable region of the anti-GPC3 antibody is set forth in SEQ ID NO: 8, and the sequence of the light chain variable region of the anti-GPC3 antibody is set forth in SEQ ID NO: 16.

[0043] In some embodiments, the sequence of the heavy chain variable region of the anti-GPC3 antibody is set forth in SEQ ID NO: 8, and the sequence of the light chain variable region of the anti-GPC3 antibody is set forth in SEQ ID NO: 18.

[0044] In some embodiments, the heavy chain constant region of the anti-GPC3 antibody is selected from the constant regions of human IgG, IgM, IgA, IgD, and IgE, or mutants of these constant regions.

[0045] In some embodiments, the IgG is selected from IgG1, IgG2, IgG3, and IgG4.

[0046] In some embodiments, the light chain constant region of the anti-GPC3 antibody is a human-derived lambda constant region, a human kappa constant region, or a mutant of these constant regions.

[0047] In some embodiments, the sequence of the heavy chain constant region of the anti-GPC3 antibody comprises the sequence set forth in SEQ ID NO: 19, or SEQ ID NO: 19. More than 70%, preferably more than 75%, 80%, 85%, 90%, 95%, 99% homology to the sequence set forth in SEQ ID NO: 19.

[0048] In some embodiments, the sequence of the heavy chain constant region of the anti-GPC3 antibody is set forth in SEQ ID NO:19.

[0049] In some embodiments, the sequence of the light chain constant region of the anti-GPC3 antibody comprises the sequence set forth in SEQ ID NO: 20, or SEQ ID NO: 20. More than 70%, preferably more than 75%, 80%, 85%, 90%, 95%, 99% homology to the sequence set forth in SEQ ID NO: 20.

[0050] In some embodiments, the sequence of the light chain constant region of the anti-GPC3 antibody is set forth in SEQ ID NO:20.

[0051] In some embodiments, p is any value between 2 and 8.

[0052] In some embodiments, p is any value between 2 and 6.

[0053] In some embodiments, p is any value between 2 and 5 (eg, 2.3, 3.9, 4.0, 4.2).

[0054] In a second aspect, the present invention provides a drug composition comprising the antibody-drug conjugate described above, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof.

[0055] In some embodiments, the pharmaceutical composition further comprises at least one of a chemotherapeutic agent, an immunotherapeutic agent, and an immunosuppressant used in the treatment of tumors.

[0056] In some embodiments, the chemotherapeutic agent is, for example, Adriamycin, cyclophosphamide, taxanes (e.g., paclitaxel (Taxol), Taxotere), capecitabine (Xeloda), gemcitabine (Gemzar), navelbine, tamoxifen, aromatase inhibitors (Arimidex, Femara, Aromasin), 5-FU leucovorin, irinotecan (Camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, etc., or a combination thereof.

[0057] In some embodiments, the immunotherapeutic agent is, for example, a PD-1 monoclonal antibody (e.g., pembrolizumab, nivolumab), a PD-L1 monoclonal antibody (e.g., atezolizumab), a TIGIT monoclonal antibody, a 4-1BB monoclonal antibody, a VEGFR2 monoclonal antibody (e.g., ramucirumab, apatinib), a HER2 monoclonal antibody (e.g., trastuzumab, trastuzumab biosimilar, trastuzumab-dkst), or the like, or a combination thereof.

[0058] In some embodiments, the immunosuppressant is selected from (1) glucocorticoids (e.g., cortisone and prednisone), (2) microbial metabolites (e.g., cyclosporine and tacrolimus), (3) antimetabolites (e.g., azathioprine and 6-mercaptopurine), (4) polyclonal and monoclonal antilymphocyte antibodies (e.g., antilymphocyte globulin and OKT3), and (5) alkylating agents (e.g., cyclophosphamide). In some specific embodiments, the immunosuppressant is, for example, methylprednisone, prednisone, azathioprine, Prograf, Zenapax, Simulect, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, mizoribine, cyclophosphamide, fingolimod, etc.

[0059] In some embodiments, the drug composition further comprises at least one pharmaceutical excipient.

[0060] In a third aspect, the present invention provides use of the antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, or the pharmaceutical composition in the manufacture of a drug for preventing and / or treating a GPC3-associated disease. Preferably, the GPC3-associated disease is a GPC3-positive disease.

[0061] In some embodiments, the GPC3-positive associated disease includes but is not limited to liver cancer, lung cancer, gastric cancer, head and neck cancer, esophageal cancer, Merkel cell carcinoma, or liposarcoma.In some embodiments, the GPC3-positive associated disease is liver cancer.

[0062] In a fourth aspect, the present invention provides the antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, or the pharmaceutical composition for preventing and / or treating a GPC3-associated disease, preferably a GPC3-positive disease.

[0063] In some embodiments, the GPC3-positive associated disease includes but is not limited to liver cancer, lung cancer, gastric cancer, head and neck cancer, esophageal cancer, Merkel cell carcinoma, or liposarcoma.In some embodiments, the GPC3-positive associated disease is liver cancer.

[0064] In a fifth aspect, the present invention provides a method for treating and / or preventing a GPC3-related disease. The method comprises administering to a subject in need thereof the antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, or the pharmaceutical composition in a therapeutically and / or prophylactically effective amount. Preferably, the GPC3-related disease is a GPC3-positive disease.

[0065] In some embodiments, the GPC3-positive associated disease includes but is not limited to liver cancer, lung cancer, gastric cancer, head and neck cancer, esophageal cancer, Merkel cell carcinoma, or liposarcoma.In some embodiments, the GPC3-positive associated disease is liver cancer.

[0066] Beneficial effects 1. The novel antibodies Hu 52H5D3B8-7 and Hu 52H5D3B8-8 of the present invention are developed anti-GPC3 monoclonal antibodies that can be conjugated via linkers to various potent cytotoxic small molecule drugs, such as MMAE (methylauristatin E), MMAF, and Duo, to obtain GPC3-ADCs. GPC3-ADCs can effectively inhibit tumor cell proliferation and cause tumor cell death by binding to the GPC3 receptor on the tumor cell surface, endocytosis, and release of small molecule toxins.

[0067] 2. Compared with known antibodies such as Y035 and GC33, the novel antibodies Hu 52H5D3B8-7 and Hu 52H5D3B8-8 of the present invention have higher cell binding affinity and higher internalization efficiency. Furthermore, the antibodies conjugated with cytotoxins have no change in affinity for HepG2 cells expressing GPC3.

[0068] 3. The newly developed antibodies Hu 52H5D3B8-7 and Hu 52H5D3B8-8 of the present invention can be coupled to a variety of different linker-payloads and exhibit significantly more potent cell-killing effects in liver cancer cells than GC33-ADC with the same linker-payload.

[0069] 4. The newly developed GPC3-ADC of the present invention exhibits significant efficacy in suppressing tumor cell proliferation in a human liver cancer PDX tumor model. [Brief explanation of the drawings]

[0070] [Figure 1] FIG. 1 shows the affinity of each GPC3 antibody for GPC3-CHO-K1 cells. [Figure 2] FIG. 2 shows the affinity of each GPC3 antibody for HepG2 cells. [Figure 3] FIG. 3 shows the affinity of each GPC3 antibody for Huh-7 cells. [Figure 4] FIG. 4 shows the internalization of each GPC3 antibody in HepG2 cells. [Figure 5] FIG. 5 shows the hydrophobicity effect chromatogram of Hu 52H5D3B8-7-L1-D1. [Figure 6] FIG. 6 shows the hydrophobicity effect chromatogram of Hu 52H5D3B8-7-L3-D3. [Figure 7] FIG. 7 shows the hydrophobicity effect chromatogram of Hu 52H5D3B8-7-L4-D4. [Figure 8] FIG. 8 shows the hydrophobicity effect chromatogram of Hu 52H5D3B8-8-ZLA. [Figure 9] Figure 9 shows the affinity of the GPC3 antibody and ADC for HepG2 cells. [Figure 10] FIG. 10 shows the cell-killing effects of Hu 52H5D3B8-8-L1-D1 and GC33-L1-D1 on the Huh7 cell line. [Figure 11]FIG. 11 shows the cell-killing effects of Hu 52H5D3B8-8-L1-D1 and GC33-L1-D1 on the HepG2 cell line. [Figure 12] FIG. 12 shows the cell-killing effects of Hu 52H5D3B8-8-L3-D3 and GC33-L3-D3 on the Huh7 cell line. [Figure 13] FIG. 13 shows the cell-killing effects of Hu 52H5D3B8-7-L4-D4 and GC33-L4-D4 on the Huh7 cell line. [Figure 14] FIG. 14 shows the cell-killing effects of Hu 52H5D3B8-8-ZLA and GC33-ZLA in the Huh7 cell line. [Figure 15] FIG. 15 shows the cell-killing effects of Hu 52H5D3B8-8-ZLA and GC33-ZLA in the HepG2 cell line. [Figure 16] Figure 16 shows the tumor growth-suppressing activity of GPC3-ADC in the human liver cancer nude mouse PDX model LIV#219. [Figure 17] Figure 17 shows the effect of GPC3-ADC on animal body weight in LIV#219, a nude mouse PDX model of human liver cancer. DETAILED DESCRIPTION OF THE INVENTION

[0071] The following describes embodiments of the present invention in detail with reference to examples. However, those skilled in the art should understand that the following examples are provided merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention. Unless specific conditions are specified in the examples, they are carried out according to conventional conditions or manufacturer's recommended conditions. Unless the manufacturer of the reagent or equipment used is specified, the items in question refer to commercially available general-purpose products.

[0072] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory procedures used herein are all terms and routine procedures widely used in the corresponding fields. Meanwhile, to help further understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0073] For purposes of the present invention, unless otherwise specified, any numerical range should be understood to include any value or any subrange within that range.

[0074] In the present invention, the term "antibody" refers to an immunoglobulin molecule that typically consists of two pairs of identical polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be divided into two classes: kappa and lambda. Heavy chains can be divided into five classes: μ, delta, gamma, alpha, or epsilon, and antibodies can be classified into five heavy chain classes: IgM, IgD, IgG, IgA, and IgE. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with heavy chains also containing a "D" region of about 3 or more amino acids. Each heavy chain contains a heavy chain variable region (V H ) and heavy chain constant region ( C H The heavy chain constant region consists of three domains (C H 1. C H 2, and C H Each light chain consists of a light chain variable region (V L ) and light chain constant region ( C L The light chain constant region consists of one domain, C L The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the component C1q of the complement system. H and V LThe region may also be subdivided into regions of high variability (called complementarity determining regions (CDRs)), interspersed with relatively conserved regions called framework regions (FRs). H and V L The variable region (V) of each heavy / light chain pair consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. H and V L ) each form an antibody binding site. The assignment of amino acids to each region or domain follows the definitions of the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0075] In the present invention, algorithms used to determine percentages of sequence identity and sequence similarity include, for example, the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acid. Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used to determine percentage amino acid sequence identity in the present invention by employing the parameters described in the literature or the default parameters. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI).

[0076] In the present invention, an amino acid sequence having at least 70% sequence identity to the amino acid sequence includes a polypeptide sequence that is essentially identical to the amino acid sequence, e.g., a sequence having at least 70% sequence identity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a polypeptide sequence of the present invention, as determined, for example, by a method described herein (e.g., BLAST analysis using standard parameters). In the present invention, the variant of the amino acid sequence refers to a sequence having a homology of more than 70%, for example, more than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the amino acid sequence, and includes a sequence having, for example, three, two, or one substituted, deleted, or added amino acids. Preferably, the number of substituted, added, or deleted amino acids is three or less. More preferably, the number of substituted, added, or deleted amino acids is two or less. Most preferably, the number of substituted, added, or deleted amino acids is one or less.

[0077] "Substitutional" variants are those in which at least one amino acid residue in a native sequence has been removed and a different amino acid inserted in the same position. The substitutions may be single, where only one amino acid in the molecule is substituted, or multiple, where two or more amino acids in the same molecule are substituted. The substitutions may be at consecutive positions. Similarly, an amino acid may be substituted with multiple residues; such variants include both substitutions and insertions. "Insertional" (or "additional") variants are those in which one or more amino acids are inserted adjacent to a particular position in a native sequence. "Adjacent" means attached to the α-carboxy or α-amino functionality of that amino acid. "Deletional" variants are those in which one or more amino acids are deleted from the native amino acid sequence. Generally, deletional variants have one or two amino acids deleted within a specific region of the molecule.

[0078] In some embodiments, less than the theoretical maximum number of drug moieties are coupled to the antibody in the coupling reaction. Generally, antibodies do not contain many free and reactive cysteine ​​thiol groups that can be bound to drug moieties. In practice, most of the cysteine ​​thiol groups in antibodies exist as disulfide bridges. In some embodiments, antibodies can be reduced under partial or complete reduction conditions using a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine ​​thiol groups.

[0079] In the present invention, the term "pharmaceutically acceptable salt" refers to (i) a salt formed from an acidic functional group present in the complex provided by the present invention and a suitable inorganic or organic cation (base), including, but not limited to, alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; other metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic base salts such as ammonium salt; and organic base salts such as t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylamine salt, and tris(hydroxymethyl)aminomethane salt. and (ii) salts formed from a basic functional group present in the complex provided by the present invention and a suitable inorganic or organic anion (acid), including, but not limited to, hydrohalides such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-benzenesulfonate; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as glycine, trimethylglycine, arginine, ornithine, glutamate, and aspartate.

[0080] Pharmaceutically acceptable salts can be obtained by standard procedures well known in the art, for example, by reacting a sufficient amount of a basic material with a suitable acid to provide a pharmaceutically acceptable anion, or by reacting a sufficient amount of an acidic material with a suitable base to provide a pharmaceutically acceptable cation.

[0081] In the present invention, a solvate refers to the antibody-drug conjugate of the present invention in the solid or liquid form of a complex formed by coordination with solvent molecules. A hydrate is a specific form of a solvate having coordinated water molecules. In the present invention, a hydrate is a preferred solvate.

[0082] Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredient are known or will be apparent to those skilled in the art based on the disclosure of the present invention. As described in REMINGTON'S PHARMACEUTICAL SCIENCES, Martin, EW, Mack Publishing Company, 19th ed. (1995), methods for preparing such pharmaceutical compositions include incorporating suitable pharmaceutical additives, carriers, diluents, etc. that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations used.

[0083] In the present invention, the term "pharmaceutical additives" refers to excipients and additives used in the manufacture and formulation of drugs, and refers to substances other than active ingredients whose safety has been reasonably evaluated and that are included in drug formulations. Pharmaceutical additives are important components that can affect the quality, safety, and efficacy of drugs, serving as excipients, carriers, and stabilizers, as well as improving dissolution, solubilizing, and sustained-release properties. Depending on their origin, they can be classified into natural products, semi-synthetic products, and fully synthetic products. Depending on their function and use, they can be classified into solvents, propellants, solvent enhancers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, humectants, osmotic pressure regulators, stabilizers, flow improvers, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, coating agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, and the like. According to the route of administration, they can be classified as oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration, ocular administration, etc. The same pharmaceutical excipients can be used in drug formulations with different routes of administration and have different actions and uses.

[0084] In the present invention, the pharmaceutical composition can be prepared in various appropriate dosage forms depending on the route of administration, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, eye drops, pills, implants, aerosols, powders, sprays, etc. Here, the pharmaceutical composition or appropriate dosage form may contain 0.01 mg to 1000 mg of the antibody-drug conjugate of the present invention, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof.

[0085] As used herein, the term "treatment" generally refers to obtaining a desired pharmacological and / or physiological effect. This effect can be preventative, by completely or partially preventing a disease or its symptoms, and / or therapeutic, by partially or completely stabilizing or treating a disease and / or its side effects. As used herein, "treatment" encompasses any treatment of a disease in a patient, including (a) preventing a disease or condition from occurring in a patient who is susceptible to the disease or condition but has not yet been diagnosed with the disease, (b) inhibiting a symptom of the disease, i.e., inhibiting the progression of the disease, or (c) alleviating a symptom of the disease, i.e., causing regression of the disease or condition.

[0086] In the present invention, "subject" refers to a vertebrate. In some embodiments, a vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cows), pets (such as cats, dogs, and horses), primates, mice, and rats. In some embodiments, a mammal refers to a human.

[0087] In the present invention, the term "effective amount" refers to an amount that effectively achieves the desired therapeutic or preventive effect at the required dosage and for the required period of time. The "therapeutically effective amount" of a substance / molecule of the present invention may vary depending on factors such as the individual's disease state, age, sex, and weight, and the ability of the substance / molecule to induce the desired response in the individual. A therapeutically effective amount also encompasses an amount in which the therapeutically beneficial effects of the substance / molecule are greater than any toxic or harmful consequences. A "prophylactically effective amount" refers to an amount that effectively achieves the desired preventive effect at the required dosage and for the required period of time. Although not necessarily the case, a prophylactic dose is administered to a subject before the onset of disease or at an early stage of disease, so the prophylactically effective amount will be less than the therapeutically effective amount. In the case of cancer, a therapeutically effective amount of a drug includes reducing the number of cancer cells, shrinking tumor volume, inhibiting (i.e., slowing down, preferably stopping) cancer cell invasion into surrounding organs (i.e., slowing down, preferably stopping) tumor metastasis (i.e., slowing down, preferably stopping) tumor growth to some extent, and / or alleviating one or more symptoms associated with cancer to some extent.

[0088] In the present invention, the 20 common amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd ed., E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0089] The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0090] "Humanized" antibodies refer to non-human (e.g., murine) antibody forms, including chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibodies) in which complementarity-determining region (CDR) residues of the recipient antibody are substituted by CDR residues from a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity.

[0091] Furthermore, humanization may involve the introduction of mutations into amino acid residues within the CDR1, CDR2, and / or CDR3 regions of VH and / or VL, thereby improving one or more binding characteristics (e.g., affinity) of the antibody. Mutations may be introduced, for example, by PCR mutagenesis, and their effect on antibody binding or other functional properties may be assessed by in vitro or in vivo testing as described herein. Conservative mutations are typically introduced. Such mutations may be amino acid substitutions, additions, or deletions. Also, mutations within a CDR typically do not exceed one or two. [Example]

[0092] The present invention will be further explained below by showing specific examples, but these examples are not intended to limit the scope of the present invention.

[0093] Example 1: Production, sequence and expression of humanized GPC3 antibodies 1. Production of humanized GPC3 antibody To obtain a mouse monoclonal antibody targeting human GPC3, BALB / C and C57BL / 6 mice were immunized with human GPC3 protein, and serum antibody titers were detected using ELISA (human GPC3 protein) and FACS (human GPC3-overexpressing CHO-K1 cells). After multiple immunizations, mice with high titers were selected to generate anti-human GPC3 hybridoma cell lines. Positive clones were identified by ELISA and FACS. Subcloning screening was then performed to obtain the monoclonal antibody 52H5D3B8. Finally, new humanized antibodies Hu 52H5D3B8-7 and Hu 52H5D3B8-8 were obtained through antibody humanization design.

[0094] II. Sequence and Expression of Humanized GPC3 Antibody 1. Antibody sequence Hu 52H5D3B8-7: VH: EVQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPGQGLEWMG AIHPGSGGTAYNQKFKG RVTMTADKSISTAYMELSRLRSDDTAVYYCTR FYSYAY WGQGTLVTVSS(SEQ ID NO:8) The underlined parts are, from left to right, CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3). The non-underlined portions are, from left to right, FR1 (SEQ ID NO:4), FR2 (SEQ ID NO:5), FR3 (SEQ ID NO:6), and FR4 (SEQ ID NO:7). VL: DVVMTQTPLSLSVTPGQPASISC RSSQSLVHSNGNTYLQ WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC SQSIHVPYT FGGGTKVEIK (SEQ ID NO: 16) The underlined parts are, from left to right, CDR1 (SEQ ID NO: 9), CDR2 (SEQ ID NO: 10), and CDR3 (SEQ ID NO: 11). The non-underlined portions are, from left to right, FR1 (SEQ ID NO:12), FR2 (SEQ ID NO:13), FR3 (SEQ ID NO:14), and FR4 (SEQ ID NO:15). Hu 52H5D3B8-8: VH: EVQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPGQGLEWMG AIHPGSGGTAYNQKFKG RVTMTADKSISTAYMELSRLRSDDTAVYYCTR FYSYAY WGQGTLVTVSS(SEQ ID NO:8) The underlined parts are, from left to right, CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3). The non-underlined portions are, from left to right, FR1 (SEQ ID NO:4), FR2 (SEQ ID NO:5), FR3 (SEQ ID NO:6), and FR4 (SEQ ID NO:7). VL: DVVMTQTPLSLSVTPGQPASISC RSSQSLVHSNGNTYLQ WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYFC SQSIHVPYT FGGGTKVEIK (SEQ ID NO: 18) The underlined parts are, from left to right, CDR1 (SEQ ID NO: 9), CDR2 (SEQ ID NO: 10), and CDR3 (SEQ ID NO: 11). The non-underlined portions are, from left to right, FR1 (SEQ ID NO: 12), FR2 (SEQ ID NO: 13), FR3 (SEQ ID NO: 17), and FR4 (SEQ ID NO: 15). The heavy chain constant regions of both Hu 52H5D3B8-7 and Hu 52H5D3B8-8 are as follows: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:19) The light chain constant regions of both Hu 52H5D3B8-7 and Hu 52H5D3B8-8 are as follows: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:20) Control antibody: (1)GC33 antibody (Chugai Pharmaceutical) A method for producing the GC33 antibody is described in US Pat. No. 7,919,086B2. The CDR information for the heavy and light chains of the GC33 antibody is as follows:

[0095] [Table 4]

[0096] The FR information for the heavy and light chains of the GC33 antibody is as follows:

[0097] [Table 5]

[0098] (2)Y035 antibody (CARSGEN Therapeutics) The method for producing the Y035 antibody is described in CN106397593B. The amino acid sequences of the heavy and light chain variable regions of the humanized Y035 antibody are as follows: Humanized Y035 heavy chain variable region (SEQ ID NO:42): EVQLVQSGAEVKKPGASVKVSCKASGYTHS DYEMH WVRQAPGQGLEWMG AIHPGSGDTAYNQRFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR FYSYAY WGQGTLVTVSA The underlined parts are, from left to right, CDR1 (SEQ ID NO: 35), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 37). The non-underlined portions are, from left to right, FR1 (SEQ ID NO:38), FR2 (SEQ ID NO:39), FR3 (SEQ ID NO:40), and FR4 (SEQ ID NO:41). Humanized Y035 light chain variable region (SEQ ID NO:50): DIVMTQTPLSLPVTPGEPASISC RSSQSLVHSNGNTYLQ WYLQWYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC SQSIYVPYT FGQGTKLEIKR The underlined parts are, from left to right, CDR1 (SEQ ID NO: 43), CDR2 (SEQ ID NO: 44), and CDR3 (SEQ ID NO: 45). The non-underlined portions are, from left to right, FR1 (SEQ ID NO: 46), FR2 (SEQ ID NO: 47), FR3 (SEQ ID NO: 48), and FR4 (SEQ ID NO: 49). The heavy chain constant regions of both the GC33 antibody and the Y035 antibody are as follows: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:19) The light chain constant regions of both the GC33 antibody and the Y035 antibody are as follows: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:20)

[0099] 2. Cell binding of humanized GPC3 antibody The present inventors evaluated the binding activity of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 to CHO-K1 cells, HepG2 cells, and HuH-7 cells overexpressing human GPC3 by FACS and compared them with the known anti-GPC3 antibodies GC33 and Y035. To evaluate the CDR regions of the two antibodies, Hu 52H5D3B8-7 and Hu 52H5D3B8-8, and the FR region of the Hu 52H5D3B8-7 antibody, the present inventors prepared two recombinant antibodies. "Hu 52H5D3B8-7 / 8 CDRs with Y035 FRs" means that the antibody was obtained by recombination of the CDR region of the Hu 52H5D3B8-7 antibody or Hu 52H5D3B8-8 antibody with the FR region of the Y035 antibody. The term "Y035 CDRs with Hu 52H5D3B8-7 FRs" antibody means that the antibody is obtained by recombination of the CDR regions of the Y035 antibody with the FR regions of the Hu 52H5D3B8-7 antibody. GPC3-CHO-K1, HepG2, and Huh-7 cells were first incubated with Fc-blocking agents at 4°C for 15 minutes. The cells were washed with FACS buffer (PBS + 2% FBS), and 5-fold serial dilutions of Hu 52H5D3B8-7, Hu 52H5D3B8-8, Hu 52H5D3B8-7 / 8 CDRs with Y035FRs, Y035 CDRs with Hu 52H5D3B8-7FRs, or Y035 mAb (initial concentration 100 nM) were added to each well and incubated at 4°C for 60 minutes. The cells were then washed with FACS buffer and fixed with 2% PFA at room temperature for 15 minutes. After washing with FACS buffer, AlexaFluor® 647 AffiniPure goat anti-human IgG was added to each well and incubated at room temperature for 30 minutes. The samples were washed twice with FACS buffer. The fluorescence signals of the cell samples were analyzed using a MACSQuant Analyzer16 and expressed as MFI (GeoMean Fluorescence Intensity). The experimental results are shown in Figures 1-3. Hu 52H5D3B8-7 and Hu 52H5D3B8-8 had similar binding affinities to GPC3-CHO-K1, HepG2, and Huh-7 cells, and were higher than those of other antibodies. Furthermore, both the antibody "Hu 52H5D3B8-7 / 8 CDRs with Y035 FRs" and the antibody "Y035 CDRs with Hu 52H5D3B8-7 FR" showed higher cell affinity than Y035, indicating that the CDR regions of the Hu 52H5D3B8-7 and Hu 52H5D3B8-8 antibodies and the FR region of the Hu 52H5D3B8-7 antibody are superior to the Y035 antibody. EC of each test antibody in GPC3-CHO-K1 cells, HepG2 cells, and Huh-7 cells 50 The values ​​are shown in Table 1 below.

[0100] [Table 6]

[0101] 3. Cellular internalization of humanized GPC3 antibody We evaluated the internalization of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 in HepG2 cells and compared them with the known GPC3 antibody Y035 (CARSGEN Therapeutics). In addition, to evaluate the CDRs and FRs of the Hu 52H5D3B8-7 and Hu 52H5D3B8-8 antibodies, we also evaluated the antibody "Hu 52H5D3B8-7 / 8 CDRs with Y035 FRs" and the antibody "Y035 CDRs with Hu 52H5D3B8-7 FRs" in in vitro internalization experiments. pHAb thiol dye is a pH sensor dye that has very low fluorescence at pH > 7 and exhibits a significant increase in fluorescence upon internalization into endosomes or lysosomes (pH 6.3 or 4.7, respectively). Briefly, pHAb thiol dye-labeled α-hIgG secondary antibody (10 μg / mL) was incubated with antibody (20 μg / mL) for 30 min. After incubation, the mixed antibody was serially diluted two-fold, and the diluted mixture was diluted to 2 × 10 4 The antibody was added to each well of a 96-well assay plate pre-seeded with HepG2 cells. After 48 hours of culture, the fluorescent signal (Envision® 2105) was captured using a multimodal reader. As shown in Figure 4, the experimental results demonstrate that both Hu 52H5D3B8-7 and Hu 52H5D3B8-8 exhibited higher internalization efficiency than the other antibodies. Furthermore, the antibody "Hu 52H5D3B8-7 / 8 CDRs with Y035 FRs" exhibited a higher internalization efficiency (higher Top value and corresponding EC 50 ) which means that the CDRs of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 are superior to Y035. 50 and Top values ​​are shown in Table 2 below.

[0102] [Table 7]

[0103] Example 2: Preparation of GPC3-ADC 1. Preparation of Antibody-Drug Conjugate Anti-GPC3-L1-D1 a: 10 mg of GPC3 antibody (Hu 52H5D3B8-7, Hu 52H5D3B8-8, or GC33) was taken, the antibody was adjusted to pH 7.5 with 0.7 M Tris-0.5 M EDTA solution, weighed, the protein concentration was measured, and the total protein amount was calculated. TCEP was added to the antibody in a 2.8-fold molar amount, and the mixture was placed on a 3D shaker and reacted at 22°C for 105 minutes with continuous mixing. b: L1-D1 (i.e., MC-vc-PAB-MMAE) (manufactured by Shanghai Miracogen Inc. on behalf of Hubei Huashitong Biomedical Science and Technology Co., Ltd. and dissolved in DMSO) was added to the reduced antibody in an amount 1.6 times the free mercapto moles, mixed, placed on a 3D shaker, and reacted at 22°C for 20 minutes with continuous mixing. N-acetylcysteine ​​was added in an amount 3 times the molar amount of L1-D1 (i.e., MC-vc-PAB-MMAE) added to the reaction system, mixed, placed on a 3D shaker, and reacted at 22°C for 5 minutes with continuous mixing. The solution was replaced with conjugate stock solution (10 mM histidine, 3% sucrose, 0.03% Tween®-80, pH 5.6) using a 15 mL 30KD ultrafiltration device for a total of five replacements, and then filtered through a 0.22 μm filter membrane to obtain the antibody-drug conjugate product Anti-GPC3-L1-D1 (a collective term for Hu 52H5D3B8-7-L1-D1, Hu 52H5D3B8-8-L1-D1, and GC33-L1-D1), which was stored at 4°C. 2. Preparation of Antibody-Drug Conjugate Anti-GPC3-L3-D3 a: 2 mg of GPC3 antibody (Hu 52H5D3B8-7, Hu 52H5D3B8-8, or GC33) was taken, the antibody was adjusted to pH 7.5 with 0.7 M Tris-0.5 M EDTA solution, weighed, the protein concentration was measured, and the total protein amount was calculated. TCEP was added to the antibody in a 1.3-fold molar amount, and the mixture was placed on a 3D shaker and reacted at 22°C for 105 minutes with continuous mixing. b: To the reduced antibody, L3-D3 (i.e., MA-PEG4-VC-PAB-DMEA-Duocarmycin SA) (purchased from Levena (Suzhou) Biopharma Co., Ltd., dissolved in DMSO) was added in an amount twice the number of free mercaptomoles. After mixing, the mixture was placed on a 3D shaker and incubated at 22°C for 20 minutes with continuous mixing. N-acetylcysteine ​​was added in an amount three times the molar amount of L3-D3 (i.e., MA-PEG4-VC-PAB-DMEA-Duocarmycin SA) added to the reaction system. After mixing, the mixture was placed on a 3D shaker and incubated at 22°C for 5 minutes with continuous mixing. Using a 0.5 mL 30KD ultrafiltration device, the solution was replaced with conjugate stock solution (10 mM histidine, 3% sucrose, 0.03% Tween®-80, pH 5.6) five times in total, and then filtered through a 0.22 μm filter membrane to obtain the antibody-drug conjugate product Anti-GPC3-L3-D3 (a collective term for Hu 52H5D3B8-7-L3-D3, Hu 52H5D3B8-8-L3-D3, and GC33-L3-D3), which was stored at 4°C. 3. Preparation of Antibody-drug Conjugate Anti-GPC3-L4-D4 a: 10 mg of GPC3 antibody (Hu 52H5D3B8-7, Hu 52H5D3B8-8, or GC33) was taken, the antibody was adjusted to pH 7.5 with 0.7 M Tris-0.5 M EDTA solution, weighed, the protein concentration was measured, and the total protein amount was calculated. TCEP was added to the antibody in a 2.7-fold molar amount, and the mixture was placed on a 3D shaker and reacted at 22°C for 105 minutes with continuous mixing. b: L4-D4, i.e., MC-MMAF (purchased from BrightGene Bio-Medical Technology (Suzhou) Co., Ltd., dissolved in DMSO), was added to the reduced antibody in an amount twice the number of free mercaptomoles. After mixing, the mixture was placed on a 3D shaker and reacted at 22°C for 20 minutes with continuous mixing. N-acetylcysteine ​​was added in an amount three times the molar amount of the L4-D4 (i.e., MC-MMAF) added to the reaction system. After mixing, the mixture was placed on a 3D shaker and reacted at 22°C for 5 minutes with continuous mixing. Using a 15 mL 30KD ultrafiltration device, the solution was replaced with conjugate stock solution (10 mM histidine, 3% sucrose, 0.03% Tween®-80, pH 5.6) five times in total, and then filtered through a 0.22 μm filter membrane to obtain the antibody-drug conjugate product Anti-GPC3-L4-D4 (a collective term for Hu 52H5D3B8-7-L4-D4, Hu 52H5D3B8-8-L4-D4, and GC33-L4-D4), which was stored at 4°C. 4. Preparation of Antibody-drug Conjugate Anti-GPC3-ZLA a: 10 mg of GPC3 antibody (Hu 52H5D3B8-7, Hu 52H5D3B8-8, or GC33) was dissolved in 40 mM PB + 2 mM EDTA (pH 7.0). The antibody was diluted to a concentration of 5 mg / mL with 40 mM PB + 2 mM EDTA (pH 6.99), weighed, and the protein concentration was determined and the total protein amount was calculated. TCEP was added in a 2.25-fold molar ratio to the antibody, and the sample was mixed and placed in an incubator at 22°C for 3 hours, with continuous mixing at 200 rpm. b: The reduced antibody solution was cooled on ice, and ZLA (dissolved in DMA, the total DMA content was 10% of the reaction system; ZLA is the compound described in Example 2.1 of Patent Publication CN107001415B, structural formula is as follows) was added to the cooled antibody in an amount of 7 times the moles of antibody, mixed quickly, and then placed in an incubator at 4°C for 2 hours with continuous mixing at a mixer speed of 200 rpm. N-acetylcysteine ​​was added to the reaction system in an amount of 6 times the moles of antibody added, mixed, and then placed in an incubator at 22°C for 15 minutes with continuous mixing at a mixer speed of 200 rpm. The solution was then substituted with a 15 mL 50 kD ultrafiltration device containing a conjugate storage solution (20 mM histidine / histidine-HCl, pH 5.41), filtered through a 0.22 μm membrane for sterilization, and the protein concentration was determined. The antibody-drug conjugate products, Anti-GPC3-ZLA (collectively Hu 52H5D3B8-7-ZLA, Hu 52H5D3B8-8-ZLA, and GC33-ZLA), were obtained and stored at 4°C.

[0104] [ka] Structural formula of ZLA

[0105] The drug loading (DAR) of the above GPC3-ADC was detected by hydrophobic interaction chromatography (HIC-HPLC), and typical charts are shown in Figures 5 to 8. The average drug loading (DAR) of Hu 52H5D3B8-7-L1-D1, Hu 52H5D3B8-7-L3-D3, Hu 52H5D3B8-7-L4-D4, and Hu 52H5D3B8-8-ZLA was calculated from the spectral peak areas to be 4.2, 2.3, 4.0, and 3.9, respectively.

[0106] Example 3 Pharmacological and pharmacodynamic studies of GPC3-ADC 1. Cell Binding of GPC3-ADC FACS experiments confirmed that the binding of the ADCs was not affected by Cys coupling and simultaneously compared the cell binding of the Hu 52H5D3B8-8 ADC with the GC33 ADC. After trypsin-EDTA digestion, cultured HepG2 cells were harvested and centrifuged. The cells were resuspended in FACS buffer (PBS + 2% FBS) to adjust the cell density. Then, the cells were sorted into a 96-well U-bottom cell culture plate at 200,000–500,000 cells per well. Serially diluted antibodies or ADCs were added in FACS buffer at a final concentration of 10 μg / mL. The 96-well plate was incubated at 4°C for 60 minutes. The cells were thoroughly washed with FACS buffer to remove unbound antibodies and ADCs. A 1:1000 dilution of Goat anti-Human IgG (H + L) secondary antibody, Alexa Fluor 488 (Invitrogen) in FACS buffer was added and incubated at 4°C for 30 minutes, protected from light. The cells were then washed with FACS buffer to remove unbound secondary antibodies. The cell samples were subjected to fluorescent signal analysis using a CytoFLEX flow cytometer (Beckman Coulter). The experimental results, shown in Figure 9, indicate that the affinity of the antibodies Hu 52H5D3B8-8 and GC33 for HepG2 cells remains unchanged before and after conjugation with the cytotoxin. Furthermore, the Hu 52H5D3B8-8 antibody and Hu 52H5D3B8-8 ADC have stronger affinity for HepG2 cells than the GC33 antibody and GC33 ADC. The affinity of each test compound for HepG2 cells is shown in Table 3.

[0107] [Table 8]

[0108] 2. Antibody internalization in GPC3-ADC To confirm that the internalization of the ADC was not inferior to that of the monoclonal antibody, the internalization of the Hu 52H5D3B8-7 ADC, Hu 52H5D3B8-8 ADC, and GC33 ADC was also compared. A 96-well U-bottom cell culture plate was blocked with 5% nonfat dry milk dissolved in PBS for 60 minutes. Cultured Huh7 hepatoma cells were harvested after treatment with non-pancreatic enzyme digestion solution (Sigma-Aldrich). After centrifugation, the cells were resuspended in pre-chilled DMEM medium, adjusted to a cell density of 500,000–800,000 cells per well, and then aliquoted into the blocked 96-well U-bottom cell culture plate. Antibodies and ADCs diluted in 5% FBS-containing DMEM medium were added and mixed to a final concentration of 10 μg / mL. The 96-well plate was placed on ice and incubated for 60 minutes. The cells were thoroughly washed with pre-chilled FACS buffer (PBS + 2% FBS) to remove unbound antibodies and ADCs. The cells were equally divided between two blocked 96-well U-plates, centrifuged, and resuspended in DMEM medium. One 96-well plate was placed on ice and the other in a 37°C cell incubator. After 2 hours, the 37°C plate was transferred to ice and left for an additional 5–10 minutes. Diluted goat anti-human IgG (H + L) secondary antibody, Alexa Fluor 488 (Invitrogen), was added and incubated on ice for 30 minutes protected from light. Cells in the wells were washed with pre-chilled FACS buffer to remove unbound secondary antibody. Cell samples were subjected to fluorescent signal analysis using a CytoFLEX flow cytometer (Beckman Coulter) and expressed as MFI (Geomean fluorescence intensity). Except for the 37°C incubation, the entire experimental process was maintained on ice or at 4°C. The rate of cell surface molecule depletion is calculated as follows: Cell surface molecule reduction % = (MFI 氷上 - MFI 37℃ ) / MFI 氷上 * 100%. The experimental results are shown in Table 4. There was no change in the internalization ability of the antibodies Hu 52H5D3B8-8 and GC33 in Huh7 cells before and after conjugation with a cytotoxin, and the internalization ability of the Hu 52H5D3B8-8 antibody and Hu 52H5D3B8-8 ADC was higher than that of the GC33 antibody and GC33 ADC. The internalization results of each test compound in Huh7 cells are shown in Table 4.

[0109] [Table 9]

[0110] Example 4 In vitro pharmacodynamic studies The cytotoxicity of Hu 52H5D3B8-7 or Hu 52H5D3B8-8 ADCs conjugated with various linker-payloads (i.e., LDs) was compared with that of the GC33 ADC. After digestion with trypsin-EDTA, the cultured hepatoma cells were harvested. After centrifugation, the cells were resuspended in fresh culture medium and counted. The cells were seeded into 96-well clear-bottom black cell culture plates (Costar) and cultured overnight in a cell incubator. The next day, antibodies and ADCs were serially diluted 4-fold with culture medium, and the dilutions were carefully transferred to the black plate so that the final initial concentration for each sample was 10 μg / mL. After 96 hours of incubation in the cell incubator, PrestoBlue reagent (Invitrogen) was added at 1 / 10 the well volume, followed by incubation in the cell incubator for 1 hour. Fluorescence signals were read using a SpectraMax M5 plate reader, with the excitation and emission wavelengths set to 560 nm and 590 nm, respectively. The resulting fluorescence signal data were analyzed using SoftMax Pro 6.5 software. 1. Experimental reagents and sources:

[0111] [Table 10]

[0112] 2. Experimental results: EC of cytotoxicity of each GPC3-ADC 50 The mean values ​​are shown in Table 5. Figures 10-15 are representative images of the killing of Huh7 / HepG2 cells by different GPC3-ADCs.

[0113] [Table 11]

[0114] As can be seen from the results in Table 5 and Figures 10-15, the different GPC3-ADCs of the present invention exhibited significant cell-killing activity in cell lines with moderate to high GPC3 expression, and were stronger than GC33-ADC.

[0115] Example 5 In vivo Pharmacodynamic Studies The antitumor activity of GPC3-ADC was assayed in the LIV#219 liver cancer PDX model, which has relatively high GPC3 RNA expression levels and confirmed positive GPC3 expression by FACS analysis. The process for creating a PDX model of human liver cancer in nude mice is as follows: 3 The tumor tissue was subcutaneously transplanted into the dorsal skin of BALB / c nude mice. 3 When tumor volume reached 1000 mg / kg, mice were randomly divided into four groups, each with six mice, according to body weight to ensure uniform tumor volume across the groups: vehicle, 3 mg / kg GC33-L1-D1, 3 mg / kg Hu 52H5D3B8-7-L1-D1, and 3 mg / kg Hu 52H5D3B8-8-L1-D1. Data analysis: Tumor volume was measured twice a week during the experimental period. Tumor volume (TV) was calculated using the formula TV = l × w 2 / 2, where l and w are the length and width of the tumor measurement, respectively. The relative tumor volume (RTV) was calculated from the measurement results, and RTV = V f / V0, where V0 is the tumor volume measured at the split dose (i.e., Day 0), and V f is the tumor volume measured on the final day. Relative tumor growth rate T / C (%) = (treatment group RTV / vehicle group RTV) × 100%. Tumor growth inhibition rate TGI% = (vehicle group mean tumor volume - drug treatment group mean tumor volume) / vehicle group mean tumor volume × 100%. If T / C (%) ≦ 40% and P < 0.05, the test substance was considered to have a significant inhibitory effect on tumor growth. The experimental results are shown in Figures 16-17. After administration of GC33-L1-D1 at a dose of 3 mg / kg, the relative tumor growth rate (T / C%) on Day 26 was 54.90%, and the tumor growth inhibition rate (TGI%) was 45.10%. In the Hu 52H5D3B8-7-L1-D1 (3 mg / kg) group, the T / C% was 33.58%, the TGI% was 66.42%, and in the Hu 52H5D3B8-8-L1-D1 (3 mg / kg) group, the T / C% was 39.93%, the TGI% was 60.07%. The experimental results showed that both Hu 52H5D3B8-7-L1-D1 (3 mg / kg) and Hu 52H5D3B8-8-L1-D1 (3 mg / kg) had significant tumor growth inhibitory effects, superior to those of GC33-L1-D1 (3 mg / kg). Tumor-bearing mice tolerated both Hu 52H5D3B8-7-L1-D1 and Hu 52H5D3B8-8-L1-D1 well.

Claims

1. 1. An antibody-drug conjugate having the structure shown in Formula I: 【Chemical 1】 (Wherein Ab is an anti-GPC3 antibody, the anti-GPC3 antibody comprises a heavy chain and a light chain, and CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and CDR1, CDR2, and CDR3 of the light chain variable region comprise the sequences shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; L is a linker, D is a cytotoxin, p is any number between 1 and 8.)

2. The linker may be 6-maleimidohexanoyl (MC), maleimidopropionyl (MP), N-succinimido 4-(2-pyridylthio)valerate (SPP), 4-(N-maleimidomethyl)-cyclohexane-1-formyl (MCC), N-succinimido(4-iodo-acetyl)aminobenzoate (SIAB), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), MA-PEG4-VC-PAB-DMEA, 【Chemistry 2】 is selected from Preferably, the linker is 6-maleimidohexanoyl (MC), 6-maleimidohexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), MA-PEG4-VC-PAB-DMEA, 【Chemistry 3】 2. The antibody-drug conjugate according to claim 1, wherein the antibody-drug conjugate is selected from the group consisting of:

3. The cytotoxin may be SN-38, gemcitabine, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), maytansinoids (e.g., Maytansine DM1, Maytansine DM4), calicheamicin, MGBA (e.g., duocarmycin), doxorubicin, ricin, diphtheria toxin, duocarmycin SA, 【Chemistry 4】 selected from I131, interleukins, tumor necrosis factors, chemokines and nanoparticles; Preferably, the cytotoxin is monomethylauristatin E (MMAE), duocarmycin SA, monomethylauristatin F (MMAF), 【Chemistry 5】 The antibody-drug conjugate according to any one of claims 1 to 2, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, which is selected from the group consisting of:

4. LD is MC-vc-PAB-MMAE, MA-PEG4-VC-PAB-DMEA-Duocarmycin SA, MC-MMAF, 【Chemistry 6】 The antibody-drug conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, which is selected from the group consisting of:

5. The anti-GPC3 antibody has one or more of the following characteristics 1) to 2): 1) The FR1, FR2, FR3, and FR4 regions of the heavy chain variable region of the anti-GPC3 antibody comprise the sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; 2) The antibody-drug conjugate according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, wherein the FR1, FR2, FR3, and FR4 regions of the light chain variable region of the anti-GPC3 antibody comprise the sequences shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, or the FR1, FR2, FR3, and FR4 regions of the light chain variable region of the GPC3 antibody comprise the sequences shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, and SEQ ID NO: 15, respectively.

6. The anti-GPC3 antibody has one or more of the following characteristics 1) to 2): 1) The sequence of the heavy chain variable region of the anti-GPC3 antibody comprises the sequence shown in SEQ ID NO: 8, 2) The antibody-drug conjugate according to any one of claims 1 to 5, wherein the sequence of the light chain variable region of the anti-GPC3 antibody comprises the sequence shown in SEQ ID NO: 16 or SEQ ID NO: 18, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof.

7. The anti-GPC3 antibody has one or more of the following characteristics 1) to 2): 1) The heavy chain constant region of the anti-GPC3 antibody is selected from the constant regions of human IgG, IgM, IgA, IgD, and IgE, or mutants of these constant regions; Preferably, the IgG is selected from IgG1, IgG2, IgG3 and IgG4; 2) The antibody-drug conjugate according to any one of claims 1 to 6, wherein the light chain constant region of the anti-GPC3 antibody is a human-derived lambda constant region, a kappa constant region, or a mutant of these constant regions, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof.

8. The anti-GPC3 antibody has one or more of the following characteristics 1) to 2): 1) The sequence of the heavy chain constant region of the anti-GPC3 antibody comprises the sequence shown in SEQ ID NO: 19 or a sequence having greater than 70%, preferably greater than 75%, 80%, 85%, 90%, 95%, or 99% homology to the sequence shown in SEQ ID NO: 19; Preferably, the sequence of the heavy chain constant region of the anti-GPC3 antibody is shown in SEQ ID NO: 19, 2) The sequence of the light chain constant region of the anti-GPC3 antibody comprises the sequence shown in SEQ ID NO: 20 or a sequence having greater than 70%, preferably greater than 75%, 80%, 85%, 90%, 95%, or 99% homology to the sequence shown in SEQ ID NO: 20; Preferably, the sequence of the light chain constant region of the anti-GPC3 antibody is that shown in SEQ ID NO:

20. The antibody-drug conjugate according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof.

9. p is any number between 2 and 8, Preferably, p is any number between 2 and 6; More preferably, the antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof according to any one of claims 1 to 8, wherein p is any number from 2 to 5 (e.g., 2.3, 3.9, 4.0, 4.2).

10. A drug composition comprising the antibody-drug conjugate according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, Optionally, further comprising at least one of a chemotherapeutic agent, an immunotherapeutic agent, and an immunosuppressant agent for tumor treatment; Or optionally, a pharmaceutical composition further comprising at least one pharmaceutical additive.

11. Use of the antibody-drug conjugate according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, or the drug composition according to claim 10, in the manufacture of a drug used for the prevention and / or treatment of a GPC3-positive associated disease, Preferably, the GPC3-positive associated disease comprises liver cancer, lung cancer, gastric cancer, head and neck cancer, esophageal cancer, Merkel cell carcinoma, or liposarcoma.

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