GPC3 antibody drug conjugate and application thereof

Abstract

The invention provides an antibody drug conjugate and application thereof, the antibody drug conjugate has a structure as shown in formula I, i.e., Ab-(L-D) p, Ab is an anti-GPC3 antibody, L is a linker, and D is cytotoxin. The antibody drug conjugate provided by the invention has good activity of inhibiting the growth of tumor cells in vivo and in vitro, and has relatively low cytotoxicity.

Description

GPC3 antibody-drug conjugates and their applications Technical Field

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

[0002] Liver cancer is the fifth most prevalent cancer worldwide and the third most common cause of cancer-related death. Hepatocellular carcinoma (HCC) is the predominant form of liver cancer, accounting for 90% of all liver cancers. The currently accepted treatment is a multidisciplinary approach centered around surgical resection. While short-term efficacy has improved, long-term outcomes remain suboptimal. Therefore, exploring new, more effective strategies for treating liver cancer is a current research hotspot both domestically and internationally, and the development of new drugs for the treatment of liver cancer remains urgent.

[0003] GPC3, the full name of which is Glypican-3, is a member of the heparan sulfate proteoglycan family and is connected to the cell surface through a glycosylphosphatidylinositol anchor on the cell membrane. The protein core of GPC3 consists of two subunits, of which the N-terminal subunit is approximately 40kDa and the C-terminal subunit is approximately 30kDa. 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 to form a complex and trigger downstream signals. The core protein of GPC3 can serve as a co-receptor or receiver for Wnt.

[0004] In the human body, the GPC3 protein expressed by the GPC3 gene exhibits significant differences in expression at different developmental stages and in different tissues. For example, it is present in very low levels in normal adult tissues, is lowly expressed or absent in cancers such as gastric cancer, breast cancer, and ovarian cancer, and is often overexpressed (70% to 80%) in hepatocellular carcinoma (HCC). Studies have shown that GPC3 is a liver cancer-specific antigen that can help confirm the nature of HCC lesions in the early stages, significantly improving diagnostic accuracy. Therefore, GPC3 is considered to have great potential in liver cancer immunotherapy.

[0005] GPC3-targeted therapies have gradually attracted attention in recent years. Currently, therapeutic strategies based on the GPC3 target mainly focus on antibody drugs, cell therapy, and vaccines. Therapeutic anti-GPC3 antibodies that have been developed include antibodies such as GC33, YP7, and HN3, as well as bispecific antibodies (such as ERY974 and CM350 in clinical trials). Some of these antibodies inhibit Wnt signaling in liver cancer cells. In addition, chimeric antigen receptor (CAR) T cell immunotherapy has been developed at various stages for the treatment of liver cancer. In mice with xenografts or orthotopic liver tumors, CAR-T cells can eliminate GPC3-positive cancer cells by inducing perforin- and granzyme-mediated cell death and reducing WNT signaling in tumor cells.

[0006] Summary of the Invention

[0007] The inventors of the present application prepared an anti-GPC3 antibody-drug conjugate (ADC) through extensive experiments and creative work, and confirmed that it has good biological activity, thereby completing the present invention.

[0008] To this end, in the first aspect of the present invention, the present invention provides an antibody drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the antibody drug conjugate has the structure shown in Formula I, Ab-(LD) p Formula I

[0009] in:

[0010] Ab is an anti-GPC3 antibody, comprising a heavy chain and a light chain, wherein the heavy chain variable region CDR1, CDR2, and CDR3 comprise the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise the sequences shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively;

[0011] L is the connector;

[0012] D is cytotoxin;

[0013] p is any value 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, 7, 7.5, or 7.5-8).

[0014] In formula I, LD represents that the linker and the cytotoxin are connected by a covalent bond to form an LD molecule; Ab-(LD) p represents p LD molecules coupled to Ab via covalent bonds.

[0015] In the present invention, the drug-antibody ratio (DAR) refers to the number of drug molecules coupled to the antibody (e.g., p in Formula I). ​​The number of drug molecules contained in the antibody-drug conjugates described herein can be an integer or a decimal. Whether it is an integer or a decimal, it refers to the average number of drug molecules coupled to each antibody. "p is any value between 1-8" means that p can be any integer selected from 1-8 (including endpoints 1 and 8), or any decimal selected from 1-8, such as 2.3, 3.9, 4.0, or 4.2. At the same time, it will be understood by those skilled in the art that even if the same preparation method is used, the DAR values ​​of the antibody-drug conjugates prepared in different batches are not necessarily exactly the same, for example, they can fluctuate within a range of no more than 0.5.

[0016] The drug-antibody ratio (DAR) can be verified by conventional means, such as mass spectrometry, ELISA assays, HIC, and HPLC. The quantitative distribution of ADC in terms of p can also be determined. In some cases, separation, purification, and verification 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.

[0017] In some embodiments, the linker is selected from: 6-maleimidocaproyl (MC), maleimidopropionyl (MP), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), 4-(N-maleimidomethyl)-cyclohexane-1-carboxyl (MCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), MA-PEG4-VC-PAB-DMEA,

[0018] In some embodiments, the linker is selected from: 6-maleimidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), MA-PEG4-VC-PAB-DMEA,

[0019] The structural formulas of MC, MC-vc-PAB and MA-PEG4-VC-PAB-DMEA are as follows:

[0020] In some embodiments, the cytotoxin is selected from the group consisting of: SN-38, gemcitabine, Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), maytansinoids (e.g., Maytansine DM1, Maytansine DM4), calicheamicin, MGBA (e.g., duocarmycin), doxorubicin, ricin, diphtheria toxin, duocarmycin SA, I131, interleukins, tumor necrosis factor, chemokines and nanoparticles.

[0021] In some embodiments, the cytotoxin is selected from the group consisting of: Monomethyl auristatin E (MMAE), Duocarmycin SA, Monomethyl auristatin F (MMAF),

[0022] The structural formulas of MMAE, MMAF and duocarmycin SA are shown below:

[0023] In some embodiments, LD is selected from the group consisting of MC-vc-PAB-MMAE, MA-PEG4-VC-PAB-DMEA-Duocarmycin SA, MC-MMAF,

[0024] The structural formulas of MC-vc-PAB-MMAE, MA-PEG4-VC-PAB-DMEA-Duocarmycin SA, and MC-MMAF are shown below:

[0025] When the four LDs are covalently coupled to the Ab, the coupling method and site are the same, that is, they are all formed by coupling the succinimide at the end of the LD to the sulfhydryl group in the antibody. For example, when MC-vc-PAB-MMAE is covalently coupled to the Ab, the structural formula of the resulting ADC is as follows:

[0026] Among them, in the ADC formed above, the antibody Ab is connected to the carbon atom of the succinimide at the end of LD through -S-. The -S- is not an additional external thiol group, but a thiol group contained in the antibody itself after the antibody Ab is reduced and the disulfide bond is opened.

[0027] In some embodiments, the sequences of the heavy chain variable region CDR1, CDR2, and CDR3 of the anti-GPC3 antibody are shown as 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 are shown as SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

[0028] In some embodiments, the sequences of the heavy chain variable regions FR1, FR2, FR3, and FR4 of the anti-GPC3 antibody are shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

[0029] In some embodiments, the sequences of the FR1, FR2, FR3, and FR4 regions of the light chain variable region of the anti-GPC3 antibody are shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; or the sequences of the FR1, FR2, FR3, and FR4 regions of the light chain variable region of the anti-GPC3 antibody are shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, and SEQ ID NO: 15, respectively.

[0030] In some embodiments, the sequence of the heavy chain variable region of the anti-GPC3 antibody is shown in SEQ ID NO:8.

[0031] In some embodiments, the sequence of the light chain variable region of the anti-GPC3 antibody is shown in SEQ ID NO: 16 or SEQ ID NO: 18.

[0032] In some embodiments, the sequence of the heavy chain variable region of the anti-GPC3 antibody is shown in SEQ ID NO: 8, and the sequence of the light chain variable region of the anti-GPC3 antibody is shown in SEQ ID NO: 16.

[0033] In some embodiments, the sequence of the heavy chain variable region of the anti-GPC3 antibody is shown in SEQ ID NO: 8, and the sequence of the light chain variable region of the anti-GPC3 antibody is shown in SEQ ID NO: 18.

[0034] In some embodiments, the heavy chain constant region of the anti-GPC3 antibody is selected from human IgG, IgM, IgA, IgD, IgE constant regions, or mutants of the aforementioned constant regions.

[0035] In some embodiments, the IgG is selected from IgG1, IgG2, IgG3, and IgG4.

[0036] In some embodiments, the light chain constant region of the anti-GPC3 antibody is a human lambda constant region, a kappa constant region, or a mutant of the above constant regions.

[0037] In some embodiments, the sequence of the heavy chain constant region of the anti-GPC3 antibody comprises the sequence shown in SEQ ID NO: 19, or comprises a sequence having greater than 70% homology to the sequence shown in SEQ ID NO: 19, preferably greater than 75%, 80%, 85%, 90%, 95%, or 99%.

[0038] In some embodiments, the sequence of the heavy chain constant region of the anti-GPC3 antibody is shown in SEQ ID NO: 19.

[0039] In some embodiments, the sequence of the light chain constant region of the anti-GPC3 antibody comprises the sequence shown in SEQ ID NO: 20, or comprises a sequence having greater than 70% homology to the sequence shown in SEQ ID NO: 20, preferably greater than 75%, 80%, 85%, 90%, 95%, or 99%.

[0040] In some embodiments, the sequence of the light chain constant region of the anti-GPC3 antibody is as shown in SEQ ID NO: 20.

[0041] In some embodiments, p is any number between 2-8.

[0042] In some embodiments, p is any number between 2-6.

[0043] In some embodiments, p is any number between 2 and 5 (eg, 2.3, 3.9, 4.0, 4.2).

[0044] In the second aspect of the present invention, the present invention provides a pharmaceutical composition comprising the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof.

[0045] In some embodiments, the pharmaceutical composition further comprises at least one of a chemotherapeutic drug, an immunotherapeutic drug, and an immunosuppressant for treating tumors.

[0046] In some embodiments, the chemotherapy drug is, for example, doxorubicin (Adriamycin), cyclophosphamide, taxanes [such as paclitaxel (Taxol), docetaxel (Taxotere)], capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (Arimidex, Felocell, Aromasin), 5-FU plus folinic acid, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, etc., or a combination thereof.

[0047] In some embodiments, the immunotherapy drug is, for example, PD-1 monoclonal antibody (e.g., pembrolizumab, nivolumab), PD-L1 monoclonal antibody (e.g., atezolizumab), TIGIT monoclonal antibody, 4-1BB monoclonal antibody, VEGFR2 monoclonal antibody (e.g., Ramucirumab, apatinib), HER2 monoclonal antibody (e.g., trastuzumab, trastuzumab biosimilar, trastuzumab-dkst), etc., or a combination thereof.

[0048] In some embodiments, the immunosuppressant is selected from the group consisting of: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporine and tacrolimus; (3) antimetabolites, such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3; (5) alkylating agents, such as cyclophosphamide. In some specific embodiments, the immunosuppressant is, for example, methylprednisolone, prednisone, azathioprine, Prograf, Zenapax, Sule, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, mizoribine, cyclophosphamide, fingolimod, etc.

[0049] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0050] In a third aspect, the present invention provides use of the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, or the aforementioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease associated with GPC3; preferably, the disease associated with GPC3 is a disease associated with GPC3 positivity.

[0051] In some embodiments, the disease associated with GPC3 positivity 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 disease associated with GPC3 positivity is liver cancer.

[0052] In a fourth aspect, the present invention provides the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, or the aforementioned pharmaceutical composition, for use in preventing and / or treating a disease associated with GPC3; preferably, the disease associated with GPC3 is a disease associated with GPC3 positivity.

[0053] In some embodiments, the disease associated with GPC3 positivity 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 disease associated with GPC3 positivity is liver cancer.

[0054] In a fifth aspect, the present invention provides a method for treating and / or preventing a disease associated with GPC3, comprising: administering to a subject in need thereof an effective therapeutic and / or preventive amount of the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, or the aforementioned pharmaceutical composition; preferably, the disease associated with GPC3 is a disease associated with GPC3 positivity.

[0055] In some embodiments, the disease associated with GPC3 positivity 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 disease associated with GPC3 positivity is liver cancer. Beneficial effects

[0056] 1. The novel antibodies Hu 52H5D3B8-7 and Hu 52H5D3B8-8 of this invention are developed anti-GPC3 monoclonal antibodies that can be conjugated to various potent cytotoxic small molecule drugs, such as MMAE (methyl auristatin E), MMAF, and Duo, via linkers to form GPC3-ADCs. GPC3-ADCs effectively inhibit tumor cell proliferation and cause cell death by binding to GPC3 receptors on the tumor cell surface, internalizing them, and releasing small molecule toxins.

[0057] 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 stronger cellular binding affinity and better internalization. In addition, the affinity of the antibodies for HepG2 cells expressing GPC3 was unchanged after conjugation with cytotoxin.

[0058] 3. The newly developed antibodies Hu 52H5D3B8-7 and Hu 52H5D3B8-8 of the present invention can be coupled with a variety of different linker-payloads and exhibit strong cell-killing effects in liver cancer cells, which are significantly stronger than GC33-ADC with the same linker-payload.

[0059] 4. The newly developed GPC3-ADC of the present invention showed significant pharmacological effects in inhibiting tumor cell growth in the human liver cancer PDX tumor model. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 shows the affinity of various GPC3 antibodies for GPC3-CHO-K1 cells;

[0061] Figure 2 shows the affinity of various GPC3 antibodies for HepG2 cells;

[0062] FIG3 shows the affinity of each GPC3 antibody for Huh-7 cells;

[0063] FIG4 shows the internalization of various GPC3 antibodies on HepG2 cells;

[0064] FIG5 shows a hydrophobic interaction chromatogram of Hu 52H5D3B8-7-L1-D1;

[0065] FIG6 shows a hydrophobic interaction chromatogram of Hu 52H5D3B8-7-L3-D3;

[0066] FIG7 shows a hydrophobic interaction chromatogram of Hu 52H5D3B8-7-L4-D4;

[0067] FIG8 shows a hydrophobic interaction chromatogram of Hu 52H5D3B8-8-ZLA;

[0068] FIG9 shows the affinity of GPC3 antibodies and ADCs for HepG2 cells;

[0069] FIG10 shows the cell killing effects of Hu 52H5D3B8-8-L1-D1 and GC33-L1-D1 in Huh7 cell lines;

[0070] FIG11 shows the cell killing effects of Hu 52H5D3B8-8-L1-D1 and GC33-L1-D1 in HepG2 cell lines;

[0071] FIG12 shows the cell killing effects of Hu 52H5D3B8-8-L3-D3 and GC33-L3-D3 in Huh7 cell lines;

[0072] FIG13 shows the cell killing effects of Hu 52H5D3B8-7-L4-D4 and GC33-L4-D4 in Huh7 cell lines;

[0073] FIG14 shows the cell killing effects of Hu 52H5D3B8-8-ZLA and GC33-ZLA in Huh7 cell lines;

[0074] FIG15 shows the cell killing effects of Hu 52H5D3B8-8-ZLA and GC33-ZLA in HepG2 cell lines;

[0075] FIG16 shows the inhibitory activity of GPC3-ADC on the tumor growth of human liver cancer nude mouse PDX model LIV#219;

[0076] FIG17 shows the effect of GPC3-ADC on the body weight of the human liver cancer nude mouse PDX model LIV#219. DETAILED DESCRIPTION

[0077] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0078] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0079] In the present invention, unless otherwise stated, any numerical range should be understood to include any value or any sub-range within the range.

[0080] In the present invention, the term "antibody" refers to an immunoglobulin molecule that is usually composed of two pairs of identical polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. The light chains of antibodies can be divided into two types: κ and λ. Heavy chains can be divided into five types: μ, δ, γ, α or ε. Depending on the heavy chain, antibodies can be divided into five types: IgM, IgD, IgG, IgA and IgE. Within the light chain and heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of 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 3). Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the C1q component of the complement system. H and V L The V domain can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L) respectively form the antibody binding site. The assignment of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or the definitions of Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0081] In the present invention, algorithms used to determine percentage sequence homology and sequence similarity are, for example, 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 homology of the present invention using, for example, parameters described in the literature or using default parameters. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI).

[0082] In the present invention, the amino acid sequence having at least 70% sequence identity to the amino acid sequence includes polypeptide sequences that are substantially identical to the amino acid sequence, for example, those sequences that contain 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, when compared to the polypeptide sequence of the present invention using the methods described herein (e.g., BLAST analysis using standard parameters).

[0083] In the present invention, a mutant of an amino acid sequence refers to a sequence having greater than 70% homology to the amino acid sequence, such as greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, such as a sequence having 3, 2, or 1 amino acid substitutions, deletions, or additions. Preferably, the number of amino acids substituted, added, or deleted does not exceed 3. More preferably, the number of amino acids substituted, added, or deleted does not exceed 2. Most preferably, the number of amino acids substituted, added, or deleted does not exceed 1.

[0084] "Substitution" variants are variants in which at least one amino acid residue in the native sequence has been removed and a different amino acid has been inserted in the same position. The substitutions can be single, in which only one amino acid is substituted in the molecule, or multiple, in which two or more amino acids are substituted in the same molecule. Multiple substitutions can be located at consecutive sites. Likewise, one amino acid can be substituted by multiple residues, in which case such variants include both substitutions and insertions. "Insertion" (or "addition") variants are variants in which one or more amino acids are inserted adjacent to an amino acid at a specific position in a native sequence. Adjacent to an amino acid means linked to the α-carboxyl or α-amino functional group of the amino acid. "Deletion" variants are variants in which one or more amino acids in the native amino acid sequence have been removed. Typically, deletion variants have one or two amino acids deleted in a specific region of the molecule.

[0085] In certain embodiments, less than the theoretical maximum number of drug moieties are coupled to the antibody in the coupling reaction. Generally speaking, antibodies do not contain many free and reactive cysteine ​​thiol groups to which drug moieties can be attached; in fact, most cysteine ​​thiol groups in antibodies exist as disulfide bridges. In certain embodiments, antibodies can be reduced under partial or complete reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine ​​thiol groups.

[0086] In the present invention, the term "pharmaceutically acceptable salt" refers to (i) a salt formed by an acidic functional group present in the conjugate provided by the present invention and a suitable inorganic or organic cation (base), and includes, but is not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts, such as ammonium salts; organic base salts, such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylamine salts, and tris(hydroxymethyl)aminomethane salts. and (ii) salts formed between the basic functional groups present in the conjugates provided by the present invention and appropriate inorganic or organic anions (acids), including but not limited to hydrohalides, such as hydrofluorides, hydrochlorides, hydrobromides, and hydroiodides; inorganic acid salts, such as nitrates, perchlorates, sulfates, and phosphates; lower alkanesulfonates, such as methanesulfonates, trifluoromethanesulfonates, and ethanesulfonates; arylsulfonates, such as benzenesulfonates and p-toluenesulfonates; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, and maleates; and amino acid salts, such as glycine, trimethylglycine, arginine, ornithine, glutamate, and aspartate.

[0087] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic substance with a suitable acid to provide a pharmaceutically acceptable anion, or by reacting a sufficient amount of an acidic substance with a suitable base to provide a pharmaceutically acceptable cation.

[0088] In the present invention, solvates refer to forms of the antibody-drug conjugates of the present invention that form complexes in solid or liquid form by coordination with solvent molecules. Hydrates are a specific form of solvates that have coordinated water molecules. In the present invention, hydrates are preferred solvates.

[0089] 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 present disclosure. As described in REMINGTON'S PHARMACEUTICAL SCIENCES, Martin, EW, ed., Mack Publishing Company, 19th ed. (1995), the method for preparing the pharmaceutical composition includes incorporating suitable pharmaceutical excipients, carriers, diluents, etc., which are non-toxic to the cells or mammals exposed thereto at the dosages and concentrations employed.

[0090] For the purposes of this invention, pharmaceutical excipients refer to excipients and additives used in the production of pharmaceuticals and formulations. These substances, in addition to the active ingredient, have been rationally evaluated for safety and are included in pharmaceutical preparations. In addition to imparting shape, acting as a carrier, and enhancing stability, pharmaceutical excipients also perform important functions such as solubilization, dissolution enhancement, and sustained-release control. They are crucial components that may impact the quality, safety, and efficacy of pharmaceuticals. Based on their source, they can be categorized as natural, semi-synthetic, or fully synthetic. According to their functions and uses, they can be divided into: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc. According to their route of administration, they can be divided into oral, parenteral, mucosal, transdermal or topical administration, nasal or oral inhalation administration, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations for different routes of administration and have different functions and uses.

[0091] In the present invention, the pharmaceutical composition can be prepared into various suitable dosage forms according to the route of administration, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder sprays, and the like. The pharmaceutical composition or suitable dosage form can 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.

[0092] As used herein, the term "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms; and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or causing side effects due to the disease. As used herein, "treat" encompasses any treatment of a disease in a patient, including: (a) preventing the onset of a disease or symptom in a patient who is susceptible to the disease or symptom but has not yet been diagnosed with the disease; (b) suppressing the symptoms of a disease, i.e., arresting its development; or (c) relieving the symptoms of a disease, i.e., causing regression of the disease or symptom.

[0093] In the present invention, "subject" refers to a vertebrate. In certain embodiments, the vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the mammal refers to a human.

[0094] In the present invention, an "effective amount" refers to an amount that is effective at the necessary dosage and time to achieve the desired therapeutic or preventive effect. The "therapeutically effective amount" of the substance / molecule of the present invention may vary according to factors such as the disease state, age, sex and weight of the individual and the ability of the substance / molecule to elicit the desired response in the individual. A therapeutically effective amount also encompasses an amount in which the therapeutically beneficial effects of the substance / molecule outweigh any toxic or deleterious consequences. A "prophylactically effective amount" refers to an amount that is effective at the necessary dosage and time to achieve the desired preventive effect. Typically, but not necessarily, a prophylactic dose is used in subjects before the onset of disease or in the early stages of the disease, so the prophylactic effective amount will be lower than the therapeutically effective amount. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down to a certain extent, preferably stop) cancer cell infiltration into surrounding organs; inhibit (i.e., slow down to a certain extent, preferably stop) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with cancer to a certain extent.

[0095] In the present invention, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0096] The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0097] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., 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 antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0098] In addition, in humanization, it is also possible to mutate amino acid residues within the CDR1, CDR2 and / or CDR3 regions of VH and / or VL to improve one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, by PCR-mediated mutations, and their effects on antibody binding or other functional properties can be assessed using in vitro or in vivo tests as described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, the mutations within the CDRs are typically no more than one or two.

[0099] The present invention will be further explained below with reference to specific examples, but these examples are not intended to limit the scope of the present invention.

[0100] Example 1 Preparation, Sequence and Characterization of Humanized GPC3 Antibody

[0101] 1. Preparation of humanized GPC3 antibody

[0102] 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 measured using ELISA (human GPC3 protein) and FACS (CHO-K1 cells overexpressing human GPC3). After multiple rounds of immunization, mice with high titers were selected for the establishment of anti-human GPC3 hybridoma cell lines, and positive clones were identified by ELISA and FACS. Subclone screening and identification were then performed, and the monoclonal antibody 52H5D3B8 was obtained. Through antibody humanization design, the new humanized antibodies Hu 52H5D3B8-7 and Hu 52H5D3B8-8 were ultimately obtained.

[0103] 2. Sequence and Characterization of Humanized GPC3 Antibody

[0104] 1. Antibody sequence

[0105] Hu 52H5D3B8-7:

[0106] VH:

[0107] The underlined parts from left to right are CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO:

[0108] 2), CDR3 (SEQ ID NO: 3);

[0109] The ununderlined parts are FR1 (SEQ ID NO: 4), FR2 (SEQ ID NO: 5), FR3 (SEQ ID NO: 6), and FR4 (SEQ ID NO: 7) from left to right.

[0110] VL:

[0111] The underlined parts from left to right are CDR1 (SEQ ID NO: 9), CDR2 (SEQ ID NO:

[0112] 10), CDR3 (SEQ ID NO: 11);

[0113] The ununderlined parts are FR1 (SEQ ID NO: 12), FR2 (SEQ ID NO:

[0114] 13), FR3 (SEQ ID NO: 14), FR4 (SEQ ID NO: 15).

[0115] Hu 52H5D3B8-8:

[0116] VH:

[0117] The underlined parts from left to right are CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO:

[0118] 2), CDR3 (SEQ ID NO: 3);

[0119] The ununderlined parts are FR1 (SEQ ID NO: 4), FR2 (SEQ ID NO: 5), FR3 (SEQ ID NO: 6), and FR4 (SEQ ID NO: 7) from left to right.

[0120] VL:

[0121] The underlined parts from left to right are CDR1 (SEQ ID NO: 9), CDR2 (SEQ ID NO: 10), and CDR3 (SEQ ID NO: 11);

[0122] The ununderlined parts are FR1 (SEQ ID NO: 12), FR2 (SEQ ID NO:

[0123] 13), FR3 (SEQ ID NO: 17), FR4 (SEQ ID NO: 15).

[0124] The heavy chain constant regions of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 are:

[0125] The light chain constant regions of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 are:

[0126] Control Antibody:

[0127] (1) GC33 antibody (Chugai Pharmaceutical)

[0128] The preparation of GC33 antibody is described in US7919086B2.

[0129] The CDR information of the heavy and light chains of the GC33 antibody are as follows:

[0130] The FR information of the heavy and light chains of the GC33 antibody are as follows:

[0131] (2) Y035 antibody (CARSGEN Therapeutics)

[0132] The preparation of the Y035 antibody is described in CN106397593B. The amino acid sequences of the variable regions of the heavy and light chains of the humanized Y035 antibody are as follows:

[0133] Humanized Y035 heavy chain variable region (SEQ ID NO: 42):

[0134] The underlined parts from left to right are CDR1 (SEQ ID NO: 35), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 37);

[0135] The ununderlined parts are FR1 (SEQ ID NO: 38), FR2 (SEQ ID NO: 39), FR3 (SEQ ID NO: 40), and FR4 (SEQ ID NO: 41) from left to right.

[0136] Humanized Y035 light chain variable region (SEQ ID NO: 50):

[0137] The underlined parts from left to right are CDR1 (SEQ ID NO: 43), CDR2 (SEQ ID NO: 44), and CDR3 (SEQ ID NO: 45);

[0138] The ununderlined parts are FR1 (SEQ ID NO: 46), FR2 (SEQ ID NO: 47), FR3 (SEQ ID NO: 48), and FR4 (SEQ ID NO: 49) from left to right.

[0139] The heavy chain constant regions of GC33 and Y035 antibodies are:

[0140] The light chain constant regions of GC33 and Y035 antibodies are:

[0141] 2. Cell Binding of Humanized GPC3 Antibody

[0142] The 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 the activity with known anti-GPC3 antibodies GC33 and Y035. To evaluate the CDR regions of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 and the FR regions of Hu 52H5D3B8-7, the inventors prepared two recombinant antibodies. "Hu 52H5D3B8-7 / 8 CDRs with Y035 FRs" means that the antibodies are composed of the CDR regions of Hu 52H5D3B8-7 or Hu 52H5D3B8-8 and the FR regions of Y035. The "Y035 CDRs with Hu 52H5D3B8-7 FRs" antibody refers to an antibody formed by the recombination of the CDR regions of the Y035 antibody and the FR regions of the Hu 52H5D3B8-7 antibody.

[0143] First, GPC3-CHO-K1 cells, HepG2 cells, and Huh-7 cells were incubated with Fc blockers 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 / 8CDRs with Y035FRs, Y035CDRs with Hu 52H5D3B8-7FRs, or Y035mAb (starting concentration 100nM) were added to each well and incubated at 4°C for 60 minutes. The cells were washed with FACS buffer and fixed with 2% PFA at room temperature for 15 minutes. After washing with FACS buffer, Alexa Fluor 500 mAb was added. 647 AffiniPure goat anti-human IgG was added to each well and incubated at room temperature for 30 minutes. Samples were washed twice with FACS buffer. Fluorescence signals from cell samples were analyzed using a MACSQuant Analyzer 16 flow cytometer, expressed as MFI (GeoMean Fluorescence Intensity). The results, as shown in Figures 1-3, show that Hu 52H5D3B8-7 and Hu 52H5D3B8-8 have comparable binding affinities to GPC3-CHO-K1 cells, HepG2 cells, and Huh-7 cells, and are higher than those of other antibodies. In addition, both the antibody "Hu 52H5D3B8-7 / 8 CDRs with Y035FRs" and the antibody "Y035CDRs with Hu 52H5D3B8-7FR" showed higher cell affinity than Y035, which means that the CDR regions of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 antibodies or the FR regions of Hu 52H5D3B8-7 antibody are superior to Y035 antibody. EC of each test antibody on GPC3-CHO-K1 cells, HepG2 cells, and Huh-7 cells 50 The values ​​are shown in Table 1 below.

[0144] Table 1: Affinity of various GPC3 antibodies for GPC3-CHO-K1 cells, HepG2 cells and Huh-7 cells (EC 50 )

[0145] 3. Cellular internalization of humanized GPC3 antibody

[0146] The internalization of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 on HepG2 cells was evaluated and compared with the internalization of the known GPC3 antibody Y035 (CARSGEN Therapeutics). In addition, to evaluate the CDRs and FRs of Hu52H5D3B8-7 and Hu 52H5D3B8-8 antibodies, the inventors 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.

[0147] pHAb thiol dye is a pH sensor dye with very low fluorescence at pH>7. When it is internalized into endosomes or lysosomes (pH 6.3 or 4.7, respectively), the fluorescence increases significantly. Briefly, pHAb thiol dye-labeled α-hIgG secondary antibody (10 μg / mL) was incubated with antibody (20 μg / mL) for 30 minutes. After incubation, the mixed antibody was serially diluted 2-fold and the diluted mixture was added to each well with 2×10 4 HepG2 cells were pre-seeded in a 96-well assay plate. After 48 hours of culture, the fluorescence signal was captured on a multi-mode reader ( 2105). The experimental results are shown in Figure 4. Both Hu 52H5D3B8-7 and Hu 52H5D3B8-8 showed higher internalization effects than other antibodies. In addition, the antibody "Hu 52H5D3B8-7 / 8CDRs with Y035FRs" showed higher internalization effect than Y035 (higher Top value and comparable EC 50 ), which means that the CDRs of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 are better than those of Y035. 50 The and Top values ​​are shown in Table 2 below.

[0148] Table 2: Endocytosis of various GPC3 antibodies on HepG2 cells (EC 50 、Top)

[0149] Example 2 Preparation of GPC3-ADC

[0150] 1. Preparation of Antibody-Drug Conjugate Anti-GPC3-L1-D1

[0151] a: Take 10 mg of GPC3 antibody (Hu 52H5D3B8-7, Hu 52H5D3B8-8, or GC33) and adjust the pH to 7.5 with 0.7M Tris-0.5M EDTA solution. Weigh the antibody, determine the protein concentration, and calculate the total protein. Add 2.8 times the molar ratio of TCEP to the antibody and incubate on a 3D shaker at 22°C for 105 minutes, mixing continuously.

[0152] b: Add 1.6 times the molar number of free thiol groups of L1-D1 (i.e., MC-vc-PAB-MMAE) (manufactured by Shanghai Meiyake Biotechnology Co., Ltd. and commissioned by Hubei Huashitong Biopharmaceutical Technology Co., Ltd., dissolved in DMSO) to the reduced antibody. Mix thoroughly, then incubate on a 3D shaker at 22°C for 20 minutes with continuous mixing. Add 3 times the molar number of L1-D1 (i.e., MC-vc-PAB-MMAE) to the reaction mixture, mix thoroughly, and incubate on a 3D shaker at 22°C for 5 minutes with continuous mixing. Use a 15 mL 30KD ultrafiltration device to replace the conjugate storage solution (10 mM histidine, 3% sucrose, 0.03% Tween-80, pH 5.6) for a total of 5 replacements, and then filter with a 0.22 μm filter membrane to obtain the antibody drug conjugate product Anti-GPC3-L1-D1 (collectively referred to as Hu 52H5D3B8-7-L1-D1, Hu 52H5D3B8-8-L1-D1 and GC33-L1-D1) and store at 4 ° C.

[0153] 2. Preparation of Antibody Drug Conjugate Anti-GPC3-L3-D3

[0154] a: Take 2 mg of GPC3 antibody (Hu 52H5D3B8-7, Hu 52H5D3B8-8, or GC33) and adjust the pH to 7.5 with 0.7M Tris-0.5M EDTA solution. Weigh the antibody, determine the protein concentration, and calculate the total protein. Add 1.3 times the molar ratio of TCEP to the antibody and incubate on a 3D shaker at 22°C for 105 minutes, mixing continuously.

[0155] b: Add L3-D3 (i.e., MA-PEG4-VC-PAB-DMEA-Duocarmycin SA, purchased from Lianning (Suzhou) Biopharmaceutical Co., Ltd., dissolved in DMSO) to the reduced antibody at a concentration twice the molar number of free thiol groups. Mix thoroughly and incubate on a 3D shaker at 22°C for 20 minutes with continuous mixing. Add N-acetylcysteine ​​(i.e., three times the molar number of L3-D3 (i.e., MA-PEG4-VC-PAB-DMEA-Duocarmycin SA) to the reaction mixture, mix thoroughly, and incubate on a 3D shaker at 22°C for 5 minutes with continuous mixing. Use a 0.5 mL 30KD ultrafiltration device to replace the conjugate storage solution (10 mM histidine, 3% sucrose, 0.03% Tween-80, pH 5.6) for a total of 5 replacements, and then filter with a 0.22 μm filter membrane to obtain the antibody drug conjugate product Anti-GPC3-L3-D3 (collectively referred to as Hu 52H5D3B8-7-L3-D3, Hu 52H5D3B8-8-L3-D3 and GC33-L3-D3) and store at 4 ° C.

[0156] 3. Preparation of Antibody Drug Conjugate Anti-GPC3-L4-D4

[0157] a: Take 10 mg of GPC3 antibody (Hu 52H5D3B8-7, Hu 52H5D3B8-8, or GC33) and adjust the pH to 7.5 with 0.7M Tris-0.5M EDTA solution. Weigh the antibody, determine the protein concentration, and calculate the total protein. Add 2.7 times the molar ratio of TCEP to the antibody and incubate on a 3D shaker at 22°C for 105 minutes, mixing continuously.

[0158] b: Add 2.0 times the molar number of free thiol groups of L4-D4 (i.e., MC-MMAF) (purchased from Borai Biopharmaceuticals (Suzhou) Co., Ltd., dissolved in DMSO) to the reduced antibody. Mix thoroughly, then incubate on a 3D shaker at 22°C for 20 minutes with continuous mixing. Add 3 times the molar number of L4-D4 (i.e., MC-MMAF) of N-acetylcysteine ​​to the reaction mixture, mix thoroughly, and incubate on a 3D shaker at 22°C for 5 minutes with continuous mixing. Use a 15 mL 30KD ultrafiltration tube to replace the solution into the conjugate storage solution (10 mM histidine, 3% sucrose, 0.03% Tween-80, pH 5.6) for a total of 5 replacements, and then filter with a 0.22 μm filter membrane to obtain the antibody drug conjugate product Anti-GPC3-L4-D4 (collectively referred to as Hu 52H5D3B8-7-L4-D4, Hu 52H5D3B8-8-L4-D4 and GC33-L4-D4) and store at 4 ° C.

[0159] 4. Preparation of Antibody-Drug Conjugate Anti-GPC3-ZLA

[0160] a: Take 10 mg of GPC3 antibody (Hu 52H5D3B8-7, Hu 52H5D3B8-8, or GC33) and exchange the antibody solution in 40 mM PB + 2 mM EDTA (pH 7.0). Dilute the antibody to 5 mg / mL in 40 mM PB + 2 mM EDTA (pH 6.99). Weigh the sample, measure the protein concentration, and calculate the total protein. Add 2.25 times the molar ratio of TCEP to the antibody, mix the sample, and incubate at 22°C for 3 hours with continuous mixing at 200 rpm.

[0161] b: Cool the reduced antibody solution on ice. Add 7 times the molar ratio of the antibody to the cooled antibody (dissolved in DMA, with the total DMA content being 10% of the reaction system; ZLA is the compound described in Example 2.1 of CN107001415B, with the structural formula shown below). Mix rapidly and place in an incubator at 4°C for 2 hours with continuous mixing at 200 rpm. Add 6 times the molar ratio of the antibody to the reaction solution with N-acetylcysteine. Mix thoroughly and place in an incubator at 22°C for 15 minutes with continuous mixing at 200 rpm. Use a 15 mL 50KD ultrafiltration tube to transfer the solution into the conjugate stock solution (20 mM histidine / histidine-HCl, pH 5.41). Sterile filter the solution through a 0.22 μm filter and measure the protein concentration. The antibody drug conjugate product Anti-GPC3-ZLA (collectively referred to as Hu 52H5D3B8-7-ZLA, Hu 52H5D3B8-8-ZLA and GC33-ZLA) was obtained and stored at 4°C.

[0162] The drug loading rates (DARs) of the aforementioned GPC3-ADCs were determined using hydrophobic interaction chromatography (HIC-HPLC). Representative chromatograms are shown in Figures 5-8. Calculated based on the peak areas, the average DARs for Hu 52H5D3B8-7-L1-D1, Hu 52H5D3B8-7-L3-D3, Hu 52H5D3B8-7-L4-D4, and Hu 52H5D3B8-8-ZLA were 4.2, 2.3, 4.0, and 3.9, respectively.

[0163] Example 3 GPC3-ADC pharmacology and pharmacodynamics studies

[0164] 1. Cell Binding of GPC3-ADC

[0165] FACS experiments confirmed that the binding of ADC was not significantly affected by Cys conjugation, and the cell binding of Hu 52H5D3B8-8 ADC was compared with that of GC33 ADC.

[0166] After trypsin-EDTA digestion, cultured HepG2 cells were harvested. After centrifugation, the cells were resuspended in FACS buffer (PBS + 2% FBS), the cell density was adjusted, and the cells were plated into 96-well U-bottom cell culture plates, with 200,000 to 500,000 cells per well. Antibodies or ADCs diluted fourfold in FACS buffer were added and mixed thoroughly, with a final concentration of 10 μg / mL for each sample. The 96-well plates were incubated at 4°C for 60 minutes. The cells were washed thoroughly with FACS buffer to remove unbound antibody and ADC. A Goat anti-Human IgG (H+L) secondary antibody, Alexa Fluor 488 (Invitrogen), was added at a 1:1,000 dilution in FACS buffer and incubated at 4°C for 30 minutes in the dark. The cells were washed with FACS buffer to remove unbound secondary antibody. Fluorescence signal analysis of cell samples was performed using a CytoFLEX flow cytometer (Beckman Coulter). The experimental results, shown in Figure 9, show that the affinity of the Hu 52H5D3B8-8 and GC33 antibodies for HepG2 cells remained unchanged before and after conjugation with the cytotoxin. Furthermore, the Hu 52H5D3B8-8 antibody and the Hu 52H5D3B8-8 ADC exhibited stronger affinity for HepG2 cells than the GC33 antibody and GC33 ADC. The affinity of each test substance for HepG2 cells is shown in Table 3.

[0167] Table 3: Binding of GPC3 antibodies and ADCs to HepG2 cells (EC 50 )

[0168] 2. Internalization of Antibodies in GPC3-ADC

[0169] To confirm that the internalization of ADC is not inferior to that of mAb, the internalization of Hu 52H5D3B8-7 ADC, Hu 52H5D3B8-8 ADC, and GC33 ADC were compared.

[0170] A 96-well U-bottom cell culture plate was blocked with 5% skim milk powder in PBS for 60 minutes. After treatment with non-trypsinized digestion buffer (Sigma-Aldrich), cultured Huh7 hepatoma cells were harvested. After centrifugation, the cells were resuspended in pre-chilled DMEM medium, adjusted to a cell density, and plated into a blocked 96-well U-bottom cell culture plate, with 500,000 to 800,000 cells per well. Antibodies and ADCs diluted in DMEM medium supplemented with 5% FBS were added and mixed to a final concentration of 10 μg / mL. The 96-well plate was incubated on ice for 60 minutes. The cells were washed thoroughly with pre-chilled FACS buffer (PBS + 2% FBS) to remove unbound antibodies and ADCs. The cells were evenly divided between two blocked 96-well U-bottom plates, centrifuged, and resuspended in DMEM medium. One 96-well plate was placed on ice, while the other was placed in a cell culture incubator at 37°C. After 2 hours, transfer the culture plate from 37°C to ice and incubate for another 5–10 minutes. Add diluted Goat anti-Human IgG (H+L) Secondary Antibody, Alexa Fluor 488 (Invitrogen), and incubate on ice for 30 minutes in the dark. Wash the cells in the wells with pre-chilled FACS buffer to remove unbound secondary antibody. Fluorescence signals from the cell samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter) and expressed as GeoMean Fluorescence Intensity (MFI). Except for the 37°C incubation, the entire experiment was maintained on ice or at 4°C.

[0171] The percentage reduction of cell surface molecules was calculated as follows: Cell surface molecule reduction % = (MFI 冰上 -MFI 37℃ ) / MFI 冰上 *100%.

[0172] The experimental results are shown in Table 4. The internalization abilities of the Hu 52H5D3B8-8 and GC33 antibodies in Huh7 cells remained unchanged before and after conjugation with the cytotoxin. Furthermore, the internalization abilities of the Hu 52H5D3B8-8 antibody and the Hu 52H5D3B8-8 ADC were stronger than those of the GC33 antibody and the GC33 ADC. The results of the internalization of each test substance in Huh7 cells are shown in Table 4.

[0173] Table 4: Internalization of GPC3 antibodies and ADCs in Huh7 cells

[0174] Example 4 In vitro pharmacodynamic study

[0175] The cytotoxicity of Hu 52H5D3B8-7 or Hu 52H5D3B8-8 ADCs conjugated with various linker-payloads (ie, LD) was compared with that of GC33 ADCs.

[0176] After trypsin-EDTA digestion, the cultured liver cancer cells were collected. After centrifugation, the cells were resuspended in new culture medium and the cells were counted. The cells were inoculated into 96-well black cell culture plates (Costar) and cultured overnight in a cell culture incubator. The next day, the antibodies and ADCs were diluted 4-fold in a gradient with culture medium, and the dilutions were carefully transferred to the black culture plates so that the final concentration of each sample was 10 μg / mL. The cells were cultured in a cell culture incubator for 96 hours, and then 1 / 10 of the well volume of PrestoBlue reagent (Invitrogen) was added and incubated in a cell culture incubator for 1 hour. The fluorescence signal was read using a SpectraMax M5 plate reader, and the excitation and emission wavelengths of the instrument were set to 560 nm and 590 nm, respectively. The obtained fluorescence signal data was analyzed using SoftMax Pro 6.5 software.

[0177] 1. Experimental reagents and sources:

[0178] Test drug

[0179] cell lines

[0180] 2. Experimental results:

[0181] EC values ​​of cytotoxicity of each GPC3-ADC 50 The average values ​​are shown in Table 5. Figures 10-15 are representative graphs showing the killing effect of different GPC3-ADCs on Huh7 / HepG2 cells.

[0182] Table 5: EC values ​​of cytotoxicity of different GPC3-ADCs in the screened cell lines 50 value

[0183] Note: “-” means not tested

[0184] From the results in Table 5 and Figures 10-15, it can be seen that the different GPC3-ADCs of the present invention exhibited significant cell killing activity in GPC3- and highly expressed cell lines, and were stronger than GC33-ADC.

[0185] Example 5 In vivo pharmacodynamic study

[0186] The anti-tumor activity of GPC3-ADC was tested in the LIV#219 liver cancer PDX model, which has high GPC3 RNA expression levels, and FACS analysis confirmed GPC3 positive expression.

[0187] The process of establishing a human liver cancer nude mouse PDX model is as follows: 3 The tumor tissue was transplanted subcutaneously on the back of BALB / c nude mice. 3 The mice were randomly divided into 4 groups, including the vehicle group, the 3 mg / kg GC33-L1-D1 group, the 3 mg / kg Hu 52H5D3B8-7-L1-D1 group, and the 3 mg / kg Hu 52H5D3B8-8-L1-D1 group.

[0188] Data analysis: During the experiment, tumor volume was measured twice a week. The calculation formula of tumor volume (TV) is: TV = l × w 2 / 2. Where l and w represent the length and width of the tumor, respectively. The relative tumor volume (RTV) is calculated based on the measurement results. RTV = V f / V0. Where V0 is the tumor volume measured at the time of group administration (i.e. Day 0), V f = tumor volume measured on the last day. Relative tumor growth rate (T / C) (%) = (RTV of the drug group / RTV of the vehicle group) × 100%. Tumor growth inhibition rate (TGI) % = (mean tumor volume of the vehicle group - mean tumor volume of the drug group) / mean tumor volume of the vehicle group × 100%. A test article was considered to have a significant inhibitory effect on tumor growth when T / C (%) ≤ 40% and P < 0.05.

[0189] The experimental results are shown in Figures 16-17. Following administration of 3 mg / kg GC33-L1-D1, the relative tumor growth rate (T / C%) on Day 26 was 54.90%, and the tumor growth inhibition rate (TGI%) was 45.10%. For the Hu 52H5D3B8-7-L1-D1 (3 mg / kg) group, the T / C (%) was 33.58%, and the TGI% was 66.42%. For the Hu 52H5D3B8-8-L1-D1 (3 mg / kg) group, the T / C (%) was 39.93%, and the TGI% was 60.07%.

[0190] 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 inhibition activity, and were superior to GC33-L1-D1 (3 mg / kg). Both Hu52H5D3B8-7-L1-D1 and Hu 52H5D3B8-8-L1-D1 were well tolerated by tumor-bearing mice.

Claims

1. An antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, wherein said antibody-drug conjugate has the structure shown in Formula I, Ab-(LD) p Formula I in: Ab is an anti-GPC3 antibody, comprising a heavy chain and a light chain, wherein the heavy chain variable region CDR1, CDR2, and CDR3 comprise the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise the sequences shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; L is the connector; D is cytotoxin; p is any value between 1 and 8.

2. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: The linker is selected from: 6-Maleimidocaproyl (MC), maleimidopropionyl (MP), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), 4-(N-maleimidomethyl)-cyclohexane-1-carboxyl (MCC), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), MA-PEG4-VC-PAB-DMEA, Preferably, the linker is selected from: 6-Maleimidocaproyl (MC), 6-Maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), MA-PEG4-VC-PAB-DMEA, 3. The antibody drug conjugate according to any one of claims 1 to 2, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: The cytotoxin is selected from the group consisting of: SN-38, Gemcitabine, Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), Maytansinoids (e.g., Maytansine DM1, Maytansine DM4), calicheamicin, MGBA (e.g., duocarmycin), doxorubicin, ricin, diphtheria toxin, Duocarmycin SA, I131, interleukins, tumor necrosis factor, chemokines, and nanoparticles; Preferably, the cytotoxin is selected from: Monomethyl auristatin E (MMAE), Duocarmycin SA (Duocarmycin SA), Monomethyl auristatin F (MMAF), 4. The antibody drug conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: LD is selected from the following: MC-vc-PAB-MMAE、MA-PEG4-VC-PAB-DMEA-Duocarmycin SA、MC-MMAF、 5. 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 anti-GPC3 antibody has one or more of the following characteristics 1) to 2): 1) The heavy chain variable regions FR1, FR2, FR3, and FR4 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 anti-GPC3 antibody light chain variable region FR1, FR2, FR3, and FR4 regions 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 anti-GPC3 antibody light chain variable region FR1, FR2, FR3, and FR4 regions 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 antibody drug conjugate according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: 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 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.

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

8. 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, wherein: 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 comprises the sequence shown in SEQ ID NO: 19, or comprises a sequence having greater than 70% homology, 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 as 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 comprises a sequence having greater than 70% homology, 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 shown in SEQ ID NO:

20.

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

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

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 the salt thereof, or the pharmaceutical composition according to claim 10 in the preparation of a medicament for preventing and / or treating a disease associated with GPC3 positivity; Preferably, the disease associated with GPC3 positivity includes liver cancer, lung cancer, gastric cancer, head and neck cancer, esophageal cancer, Merkel cell carcinoma or liposarcoma.

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