Antibody drug conjugate targeting GPC3 and use thereof

Abstract

The present invention relates to a GPC3 antibody drug conjugate and a use thereof, and specifically provides an antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof. The antibody drug conjugate has a structure as shown in formula I, Ab being an anti-GPC3 antibody. The antibody drug conjugate has good tumor cell growth inhibition activity in vivo and in vitro, and has good application prospects.

Description

Antibody-drug conjugates targeting GPC3 and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the application with CN application number 202311141587.3 and application date September 5, 2023, and the application with CN application number 202411179009.3 and application date August 26, 2024, and claims all priorities. The contents of all CN applications are hereby introduced as a whole into this application. Technical Field

[0003] The present invention relates to the field of medicinal chemistry, and in particular to a GPC3 antibody-drug conjugate and applications thereof. Background Art

[0004] 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 efficacy remains suboptimal. Therefore, exploring new, more effective treatment strategies for 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.

[0005] 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.

[0006] 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 in hepatocellular carcinoma (HCC) (with a positive percentage exceeding 70%). 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 targeted liver cancer treatment.

[0007] Targeted therapy for GPC3 has gradually gained 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 GC33 (which has completed Phase 2 clinical trials), YP7, and HN3, as well as bispecific antibodies (such as ERY974 and CM350, which are undergoing clinical trials). Some of these antibodies can inhibit Wnt signaling in liver cancer cells to exert their effects. In addition, various studies have developed chimeric antigen receptor (CAR) T cell immunotherapy targeting GPC3 for the treatment of liver cancer. In studies with mouse models of xenografted or orthotopic liver cancer, CAR-T cells can eliminate GPC3-positive cancer cells by inducing perforin- and granzyme-mediated cell death and reducing WNT signaling in tumor cells.

[0008] Summary of the Invention

[0009] The inventors of the present application prepared an anti-GPC3 antibody drug conjugate (ADC) through extensive experiments and creative work, and confirmed that the ADC has good biological activity, thereby completing the present invention.

[0010] 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,

[0011] Ab-(LD) p

[0012] Formula I

[0013] in:

[0014] Ab is an anti-GPC3 antibody or an antigen-binding fragment thereof, wherein the anti-GPC3 antibody comprises a heavy chain variable region and a light chain variable region, 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, or mutants thereof, 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, or mutants thereof, respectively;

[0015] L is a linker selected from mc-AAN, mc-AAQ, mcc-AAN, mcc-AAQ, mcc-Ala-Ala-(3-cyano-alanine), mcc-Ala-Ala-(2-amino-4-cyanobutanoic acid), and mcc-Ala-Ala-Ala-Ala-Gln;

[0016] D is a cytotoxin, which is Exatecan (abbreviated as Exa);

[0017] p is any number between 1 and 10 (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, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7. - 6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10, or such as 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, 7.5-8, 8-8.5, 8.5-9, 9-9.5 or 9.5-10).

[0018] In formula I, the structural formulas of mc and mcc are as follows:

[0019] 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.

[0020] Those skilled in the art will understand that mcc and Ab are linked by covalent bonds to form the following structural fragments:

[0021] After mc and Ab are linked by covalent bonds, the following structural fragments are formed:

[0022] The * end is connected to Ab.

[0023] In some embodiments, L is selected from mc-AAN, mc-AAQ, mcc-AAN, mcc-AAQ, mcc-Ala-Ala-(3-cyano-L-alanine), mcc-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid), mcc-Ala-Ala-Ala-Ala-Gln.

[0024] In some embodiments, in Formula I, the structural formula of LD before being coupled to Ab is as follows:

[0025] When the nine LDs are covalently coupled to the Ab, the succinimide at the end of the LDs is coupled to the sulfhydryl groups in the antibody. For example, when mc-AAN-Exatecan is covalently coupled to the Ab, the structure of the resulting ADC is as follows:

[0026] Similarly, those skilled in the art will appreciate that when mcc-AAN-Exatecan is covalently coupled to Ab, the structural formula of the resulting ADC is as follows:

[0027] Similarly, those skilled in the art will appreciate that when mc-AAQ-Exatecan is covalently coupled to Ab, the structural formula of the resulting ADC is as follows:

[0028] Similarly, those skilled in the art will appreciate that when mcc-AAQ-Exatecan is covalently coupled to Ab, the structural formula of the resulting ADC is as follows:

[0029] In addition, similarly, those skilled in the art can understand that When covalently coupled to Ab, the structure of the resulting ADC is shown below:

[0030] Similarly, those skilled in the art will understand that When covalently coupled to Ab, the structure of the resulting ADC is shown below:

[0031] Similarly, those skilled in the art will appreciate that when mcc-Ala-Ala-(3-cyano-L-alanine)-Exatecan is covalently coupled to Ab, the structural formula of the resulting ADC is as follows:

[0032] Similarly, those skilled in the art will appreciate that when mcc-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)-Exatecan is covalently coupled to Ab, the structural formula of the resulting ADC is as follows:

[0033] Similarly, those skilled in the art will appreciate that when mcc-Ala-Ala-Ala-Ala-Gln-Exatecan is covalently coupled to Ab, the structural formula of the resulting ADC is as follows:

[0034] In the ADC formed above, the antibody Ab is linked to the carbon atom of the succinimide at the end of LD via -S-. This -S- is not a thiol group introduced into the Ab, but rather a thiol group contained in the antibody itself after the antibody Ab is reduced to open the disulfide bond.

[0035] 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 1-10", which means that p can be any integer selected from 1-10 (including endpoints 1 and 10) or any decimal selected from 1-10. 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. For example, when p is 7.5, it means that its DAR value can fluctuate between 7.0 and 8.0.

[0036] The drug-antibody ratio (DAR) can be determined by conventional means such as mass spectrometry, ELISA assays, hydrophobic interaction chromatography (HIC), and HPLC. The quantitative distribution of ADCs in terms of p can also be determined. In some cases, separation, purification, and validation of homogenous ADCs with a certain p value from ADCs with other drug loads can be achieved by means such as HIC, reverse-phase HPLC, or electrophoresis.

[0037] 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.

[0038] In some embodiments, 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, or mutants thereof, respectively.

[0039] 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.

[0040] In some embodiments, the light chain variable region FR1, FR2, FR3, and FR4 regions 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, or mutants thereof, respectively; or the light chain variable region FR1, FR2, FR3, and FR4 regions of the anti-GPC3 antibody comprise the sequences shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, and SEQ ID NO: 15, or mutants thereof, respectively.

[0041] In some embodiments, the sequences of the light chain variable region FR1, FR2, FR3, and FR4 regions of the anti-GPC3 antibody are as 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 light chain variable region FR1, FR2, FR3, and FR4 regions of the anti-GPC3 antibody are as shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, and SEQ ID NO: 15, respectively.

[0042] In some embodiments, the heavy chain variable region of the anti-GPC3 antibody comprises the sequence shown in SEQ ID NO: 8 or a mutant thereof.

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

[0044] In some embodiments, 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 mutant thereof.

[0045] 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.

[0046] 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 thereof. In some embodiments, the IgG is selected from IgG1, IgG2, IgG3, and IgG4.

[0047] In some embodiments, the light chain constant region of the anti-GPC3 antibody is selected from a human lambda constant region, a kappa constant region, or mutants of the aforementioned constant regions.

[0048] In some embodiments, 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% homology to the sequence shown in SEQ ID NO: 19, preferably greater than 75%, 80%, 85%, 90%, 95%, or 99%.

[0049] In some embodiments, the sequence of the heavy chain constant region of the anti-GPC3 antibody is shown in SEQ ID NO: 19.

[0050] In some embodiments, 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%.

[0051] In some embodiments, the sequence of the light chain constant region of the anti-GPC3 antibody is as shown in SEQ ID NO: 20.

[0052] In some embodiments, p is any number between 2-8.

[0053] In some embodiments, p is any number between 4 and 8 (eg, 4.0, 7.9, or 8.0).

[0054] 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.

[0055] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0056] In some embodiments, the pharmaceutical composition further comprises at least one of a chemotherapeutic drug, an immunotherapeutic drug, and an immunosuppressant for treating tumors.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] In the third aspect of the present invention, the present invention provides the 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 positivity.

[0061] 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 diseases associated with GPC3 positivity.

[0062] In a fifth aspect, the present invention provides a method for treating and / or preventing a disease associated with GPC3 positivity, comprising administering to a subject in need thereof an effective amount of the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt, or the aforementioned pharmaceutical composition.

[0063] In some embodiments, the above-mentioned disease associated with GPC3 positivity is selected from liver cancer, lung cancer, gastric cancer, head and neck cancer, esophageal cancer, Merkel cell carcinoma, liposarcoma, breast cancer, ovarian cancer, melanoma, squamous cell carcinoma, renal cell carcinoma, pancreatic cancer, prostate cancer, colorectal cancer, glioma, kidney cancer, bladder cancer, cervical cancer, gallbladder cancer, and glioblastoma.

[0064] In some embodiments, the disease associated with GPC3 positivity is liver cancer.

[0065] In the sixth aspect of the present invention, the present invention provides an in vitro non-therapeutic method for inhibiting tumor angiogenesis, delaying tumor progression, inhibiting tumor growth or inhibiting tumor cell proliferation, comprising: contacting tumor cells with the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the aforementioned pharmaceutical composition, wherein the tumor is a GPC3-expressing tumor.

[0066] In some embodiments, the GPC3-expressing tumor is selected from liver cancer, lung cancer, gastric cancer, head and neck cancer, esophageal cancer, Merkel cell carcinoma, liposarcoma, breast cancer, ovarian cancer, melanoma, squamous cell carcinoma, renal cell carcinoma, pancreatic cancer, prostate cancer, colorectal cancer, glioma, kidney cancer, bladder cancer, cervical cancer, gallbladder cancer, glioblastoma.

[0067] In some embodiments, the GPC3-expressing tumor is liver cancer. Beneficial effects

[0068] 1. The newly developed GPC3-ADC of the present invention exhibits a strong cell-killing effect in various tumor cells (such as liver cancer).

[0069] 2. The newly developed GPC3-ADC of the present invention showed significant pharmacological effects in inhibiting tumor cell growth in multiple tumor models (such as human liver cancer PDX tumor model). BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 shows the affinity of various GPC3 antibodies for GPC3-CHO-K1 cells;

[0071] Figure 2 shows the affinity of various GPC3 antibodies for HepG2 cells;

[0072] FIG3 shows the affinity of each GPC3 antibody for Huh-7 cells;

[0073] FIG4 shows the internalization of various GPC3 antibodies on HepG2 cells;

[0074] FIG5 shows a hydrophobic interaction chromatogram of Hu 52H5D3B8-8-mc-AAN-Exatecan (ADC-1);

[0075] FIG6 shows a hydrophobic interaction chromatogram of Hu 52H5D3B8-8-mcc-AAN-Exatecan (ADC-2);

[0076] FIG7 shows a hydrophobic interaction chromatogram of Hu 52H5D3B8-8-mc-AAQ-Exatecan (ADC-3);

[0077] FIG8 shows a hydrophobic interaction chromatogram of Hu 52H5D3B8-8-mcc-AAQ-Exatecan (ADC-4);

[0078] FIG9 shows a hydrophobic interaction chromatogram of Hu 52H5D3B8-7-mcc-AAQ-Exatecan (ADC-5);

[0079] FIG10 shows a hydrophobic interaction chromatogram of Hu 52H5D3B8-8-mcc-AAQ-Exatecan (ADC-6);

[0080] FIG11 shows a hydrophobic interaction chromatogram of Hu 52H5D3B8-7-mcc-AAQ-Exatecan (ADC-7);

[0081] FIG12 shows the hydrophobic interaction chromatogram of Hu 52H5D3B8-8-mcc-AA-(3-cyano-L-alanine)-Exatecan (ADC-8);

[0082] FIG13 shows a hydrophobic interaction chromatogram of Hu 52H5D3B8-8-mcc-AAAAQ-Exatecan (ADC-9);

[0083] FIG14 shows the affinity of GPC3 antibodies and ADCs for Huh7 cells;

[0084] FIG15 shows the results of endocytosis of GPC3 antibody and ADC in Huh7 cells;

[0085] Figures 16-18 are representative graphs showing the killing effect of different GPC3-ADCs on Huh7 cells;

[0086] Figure 19 shows the tumor growth inhibitory activity of GPC3-ADC on the human liver cancer nude mouse PDX model LIV#219;

[0087] FIG20 shows the effect of GPC3-ADC on the body weight of the human liver cancer nude mouse PDX model LIV#219;

[0088] FIG21 shows the tumor growth inhibitory activity of GPC3-ADC on the human liver cancer nude mouse PDX model LI6653;

[0089] FIG22 shows the effect of GPC3-ADC on the body weight of the human liver cancer nude mouse PDX model LI6653. DETAILED DESCRIPTION

[0090] 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.

[0091] 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.

[0092] In the present invention, unless otherwise stated, any numerical range should be understood to include any value or any sub-range within the range.

[0093] 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 (CH 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.

[0094] In the present invention, "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 immunoglobulins. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementary determining regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0095] 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.

[0096] In the present invention, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0097] In the present invention, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bond linking the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and the Fd fragment (consisting of VH and CH1 domains) of the heavy chain.

[0098] In the present invention, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0099] In the present invention, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, for example, Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments of the invention, scFv can form a di-scFv, which refers to two or more single scFvs in series to form an antibody. In certain embodiments of the invention, scFv can form a (scFv)2, which refers to two or more single scFvs in parallel to form an antibody.

[0100] In the present invention, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0101] As used herein, the term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as a full-length antibody. Single-domain antibodies are also known as nanobodies.

[0102] In the present invention, the term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0103] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0104] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0105] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992); Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be directly produced by recombinant host cells (see Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture medium. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0106] 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).

[0107] 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).

[0108] 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.

[0109] "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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] Example 1 Preparation, Sequence and Characterization of Humanized GPC3 Antibody

[0123] 1. Preparation of humanized GPC3 antibody

[0124] 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.

[0125] 2. Sequence and Characterization of Humanized GPC3 Antibody

[0126] 1. Antibody sequence

[0127] Hu 52H5D3B8-7:

[0128] VH:

[0129] The underlined parts from left to right are CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3);

[0130] 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.

[0131] VL:

[0132] The underlined parts from left to right are CDR1 (SEQ ID NO: 9), CDR2 (SEQ ID NO: 10), and CDR3 (SEQ ID NO: 11);

[0133] The ununderlined parts are FR1 (SEQ ID NO: 12), FR2 (SEQ ID NO: 13), FR3 (SEQ ID NO: 14), and FR4 (SEQ ID NO: 15) from left to right.

[0134] Hu 52H5D3B8-8:

[0135] VH:

[0136] The underlined parts from left to right are CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3);

[0137] 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.

[0138] VL:

[0139] The underlined parts from left to right are CDR1 (SEQ ID NO: 9), CDR2 (SEQ ID NO: 10), and CDR3 (SEQ ID NO: 11);

[0140] The ununderlined parts are FR1 (SEQ ID NO: 12), FR2 (SEQ ID NO: 13), FR3 (SEQ ID NO: 17), and FR4 (SEQ ID NO: 15) from left to right.

[0141] The heavy chain constant regions of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 are:

[0142] The light chain constant regions of Hu 52H5D3B8-7 and Hu 52H5D3B8-8 are:

[0143] Control Antibody:

[0144] (1) GC33 antibody (Chugai Pharmaceutical)

[0145] The preparation of GC33 antibody is described in US7919086B2.

[0146] The CDR information of the heavy and light chains of the GC33 antibody are as follows:

[0147] The FR information of the heavy and light chains of the GC33 antibody are as follows:

[0148] (2) Y035 antibody (CARSGEN Therapeutics)

[0149] 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:

[0150] Humanized Y035 heavy chain variable region (SEQ ID NO: 42):

[0151] The underlined parts from left to right are CDR1 (SEQ ID NO: 35), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 37);

[0152] 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.

[0153] Humanized Y035 light chain variable region (SEQ ID NO: 50):

[0154] The underlined parts from left to right are CDR1 (SEQ ID NO: 43), CDR2 (SEQ ID NO: 44), and CDR3 (SEQ ID NO: 45);

[0155] 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.

[0156] The heavy chain constant regions of GC33 and Y035 antibodies are:

[0157] The light chain constant regions of GC33 and Y035 antibodies are:

[0158] 2. Cell Binding of Humanized GPC3 Antibody

[0159] 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.

[0160] First, GPC3-CHO-K1 cells, HepG2 cells, and Huh-7 cells were incubated with Fc blocker 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 Y035 FRs, Y035 CDRs with Hu52H5D3B8-7 FRs, or Y035 mAb (starting concentration 100 nM) 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 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 Y035 FRs" and the antibody "Y035 CDRs with Hu 52H5D3B8-7 FRs" 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 Hu52H5D3B8-7 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.

[0161] Table 1: Affinity of various GPC3 antibodies for GPC3-CHO-K1 cells, HepG2 cells and Huh-7 cells (EC 50 )

[0162] 3. Cellular internalization of humanized GPC3 antibody

[0163] 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.

[0164] 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 / 8 CDRs with Y035 FRs" 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 top values ​​are shown in Table 2 below.

[0165] Table 2: Endocytosis of various GPC3 antibodies on HepG2 cells (EC 50 、Top)

[0166] Example 2 Preparation of Linker-Payload

[0167] 1. Preparation of mc-AAN-Exatecan

[0168] 1. Synthesis of Intermediate 1

[0169] Fmoc-Ala-OH (N-fluorenylmethoxycarbonyl-L-alanine, CAS No.: 35661-39-3) was activated with HOSu (N-hydroxysuccinimide, CAS No.: 6066-82-6) and then reacted with L-Ala (L-alanine, CAS No.: 56-41-7) to obtain intermediate 1; the specific steps were: Fmoc-Ala-OH (3 g, 1.0 eq) and HOSu (1.45 g, 1.3 eq) were added to the reaction flask, 21 mL of THF was added, the temperature was controlled at room temperature, DCC (2.59 g, 1.3 eq) was slowly added under stirring, and the reaction was carried out at room temperature and monitored by HPLC. After the reaction was completed, the reaction solution was filtered and the filter cake was rinsed with THF (6 mL). Purified water (15 mL) was added to the filtrate, followed by L-Ala (1.12 g, 1.3 eq) and solid sodium bicarbonate (0.81 g, 1.0 eq). The reaction was stirred at room temperature and monitored by HPLC. After the reaction was completed, citric acid (2.02 g, 1.0 eq) was added and stirred. The reaction solution was then extracted with ethyl acetate, the organic phase was concentrated, and DMF (12 mL) was added to dissolve the product. After filtration, the product was passed through prep-HPLC (preparative high performance liquid chromatography). The preparative solution was concentrated until no obvious droplets flowed out, and then extracted with ethyl acetate. The ethyl acetate phase was concentrated to dryness to obtain intermediate 1. The reaction formula is as follows:

[0170] 2. Synthesis of Intermediate 2

[0171] Intermediate 1 (N-[Fluorenylmethoxycarbonyl]-L-alanyl-L-alanine, CAS No.: 87512-31-0) was activated with HOSu (N-hydroxysuccinimide, CAS No.: 6066-82-6) and then reacted with L-Asn (L-asparagine, CAS No.: 70-47-3) to obtain intermediate 2. The specific steps are as follows: intermediate 1 (0.87 g, 1.0 eq), HOSu (0.34 g, 1.3 eq), and THF (9 mL) were added to a reaction flask, the temperature was controlled at room temperature, DCC (0.61 g, 1.3 eq) was slowly added under stirring, the reaction was carried out at room temperature, and HPLC was used for monitoring. After the reaction was completed, the reaction solution was filtered and the filter cake was rinsed with THF (2 mL). Purified water (10 mL) was added to the filtrate, followed by L-Asn (0.34 g, 1.1 eq) and solid sodium bicarbonate (0.19 g, 1.0 eq). The reaction was stirred at room temperature and monitored by HPLC. After the reaction was completed, citric acid monohydrate (0.48 g, 1.0 eq) was added and stirred. The reaction solution was concentrated to remove most of the solvent, and a purified residue was prepared. The prepared solution was concentrated until no obvious droplets flowed out, and then extracted with ethyl acetate. The organic phase was concentrated to dryness to obtain Intermediate 2. The reaction formula is as follows:

[0172] 3. Synthesis of Intermediate 3

[0173] Intermediate 2 was added with DEA ​​(diethylamine, CAS No.: 109-89-7) to remove Fmoc to obtain Intermediate 3. The specific steps are as follows: Intermediate 2 (100 mg) and DMF (1.5 mL) were added to a reaction flask, the temperature was controlled at room temperature, DEA (300 μL) was added dropwise, and the reaction was carried out at room temperature. HPLC monitoring was performed until no residue of Intermediate 2 was left. DMF was removed by concentration, DCM (4 mL) and purified water (4 mL) were added, and the mixture was stirred and separated. The aqueous phase was concentrated to dryness to obtain Intermediate 3. The reaction formula is as follows:

[0174] 4. Synthesis of Intermediate 4

[0175] Intermediate 3 reacts with 6-(maleimido)hexanoic acid succinimidyl ester (CAS No.: 55750-63-5) to obtain intermediate 4; the specific steps are: adding intermediate 3 (92 mg, 1.0 eq), 6-(maleimido)hexanoic acid succinimidyl ester (135 mg, 1.3 eq), DMF (1.5 mL), and DIPEA (0.059 mL, 1.0 eq) to a reaction flask, and monitoring by HPLC. After the reaction is completed, the reaction is prepared and purified, and the prepared solution is concentrated to obtain intermediate 4. The reaction formula is as follows:

[0176] 5. Synthesis of Products

[0177] Intermediate 4 was reacted with isotecan mesylate (CAS No. 169869-90-3) to obtain the product through a condensation reaction. Specifically, intermediate 4 (26 mg, 1.0 eq) and DMF (1.5 mL) were added to a reaction flask at room temperature. Then, isotecan mesylate (29.6 mg, 1.0 eq), EEDQ (20.7 mg, 1.5 eq), HATU (31.8 mg, 1.5 eq), DMAP (0.7 mg, 0.1 eq), and DIPEA (29.2 μL, 3.0 eq) were added in sequence. The reaction was allowed to proceed at room temperature. After completion of the reaction, the product was purified and the prepared solution was concentrated to obtain the product. The reaction formula is as follows:

[0178] 1H NMR (400MHz, DMSO-d6) δ8.22-8.29(d,J=2.0Hz,1H),7.94-8.05(d,J=2.0Hz,3H), 7.83-7.88(d,J=2.0Hz,1H), 7.75-7.82(d,J=2.0Hz ,1H), 7.34-7.40(m,1H), 7.28-7.33(m,1H), 6.96-7.02(d,J=8.0Hz,2H), 6.87-6.94(m,1H), 5.42-5.55(m,1H), 5.41-5.46(m,2H), 5. 21-5.26(m,1H), 4.41-4.49(m,1H), 4.00-4.12(m,2H), 3.32-3.40(m,2H), 3.12-3.17(m,2H), 2.86-2.96(m,2H), 2.71-2.76(m,2H), 2 .36-2.43(m,2H), 2.14-2.24(m,1H), 1.97-2.07(m,3H), 1.81-1.93(m,2H), 1.36-1.52(m,4H), 1.05-1.25(m,8H), 0.83-0.93(m,3H).

[0179] 2. Preparation of mc-AAQ-Exatecan

[0180] 1. Synthesis of Intermediate 1: Fmoc-Gln-Exatecan

[0181] Fmoc-Gln-OH (N-fluorenylmethoxycarbonyl-L-glutamine) (76.3 mg 1.1 eq.) and Exatecan (100 mg 1.0 eq.) were added to a reaction flask, followed by DMF (1 ml), DIEA (29 mg 1.5 eq.), and TBTU (72.5 mg 1.1 eq.). The reaction was allowed to proceed at room temperature. HPLC monitoring indicated no residual starting material. After completion of the reaction, silica gel was added for concentration and sample purification by column chromatography. The product was collected and concentrated to dryness to yield Intermediate 1.

[0182] The reaction formula is as follows:

[0183] 2. Synthesis of Intermediate 2 Gln-Exatecan

[0184] Intermediate 1, Fmoc-Gln-Exatecan (130 mg, 1.0 eq.), was added to a reaction flask, and DCM (2 ml) was added and stirred at room temperature. The mixture did not dissolve. DEA (0.5 ml) was added and allowed to react at room temperature. HPLC monitoring indicated no residual starting material. The reaction was stopped, and MTBE (10 ml) was slowly added. A large amount of solid precipitated. The mixture was stirred for 30 min. Filtering afforded an off-white solid, which was then dried to yield Intermediate 2.

[0185] The reaction formula is as follows:

[0186] 3. Synthesis of Intermediate 3: Fmoc-Ala-Ala-Gln-Exatecan

[0187] Intermediate II, Gln-Exatecan (100 mg, 1.0 eq.), was added to a reaction flask along with DMF (1 mL), Fmoc-Ala-Ala-OH (67.8 mg, 1.0 eq.), TBTU (68.4 mg, 1.2 eq.), and DIEA (34.4 mg, 1.5 eq.) and allowed to react at room temperature. HPLC monitoring indicated no residual intermediate II, and the reaction was stopped. Silica gel was added to mix the sample, and column purification was performed to collect the product to yield intermediate III.

[0188] The reaction formula is as follows:

[0189] 4. Synthesis of Intermediate 4: Ala-Ala-Gln-Exatecan

[0190] Intermediate 3, Fmoc-Ala-Ala-Gln-Exatecan (140 mg, 1.0 eq.), was added to a reaction flask, followed by DCM (2 ml) and DEA (0.5 ml). The reaction was allowed to react at room temperature. HPLC monitoring indicated no residual starting material. The reaction was stopped, and MTBE (10 ml) was slowly added to precipitate a large amount of solid. The mixture was stirred for 30 min. Filtration afforded an off-white solid, which was then dried to yield Intermediate 4.

[0191] The reaction formula is as follows:

[0192] 5. Product Synthesis mc-Ala-Ala-Gln-Exatecan

[0193] Intermediate 4, Ala-Ala-Gln-Exatecan (50 mg, 1.0 eq.), was added to a reaction flask with DMF (1 mL). 6-(Maleimido)hexanoic acid succinimidyl ester (21.8 mg, 1.1 eq.) and DIEA (12.1 mg, 1.5 eq.) were then added and allowed to react at room temperature. HPLC monitoring was used to stop the reaction until no intermediate 4 remained. The product was collected and concentrated to dryness to obtain the product.

[0194] The reaction formula is as follows:

[0195] 1 H NMR (400MHz, DMSO-d6) δ8.28-8.36(d,J=2.0Hz,1H),7.90-7.96(d,J=2.0Hz,2H), 7.75-7.87(d,J=2.0Hz,2H), 7.27-7.33(d,J=2.0Hz,1H) , 7.20-7.25(m,1H), 6.97-7.03(d,J=8.0Hz,2H), 6.69-6.75(m,1H), 6.49-6.53(m,1H), 5.46-5.55(m,1H), 5.40-5.45(m,2H), 5.24-5.34( m,1H), 5.04-5.14(m,1H), 4.31-4.38(m,1H), 4.02-4.23(m,2H), 3.86-3.96(m,1H), 3.40-3.49(m,1H), 3.33-3.40(m,2H), 3.12-3.20(m,1 H), 2.36-2.42(m,2H), 2.15-2.25(m,1H), 2.00-2.12(m,4H), 1.71-1.93(m,3H), 1.36-1.52(m,4H), 1.01-1.20(m,10H), 0.83-0.92(m,3H).

[0196] 3. Preparation of mcc-AAN-Exatecan

[0197] 1. Synthesis of Intermediate 1

[0198] Fmoc-Ala-Ala-OH (N-[fluorenylmethoxycarbonyl]-L-alanyl-L-alanine) is activated with HOSu (N-hydroxysuccinimide) and then reacted with L-Asn (L-asparagine) to obtain intermediate 1.

[0199] The specific steps are as follows: Fmoc-Ala-Ala-OH (1.0 eq), HOSu (1.3 eq), and THF (10 v / w) are added to a reaction flask at room temperature, the temperature is controlled at room temperature, DCC (1.3 eq) is slowly added under stirring, the reaction is carried out at room temperature, and the reaction is detected by HPLC until the residual Fmoc-Ala-Ala-OH is small (area normalization method), the reaction solution is filtered, and the filter cake is rinsed with THF (2.5 v / w). Purified water (11.5 v / w) was added to the filtrate, followed by L-Asn (1.1 eq) and solid sodium bicarbonate (1.0 eq). The mixture was stirred at room temperature and detected by HPLC until a small amount of Fmoc-Ala-Ala-OSu residue remained. Then, citric acid monohydrate (1.0 eq) was added and stirred. The reaction solution was concentrated to remove most of the solvent, and DMF (4 v / w) was added to dissolve the product. After filtration, the product was prepared by reverse phase preparation using a preparative liquid phase. The preparative liquid was concentrated until no obvious droplets flowed out, and then extracted with ethyl acetate. The ethyl acetate phase was concentrated to dryness to obtain intermediate 1. The reaction formula is as follows:

[0200] 2. Synthesis of Intermediate 2

[0201] Intermediate 1 was added with DEA ​​(diethylamine) to remove Fmoc to obtain intermediate 2.

[0202] The specific steps are: adding intermediate 1 (1.0 eq) to a reaction flask at room temperature, adding DMF (15 v / w), controlling the temperature at room temperature, adding DEA (3 v / w) dropwise, reacting at room temperature, detecting by HPLC until no residue of intermediate 1 remains, concentrating to remove DMF, adding DCM (40 v / w) and purified water (40 v / w), stirring and then separating the liquids, and concentrating the aqueous phase to dryness to obtain intermediate 2. The reaction formula is as follows:

[0203] 3. Synthesis of Intermediate 3

[0204] The intermediate 2 is reacted with 4-(N-maleimidomethyl)cyclohexanecarboxylic acid-N-succinimide ester to obtain the intermediate 3.

[0205] The specific steps are as follows: add intermediate 2 (1.0 eq) and 4-(N-maleimidomethyl)cyclohexanecarboxylic acid-N-succinimide ester (1.1 eq) to a reaction flask at room temperature, add DMF (22 v / w), water (9.5 v / w), and DIPEA (1.0 eq), and then react at room temperature until intermediate 2 remains less (QDA detection). After the reaction is completed, concentrate to remove most of the solvent, add water (50 v / w), EA (50 v / w), add 1.0 eq DIEA, stir to dissolve, and then stand for separation. The aqueous phase is extracted with EA (50 v / w*2), and the aqueous phase is concentrated to dryness to obtain intermediate 3. The reaction formula is as follows:

[0206] 4. Synthesis of Products

[0207] The intermediate 3 and isotectin mesylate (CAS No.: 169869-90-3) are subjected to a condensation reaction with HATU to obtain the product. The specific steps are as follows: Under room temperature, the intermediate 3 (1.2 eq) is added to the reaction flask, DMF (15 v / w) is added, and isotectin mesylate (1.0 eq), TBTU (1.4 eq), and DIPEA (4.5 eq) are added in sequence. The reaction is then carried out at room temperature and detected by HPLC. After completion of the reaction, the product is purified by prep-HPLC (preparative high performance liquid chromatography), the prepared solution is concentrated, and the prepared solution is extracted with DCM to obtain the product. The reaction formula is as follows:

[0208] 1 H 7 .28-7.32(m,1H), 6.98-7.05(d,J=8.0Hz,2H), 6.85-6.95(m,1H), 6.45-6.57(m,1H), 5.45-5.55(m,1H), 5.40-5.45(m,1H), 5.20-5.27(m ,1H), 4.40-4.50(m,1H), 3.95-4.05(m,1H), 3.20-3.27(m,2H), 3.10-3.18(m,2H), 2.86-2.97(m,2H), 2.36-2.43(m,3H), 2.15-2.25(m,1 H), 1.95-2.10(m,3H), 1.80-1.93(m,3H), 1.73-1.78(m,1H), 1.55-1.70(m,3H), 1.45-1.55(m,3H), 1.0-1.3(m,10H), 0.80-0.95(m,5H).

[0209] 4. Preparation of mcc-AAQ-Exatecan

[0210] 1. Synthesis of Intermediate 1: Fmoc-Gln-Exatecan

[0211] Fmoc-Gln-OH (N-fluorenylmethoxycarbonyl-L-glutamine) (76.3 mg 1.1 eq.) and Exatecan (100 mg 1.0 eq.) were added to a reaction flask, followed by DMF (1 ml) and DIEA (29 mg 1.5 eq.). TBTU (72.5 mg 1.1 eq.) was added and the reaction was allowed to proceed at room temperature. HPLC monitoring confirmed no residual starting material. After completion of the reaction, the product was purified by medium-pressure column chromatography (DCM / MeOH). The product was collected and concentrated to dryness to obtain Intermediate 1.

[0212] The reaction formula is as follows:

[0213] 2. Synthesis of Intermediate 2 Gln-Exatecan

[0214] Intermediate 1, Fmoc-Gln-Exatecan (130 mg, 1.0 eq.), was added to a reaction flask, followed by DCM (2 ml) and stirred at room temperature. DEA (0.5 ml) was added and the mixture was allowed to react at room temperature. HPLC monitoring confirmed no residual starting material. The reaction was stopped and MTBE (10 ml) was slowly added. A large amount of solid precipitated and the mixture was stirred for 30 min. Filtering and washing with MTBE afforded an off-white solid, which was then dried to yield Intermediate 2.

[0215] The reaction formula is as follows:

[0216] 3. Synthesis of Intermediate 3: Fmoc-Ala-Ala-Gln-Exatecan

[0217] Intermediate II, Gln-Exatecan (100 mg, 1.0 eq.), was added to a reaction flask along with DMF (1 mL), Fmoc-Ala-Ala-OH (67.8 mg, 1.0 eq.), TBTU (68.4 mg, 1.2 eq.), and DIEA (34.4 mg, 1.5 eq.). The reaction was allowed to react at room temperature and monitored by HPLC until completion. The product was purified by column chromatography (DCM / MeOH) at medium pressure to obtain Intermediate III.

[0218] The reaction formula is as follows:

[0219] 4. Synthesis of Intermediate 4: Ala-Ala-Gln-Exatecan

[0220] Intermediate 3, Fmoc-Ala-Ala-Gln-Exatecan (140 mg, 1.0 eq.), was added to a reaction flask, followed by DCM (2 ml) and stirred at room temperature. DEA (0.5 ml) was added and the reaction was allowed to react at room temperature. HPLC monitoring was performed until the reaction was complete. MTBE (10 ml) was slowly added to precipitate a large amount of solid. The mixture was stirred for 30 min. Filtering and washing with MTBE afforded an off-white solid, which was then dried to yield Intermediate 4.

[0221] The reaction formula is as follows:

[0222] 5. Product Synthesis MCC-Ala-Ala-Gln-Exatecan

[0223] Intermediate 4, Ala-Ala-Gln-Exatecan (50 mg, 1.0 eq.), was added to a reaction flask with DMF (1 mL), followed by MCC (18 mg, 1.1 eq.) and DIEA (12.1 mg, 1.5 eq.). The reaction was allowed to proceed at room temperature and monitored by HPLC until no residue of intermediate 4 remained. The product was then collected and concentrated to dryness to yield the product.

[0224] The reaction formula is as follows:

[0225] 1 H NMR (400MHz, DMSO-d6) δ8.17-8.26 (d, J=2.0Hz, 1H), 7.83-7.91 (d, J=2.0Hz, 2H), 7.65-7.74 (d, J=2.0Hz, 2H), 7.21-7.28 (d,J=8.0Hz,2H), 6.98-7.04(m,2H), 5.43-5.50(m,1H), 5.36-5.43(m,2H), 5.15-5.24(m,1H), 5.01-5.11(m,1H), 4.14-4 .24(m,1H), 3.97-4.08(m,1H), 3.47-3.57(m,1H), 3.20-3.27(m,1H), 3.05-3.16(m,2H), 2.30-2.36(m,2H), 2.00-2.15(m ,4H), 1.77-1.92(m,3H), 1.65-2.00(m,5H), 1.45-1.64(m,4H), 1.09-1.24(m,5H), 0.95-1.07(m,4H), 0.79-0.94(m,5H).

[0226] 5. Preparation of mcc-Ala-Ala-(3-cyano-L-alanine)-Exatecan

[0227] 1. Synthesis of Intermediate 1 (3-cyano-L-alanine)-Exatecan

[0228] Fmoc-Asn-OH (40 mg, 1.2 eq) was added to a reaction flask, followed by DMF (1.5 ml), TBTU (45 mg, 1.5 eq), and DIEA (60 mg, 2.5 eq) and stirred until completely dissolved. After stirring for 30 min, isotecan mesylate (50 mg, 1.0 eq) was added and stirred for 4 hrs. HPLC analysis showed no reaction. TBTU (45 mg, 1.5 eq) and DIEA (60 mg, 2.5 eq) were added and the reaction continued at room temperature. HPLC monitoring showed no residual exatecan starting material, and Fmoc-(3-cyano-L-alanine)-exatecan accounted for approximately 70%. The reaction was stopped and purified under medium pressure. The product was collected.

[0229] The above product was added to a reaction flask, and DCM (4 ml) was added and stirred. DEA (1 ml) was added and stirred at room temperature until it dissolved completely. HPLC monitoring showed no residual starting material. MTBE (10 ml) was added and stirred at room temperature. A large amount of solid precipitated. Filtering gave an off-white solid, Intermediate 1.

[0230] The reaction formula is as follows:

[0231] 2. Synthesis of Intermediate 2 mcc-Ala-Ala-OH

[0232] Add SMCC (460 mg, 1.1 eq) to a reaction flask, followed by NH2-Ala-Ala-OH (200 mg, 1.0 eq) and DMF (4 ml, 20 V). Stir, then add DIPEA (161.4 mg, 1.0 eq). Heat to 60°C for reaction. As the reaction proceeds, the reaction solution gradually becomes clear. Allow to react for at least 8 hours. Samples are taken for LC-MS analysis. Upon completion of the reaction, cool to room temperature. Concentrate under reduced pressure at 35°C to remove DMF, then add purified water (4 ml, 20 V) and DIPEA (161.4 mg, 1.0 eq). Add EA (4 ml, 20 V) and stir to wash. Discard the organic phase. Extract twice with EA (2 ml, 10 V). Discard the organic phase. Retain the aqueous phase and adjust the acidity to pH 1-2 with 2 M hydrochloric acid, resulting in the precipitation of a large amount of white solid. Stir for one hour. Filter to obtain a white solid, Intermediate 2.

[0233] The reaction formula is as follows:

[0234] 3. Product Synthesis mcc-Ala-Ala-(3-cyano-L-alanine)-Exatecan

[0235] Add Intermediate 1 (3-cyano-L-alanine)-Exatecan (35 mg, 1.0 eq) to a reaction flask and DMF (1 mL). Then, add Intermediate 2 (mcc-Ala-Ala-OH) (28 mg, 1.1 eq), TBTU (25.3 mg, 1.2 eq), and DIPEA (12.8 mg, 1.5 eq). After reacting at room temperature for 2 hours, the reaction was stopped when no Intermediate 1 remained, monitored by HPLC. Purify the product by Prep-HPLC.

[0236] The reaction formula is as follows:

[0237] 1 H NMR (400MHz, DMSO-d6) δ8.45-8.55 (d, J=2.0Hz, 1H), 8.23-8.29 (d, J=2Hz, 2H), 7.95-8.03 (d, J=2.0Hz, 1H), 7.84-7.92 (d, J=8.0Hz, 1H), 7.7 6-7.84(m,1H), 7.27-7.33(d,J=8.0Hz,1H), 6.98-7.02(m,1H), 5.47-5 .56(m,1H), 5.40-5.45(m,1H), 5.26-5.36(m,1H), 5.1-5.2(m,1H), 4.4 4-4.55(m,1H), 4.07-4.19(m,1H), 3.88-4.0(m,2H), 3.19-3.26(m,2H) ), 3.13-3.18(m,1H), 2.90-3.0(m,2H), 2.76-2.88(m,2H), 2.36-2.43( m,3H), 2.00-2.12(m,2H), 1.80-1.93(m,3H), 1.80-1.92(m,2H), 1.55- 1.70(m,3H), 1.13-1.26(m,5H), 1.05-1.12(m,3H), 0.80-0.95(m,5H).

[0238] 6. Synthesis of mcc-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)-Exatecan

[0239] 1. Synthesis of Intermediate 1 ((S)-2-amino-4-cyanobutanoic acid)-Exatecan

[0240] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-cyanobutanoic acid (79.8 mg, 1.1 eq) and isotecan mesylate (110 mg, 1.0 eq) were added to a reaction flask. DMF (1.5 ml), TBTU (79.73 mg, 1.5 eq), and DIEA (80 mg, 2.5 eq) were also added. The reaction was allowed to proceed at room temperature for 3 hours, monitored by HPLC. After the reaction was completed, 9 ml of ACN was added and the mixture was stirred at room temperature. 1.0 ml of DEA was then added and the reaction was continued at room temperature for 1 hour. Sampling was performed to confirm the absence of any starting material. 10 ml of MTBE was added. The mixture was stirred, but no solid precipitation was observed. The mixture was stirred for 1 hour, with no significant change.

[0241] Concentrate under reduced pressure at 30°C to remove the solvent. A large amount of solid precipitated during concentration, and only a small amount of solvent, approximately 3 mL, was removed at this point. Stop concentration and continue stirring at room temperature for 1 hour. Filter and wash the solid with MTBE. Dry to obtain an off-white solid, Intermediate 1.

[0242] The reaction formula is as follows:

[0243] 2. Synthesis of Intermediate 2 mcc-Ala-Ala-OH

[0244] SMCC (460 mg, 1.1 eq) was added to a reaction flask, followed by NH2-Ala-Ala-OH (200 mg, 1.0 eq) and DMF (4 ml, 20 V), stirred, and then DIPEA (161.4 mg, 1.0 eq) was added. Heat to 60°C and react for 8 hours. Samples were taken for LC-MS analysis. After the reaction was complete, the mixture was cooled to room temperature. DMF was removed by vacuum concentration at 35°C, and purified water (4 ml, 20 V) and DIPEA (161.4 mg, 1.0 eq) were added. EA (4 ml, 20 V) was added and washed with stirring. The organic phase was discarded. EA (2 ml, 10 V) was added and extracted twice. The organic phase was discarded. The aqueous phase was retained and adjusted to a pH of 1-2 with 2 M hydrochloric acid, resulting in the precipitation of a large amount of white solid. Stir for one hour. Filter to obtain a white solid, Intermediate 2.

[0245] The reaction formula is as follows:

[0246] 3. Product Synthesis mcc-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)-Exatecan

[0247] Add the intermediate ((S)-2-amino-4-cyanobutanoic acid)-Exatecan (25 mg, 1.0 eq) to a reaction flask, add DMF (1 mL), then add the intermediate mcc-Ala-Ala-OH (19 mg, 1.1 eq), TBTU (17.7 mg, 1.2 eq), and DIPEA (8.88 mg, 1.5 eq). After reacting at room temperature for 2 hours, the reaction was monitored by HPLC. Prep-HPLC purification was performed and the product was collected.

[0248] The reaction formula is as follows:

[0249] 1 H NMR (400MHz, DMSO-d6) δ8.30-8.37 (d, J=2.0Hz, 1H), 7.87-7.96 (d, J=2.0Hz, 2H), 7.75-7.86 (d, J=2.0Hz, 2H), 7.27-7.33 (d,J=8.0Hz,1H), 6.98-7.02(m,2H), 5.45-5.54(m,1H), 5.40-5.45(m,2H), 5.23-5.32(m,1H), 5.04-5.13(m,1H), 4.25-4 .37(m,1H), 3.97-4.09(m,1H), 3.56-3.66(m,4H), 3.10-3.26(m,4H), 2.35-2.46(m,4H), 2.00-2.13(m,3H), 1.77-1.92(m ,3H), 1.66-1.75(m,2H), 1.56-1.64(m,3H), 1.45-1.53(m,1H), 1.12-1.26(m,4H), 1.01-1.13(m,4H), 0.80-0.94(m,4H).

[0250] 7. Synthesis of mcc-Ala-Ala-Ala-Ala-Gln-Exatecan

[0251] 1. Synthesis of intermediate 1 mcc-Ala-Ala-Ala-Ala-Gln-OH

[0252] H-Gln(Trt)-OH 2-CTC resin (1.0 g, 0.5 mmol, 0.5 mmol / g) was added to a solid phase reactor, and DCM (20 mL) was added. The resin was shaken on an oscillator for 20 min to fully swell the resin. Filter the mixture and wash the resin with DMF. Add 20 mL of a DMF solution containing Fmoc-Ala-OH (3.0 eq, 467 mg, 1.5 mmol), HBTU (3.0 eq, 569 mg, 1.5 mmol), and HOBT (3.0 eq, 202 mg, 1.5 mmol). Shake well, then add DIPEA (3.0 eq, 0.26 mL, 1.5 mmol). Incubate on an oscillator for 1 hr. The resin color was negative by Kaiser assay. Filter the mixture and wash the resin with DMF. Add 20 mL of 20% piperidine / DMF solution, react on an oscillator for 15 min, filter, and add 20 mL of 20% piperidine / DMF solution again, react on an oscillator for 15 min. The Kaiser test shows positive resin color. Wash the resin with DMF to obtain H-Ala-Gln(Trt)-2-CTC-resin.

[0253] To a reaction tube containing H-Ala-Gln(Trt)-2-CTC-resin, add 20 mL of a DMF solution containing Fmoc-Ala-OH (3.0 eq, 467 mg, 1.5 mmol), HBTU (3.0 eq, 569 mg, 1.5 mmol), and HOBT (3.0 eq, 202 mg, 1.5 mmol). Shake well, then add DIPEA (3.0 eq, 0.26 mL, 1.5 mmol). Incubate on a shaker for 1 hr. Kaiser colorimetry test reveals negative resin. Filter and wash the resin with DMF. Add 20 mL of a 20% piperidine / DMF solution, incubate on a shaker for 15 min, filter, and add another 20 mL of a 20% piperidine / DMF solution. Incubate on a shaker for 15 min. Kaiser colorimetry test reveals positive resin. Wash the resin with DMF to obtain H-Ala-Ala-Gln(Trt)-2-CTC-resin.

[0254] To a reaction tube containing H-Ala-Ala-Gln(Trt)-2-CTC-resin, add 20 mL of a DMF solution containing Fmoc-Ala-OH (3.0 eq, 467 mg, 1.5 mmol), HBTU (3.0 eq, 569 mg, 1.5 mmol), and HOBT (3.0 eq, 202 mg, 1.5 mmol). Shake well, then add DIPEA (3.0 eq, 0.26 mL, 1.5 mmol). React on a shaker for 1 hr. Kaiser color test indicates negative resin. Filter and wash the resin with DMF. Add 20 mL of a 20% piperidine / DMF solution, react on a shaker for 15 min, filter, and add another 20 mL of a 20% piperidine / DMF solution. React on a shaker for 15 min. Kaiser color test indicates positive resin. Wash the resin with DMF to obtain H-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin.

[0255] To a reaction tube containing H-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin, add 20 mL of a DMF solution containing Fmoc-Ala-OH (3.0 eq, 467 mg, 1.5 mmol), HBTU (3.0 eq, 569 mg, 1.5 mmol), and HOBT (3.0 eq, 202 mg, 1.5 mmol). Shake well, then add DIPEA (3.0 eq, 0.26 mL, 1.5 mmol). Incubate on a shaker for 1 hr. Kaiser colorimetry indicates negative resin. Filter and wash the resin with DMF. Add 20 mL of 20% piperidine / DMF solution, react on an oscillator for 15 min, filter, and add 20 mL of 20% piperidine / DMF solution again, react on an oscillator for 15 min. The resin color test by Kaiser test is positive, and the resin is washed with DMF to obtain H-Ala-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin.

[0256] To a reaction tube containing H-Ala-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin, add 20 mL of SMCC (3.0 eq, 501 mg, 1.5 mmol) in DMF. Shake well, then add DIPEA (3.0 eq, 0.26 mL, 1.5 mmol). Incubate on a shaker for 1 hr. Kaiser assay confirms negative resin. Filter, wash the resin with DMF, MeOH, and MTBE. Dry in a vacuum oven at 30°C for 1 hr to obtain mcc-Ala-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin (1.42 g, 0.5 mmol).

[0257] Transfer the obtained resin to a 50 mL centrifuge tube, add 15 mL of cold 95% TFA / Tis solution, and incubate on a shaker for 1 hour. Filter, and slowly add the filtrate to 150 mL of cold MTBE solution. Allow to stand for 1 hour to precipitate a white precipitate. Centrifuge and wash the precipitate with cold MTBE solution. Collect the washed precipitate and dry it in a vacuum oven at 30°C for 4 hours to obtain the intermediate mcc-Ala-Ala-Ala-Ala-Gln-OH as a white solid.

[0258] The reaction formula is as follows:

[0259] 2. Product Synthesis mcc-Ala-Ala-Ala-Ala-Gln-Exatecan

[0260] The intermediate mcc-Ala-Ala-Ala-Ala-Gln-OH (244 mg, 2.0 eq) was added to a reaction flask, followed by DMF (2 mL), isotecan sulfonate (100 mg, 1.0 eq), TBTU (132 mg, 2.2 eq), and DIPEA (73 mg, 3.0 eq). The reaction was stirred at room temperature for 1 hour. The product was then purified by Prep-HPLC and collected.

[0261] The reaction formula is as follows:

[0262] 1H NMR (400MHz, DMSO-d6) δ8.25-8.33(d,J=2.0Hz,1H),7.87-7.95(d,J=2.0Hz,2H), 7.76-7.86(d,J=2.0Hz,2H), 7.28-7.34(d,J=8.0Hz,1H), 7.20-7.26(m,1H), 6.98-7.02(m,1H), 6.73-6.76(m,1H), 5.47-5.55( m,1H), 5.40-5.41(m,1H), 5.26-5.36(m,2H), 5.05-5.15(m,2H), 4.12- 4.20(m,2H), 4.03-4.09(m,1H), 3.87-3.94(m,1H), 3.20-3.27(m,2H) , 3.12-3.18(m,1H), 2.65-2.68(m,1H), 2.36-2.43(m,2H), 2.31-2.35( m,1H), 2.02-2.13(m,2H), 1.78-1.93(m,3H), 1.6-1.76(m,2H), 1.56- 1.64(m,2H), 1.21-1.27(m,1H), 1.06-1.20(m,6H), 0.85-0.94(m,3H).

[0263] Example 3 Preparation of GPC3-ADC

[0264] 1. Preparation of Antibody Conjugate Hu 52H5D3B8-8-mc-AAN-Exatecan (ADC-1)

[0265] a. Dilute 10 mg of Hu 52H5D3B8-8 antibody to 5 mg / mL in PBS buffer. Adjust the pH of the antibody to 7.5 with Tris-EDTA solution. Measure the protein concentration using a Nanodrop analyzer, weigh the antibody solution, and calculate the total protein content. Add 8-fold the molar ratio of TCEP solution to the antibody. Incubate on a 3D shaker at room temperature for 120 minutes, mixing continuously.

[0266] b. Add an excess of mc-AAN-Exatecan solution (dissolved in DMSO) to the reduced antibody solution, mix thoroughly, and incubate on a 3D shaker at room temperature for 30 minutes with continuous mixing. After the reaction, add an excess of N-acetylcysteine ​​solution and incubate on a 3D shaker at room temperature for 30 minutes with continuous mixing.

[0267] c. Purify the conjugated product using a 15 mL 30KD ultrafiltration centrifuge tube and replace it with storage solution (10 mM Histidine, pH 5.5) at a displacement factor greater than 1000. Filter through a 0.22 μm sterilizing filter to obtain the antibody-drug conjugate Hu 52H5D3B8-8-mc-AAN-Exatecan, which was stored at 4°C.

[0268] d. The protein concentration of the antibody-drug conjugate was detected by UV / BCA method, and the DAR was detected by HIC (as shown in Figure 5). The DAR was 8.0, and the purity was detected by SEC.

[0269] 2. Preparation of Antibody Conjugate Hu 52H5D3B8-8-mcc-AAN-Exatecan (ADC-2)

[0270] a. Dilute 10 mg of Hu 52H5D3B8-8 antibody to 5 mg / mL in PBS buffer. Adjust the pH of the antibody to 7.5 with Tris-EDTA solution. Measure the protein concentration using a Nanodrop analyzer, weigh the antibody solution, and calculate the total protein content. Add 8-fold the molar ratio of TCEP solution to the antibody. Incubate on a 3D shaker at room temperature for 120 minutes, mixing continuously.

[0271] b. Add an excess of mcc-AAN-Exatecan solution (dissolved in DMSO) to the reduced antibody solution, mix thoroughly, and incubate on a 3D shaker at room temperature for 30 minutes with continuous mixing. After the reaction is complete, add an excess of N-acetylcysteine ​​solution to the reaction mixture and incubate on a 3D shaker at room temperature for 30 minutes with continuous mixing.

[0272] c. Purify the conjugated product using a 15 mL 30KD ultrafiltration centrifuge tube and replace it with storage solution (10 mM Histidine, pH 5.5) at a displacement factor greater than 1000. Filter through a 0.22 μm sterilizing filter to obtain the antibody-drug conjugate Hu 52H5D3B8-8-mcc-AAN-Exatecan, which was stored at 4°C.

[0273] d. The protein concentration of the antibody-drug conjugate was detected by UV / BCA method, and the DAR was detected by HIC (as shown in Figure 6). The DAR was 8.0. The purity was detected by SEC.

[0274] 3. Preparation of Antibody Conjugate Hu 52H5D3B8-8-mc-AAQ-Exatecan (ADC-3)

[0275] a. Dilute 10 mg of Hu 52H5D3B8-8 antibody with PBS buffer to a concentration of approximately 5 mg / mL. Adjust the pH of the antibody to approximately 7.5 with Tris-EDTA solution. Measure the protein concentration using a Nanodrop analyzer, weigh the antibody solution, and calculate the total protein content. Add 8-fold the molar ratio of TCEP solution to the antibody. Place the mixture on a 3D shaker and incubate at room temperature for at least 120 minutes, mixing continuously.

[0276] b. Add an excess of mc-AAQ-Exatecan solution (dissolved in DMSO) to the reduced antibody solution, mix thoroughly, and incubate on a 3D shaker at room temperature for at least 30 minutes with continuous mixing. After the reaction is complete, add an excess of N-acetylcysteine ​​solution to the reaction mixture and incubate on a 3D shaker at room temperature for at least 30 minutes with continuous mixing.

[0277] c. Purify the conjugated product using a 15 mL 30KD ultrafiltration centrifuge tube and replace it with storage solution (10 mM Histidine, pH 5.5) at a displacement factor greater than 1000. Filter through a 0.22 μm sterilizing filter to obtain the antibody-drug conjugate Hu 52H5D3B8-8-mc-AAQ-Exatecan, which was stored at 4°C.

[0278] d. The protein concentration of the antibody-drug conjugate was detected by UV / BCA method, and the DAR was detected by HIC (as shown in Figure 7). The DAR was 8.0. The purity was detected by SEC.

[0279] 4. Preparation of Antibody Conjugate Hu 52H5D3B8-8-mcc-AAQ-Exatecan (ADC-4)

[0280] a. Dilute 10 mg of Hu 52H5D3B8-8 antibody to 5 mg / mL in PBS buffer. Adjust the pH of the antibody to 7.5 with Tris-EDTA solution. Measure the protein concentration using a Nanodrop analyzer, weigh the antibody solution, and calculate the total protein content. Add 8-fold the molar ratio of TCEP solution to the antibody. Incubate on a 3D shaker at room temperature for 120 minutes, mixing continuously.

[0281] b. Add an excess of mcc-AAQ-Exatecan solution (dissolved in DMSO) to the reduced antibody solution, mix thoroughly, and incubate on a 3D shaker at room temperature for 30 minutes with continuous mixing. After the reaction is complete, add an excess of N-acetylcysteine ​​solution to the reaction mixture and incubate on a 3D shaker at room temperature for 30 minutes with continuous mixing.

[0282] c. Purify the conjugate using a 15 mL 30KD ultrafiltration centrifuge tube and replace it with storage solution (10 mM Histidine, pH 5.5) at a displacement factor greater than 1000. Filter through a 0.22 μm sterilizing filter to obtain the antibody-drug conjugate Hu 52H5D3B8-8-mcc-AAQ-Exatecan, which was stored at 4°C.

[0283] d. The protein concentration of the antibody-drug conjugate was detected by UV / BCA method, and the DAR was detected by HIC (as shown in Figure 8). The DAR was 8.0. The purity was detected by SEC.

[0284] 5. Preparation of Antibody Conjugate Hu 52H5D3B8-7-mcc-AAQ-Exatecan (ADC-5)

[0285] a. Dilute 10 mg of Hu 52H5D3B8-7 antibody to 5 mg / mL in PBS buffer. Adjust the pH of the antibody to 7.5 with Tris-EDTA solution. Measure the protein concentration using a Nanodrop analyzer, weigh the antibody solution, and calculate the total protein content. Add 10-fold the molar ratio of TCEP solution to the antibody. Incubate on a 3D shaker at room temperature for 120 minutes, mixing continuously.

[0286] b. Add an excess of mcc-AAQ-Exatecan solution (dissolved in DMSO) to the reduced antibody solution, mix thoroughly, and incubate on a 3D shaker at room temperature for 30 minutes with continuous mixing. After the reaction is complete, add an excess of N-acetylcysteine ​​solution to the reaction mixture and incubate on a 3D shaker at room temperature for 30 minutes with continuous mixing.

[0287] c. Purify the conjugated product using a 15 mL 30KD ultrafiltration centrifuge tube and replace it with storage solution (10 mM Histidine, pH 5.5) at a displacement factor greater than 1000. Filter through a 0.22 μm sterilizing filter to obtain the antibody-drug conjugate Hu 52H5D3B8-7-mcc-AAQ-Exatecan, which was stored at 4°C.

[0288] d. The protein concentration of the antibody-drug conjugate was detected by UV / BCA method, and the DAR was detected by HIC (as shown in Figure 9). The DAR was 7.9. The purity was detected by SEC.

[0289] 6. Preparation of Antibody Conjugate Hu 52H5D3B8-8-mcc-AAQ-Exatecan (ADC-6)

[0290] a. Dilute 10 mg of Hu 52H5D3B8-8 antibody to 5 mg / mL in PBS buffer. Adjust the pH of the antibody to 7.5 with Tris-EDTA solution. Measure the protein concentration using a Nanodrop analyzer, weigh the antibody solution, and calculate the total protein content. Add 2.1 times the molar ratio of TCEP solution to the antibody. Incubate on a 3D shaker at room temperature for 120 minutes, mixing continuously.

[0291] b. Add an excess of mcc-AAQ-Exatecan solution (dissolved in DMSO) to the reduced antibody solution, mix thoroughly, and incubate on a 3D shaker at room temperature for 30 minutes with continuous mixing. After the reaction is complete, add an excess of N-acetylcysteine ​​solution to the reaction mixture and incubate on a 3D shaker at room temperature for 30 minutes with continuous mixing.

[0292] c. Purify the conjugated product using a 15 mL 30KD ultrafiltration centrifuge tube and replace it with storage solution (10 mM Histidine, pH 5.5) at a displacement factor greater than 1000. Filter through a 0.22 μm sterilizing filter to obtain the antibody-drug conjugate Hu 52H5D3B8-8-mcc-AAQ-Exatecan, which was stored at 4°C.

[0293] d. The protein concentration of the antibody-drug conjugate was detected by UV / BCA method, and the DAR was detected by HIC (as shown in Figure 10), with a DAR of 4.0. The purity was detected by SEC.

[0294] 7. Preparation of Antibody Conjugate Hu 52H5D3B8-7-mcc-AAQ-Exatecan (ADC-7)

[0295] a. Dilute 10 mg of Hu 52H5D3B8-7 antibody to 5 mg / mL in PBS buffer. Adjust the pH of the antibody to 7.5 with Tris-EDTA solution. Measure the protein concentration using a Nanodrop analyzer, weigh the antibody solution, and calculate the total protein content. Add 2.5 times the molar ratio of TCEP solution to the antibody. Incubate on a 3D shaker at room temperature for 120 minutes, mixing continuously.

[0296] b. Add an excess of mcc-AAQ-Exatecan solution (dissolved in DMSO) to the reduced antibody solution, mix thoroughly, and incubate on a 3D shaker at room temperature for 30 minutes with continuous mixing. After the reaction is complete, add an excess of N-acetylcysteine ​​solution to the reaction mixture and incubate on a 3D shaker at room temperature for 30 minutes with continuous mixing.

[0297] c. Purify the conjugated product using a 15 mL 30KD ultrafiltration centrifuge tube and replace it with storage solution (10 mM Histidine, pH 5.5) at a displacement factor greater than 1000. Filter through a 0.22 μm sterilizing filter to obtain the antibody-drug conjugate Hu 52H5D3B8-7-mcc-AAQ-Exatecan, which was stored at 4°C.

[0298] d. The protein concentration of the antibody-drug conjugate was detected by UV / BCA method, and the DAR was detected by HIC (as shown in Figure 11). The DAR was 4.0. The purity was detected by SEC.

[0299] 8. Preparation of Antibody Conjugate Hu 52H5D3B8-8-MCC-AA-(3-cyano-L-alanine)-Exatecan (ADC-8)

[0300] a. Take the GPC3 antibody Hu 52H5D3B8-8 and adjust the pH of the antibody to approximately 7.5 with Tris-EDTA solution. Measure the protein concentration using a Nanodrop and weigh the antibody solution to calculate the total protein. Add TCEP solution to the antibody and place on a 3D shaker at room temperature for at least 120 minutes, mixing continuously to completely reduce the interchain disulfide bonds.

[0301] b. Add an excess of MCC-AA-(3-cyano-L-alanine)-Exatecan solution (dissolved in DMSO) to the reduced antibody solution, mix thoroughly, and incubate on a 3D shaker at room temperature for at least 30 minutes with continuous mixing. After the reaction is complete, add an excess of N-acetylcysteine ​​solution to the reaction mixture and incubate on a 3D shaker at room temperature for at least 30 minutes with continuous mixing.

[0302] c. Purify the conjugated product using a 30KD ultrafiltration centrifuge tube and replace it with a storage solution (10mM Histidine, pH 5.5) at a displacement factor greater than 1000. Filter through a 0.22μm sterilizing filter to obtain the antibody-drug conjugate Hu 52H5D3B8-8-MCC-AA-(3-cyano-L-alanine)-Exatecan, which is then stored at 4°C.

[0303] d. The protein concentration of the antibody-drug conjugate was detected by UV / BCA method, and the DAR was detected by HIC (Figure 12). The DAR was 8.0. The purity was detected by SEC.

[0304] 9. Preparation of Antibody Conjugate Hu 52H5D3B8-8-MCC-AAAAQ-Exatecan (ADC-9)

[0305] a. Take the GPC3 antibody Hu 52H5D3B8-8 and adjust the pH of the antibody to approximately 7.5 with Tris-EDTA solution. Measure the protein concentration using a Nanodrop and weigh the antibody solution to calculate the total protein. Add TCEP solution to the antibody and place on a 3D shaker at room temperature for at least 120 minutes, mixing continuously to completely reduce the interchain disulfide bonds.

[0306] b. Add an excess of MCC-AAN1-Exatecan solution (dissolved in DMSO) to the reduced antibody solution, mix thoroughly, and incubate on a 3D shaker at room temperature for at least 30 minutes with continuous mixing. After the reaction is complete, add an excess of N-acetylcysteine ​​solution to the reaction mixture and incubate on a 3D shaker at room temperature for at least 30 minutes with continuous mixing.

[0307] c. Purify the conjugated product using a 30KD ultrafiltration centrifuge tube and replace it with storage solution (10mM Histidine, pH 5.5) at a displacement factor greater than 1000. Filter through a 0.22μm sterilizing filter to obtain the antibody-drug conjugate Hu 52H5D3B8-8-MCC-AAAAQ-Exatecan, which was stored at 4°C.

[0308] d. The protein concentration of the antibody-drug conjugate was detected by UV / BCA method, and the DAR was detected by HIC (Figure 13), with a DAR of 8.0. The purity was detected by SEC.

[0309] In addition, those skilled in the art can also refer to the above method to prepare other ADCs of the present invention, such as GPC3 (such as Hu 52H5D3B8-8)-mcc-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)-Exatecan.

[0310] Example 4 GPC3-ADC pharmacology and pharmacodynamics studies

[0311] 1. Cell Binding of GPC3-ADC

[0312] FACS experiments confirmed that the cell binding of ADC was not significantly affected by Cys conjugation between antibody chains.

[0313] Collect Huh7 liver cancer cells in logarithmic phase growth. After centrifugation, resuspend the cells in FACS buffer (PBS + 3% FBS), adjust the cell density, and divide the cells into 96-well U-bottom cell culture plates so that each well contains 200,000 to 500,000 cells. Add antibodies or ADC diluted 4 times with FACS buffer and mix well so that the final concentration of each sample is 10μg / mL. Incubate the 96-well plate at 4°C for 60 minutes. Wash the cells in the wells thoroughly with FACS buffer to remove unbound antibodies or ADCs. Add Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor diluted 1:1,000 with FACS buffer. TM 647 (Invitrogen, #A-21445) was incubated at 4°C in the dark for 30-60 minutes. The cells in the wells were thoroughly washed with FACS buffer to remove unbound secondary antibody. Fluorescence signal analysis of the cell samples was performed using a CytoFLEX flow cytometer (Beckman Coulter). The experimental results are shown in Figure 14. The affinity of the GPC3 antibody for Huh7 cells did not change significantly before and after conjugation with the cytotoxin. The affinity of each test substance for Huh7 cells is shown in Table 3 and Figure 14.

[0314] Table 3: Binding of GPC3 antibodies and ADCs to Huh7 cells (EC 50 )

[0315] 2. Internalization of Antibodies in GPC3-ADC

[0316] Internalization of the ADC was confirmed and compared simultaneously for each ADC molecule.

[0317] Huh7 hepatoma cells were harvested from logarithmically growing cells. After centrifugation, the cells were washed once and resuspended in pre-chilled DMEM medium supplemented with 3% FBS. The cell density was adjusted and the cells were plated into 96-well U-bottom plates, with 500,000 to 800,000 cells per well. Antibodies or ADCs diluted in DMEM supplemented with 3% FBS were added and mixed to a final concentration of 10 μg / mL. The 96-well plates were incubated on ice for 60 minutes. The cells were washed thoroughly with pre-chilled FACS buffer (PBS + 3% FBS) to remove unbound antibody or ADC. The cells were evenly distributed into four 96-well U-bottom plates. After centrifugation, the cells were resuspended in DMEM medium. One 96-well plate was placed on ice, while the other three plates were incubated at 37°C in a cell culture incubator. After 0.5, 1, and 2 hours, one of the 37°C plates was transferred to ice. After the third culture plate is transferred to ice for 5-10 minutes, add diluted Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 647 (Invitrogen, #A-21445) and incubate on ice in the dark for 30 minutes. 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 MFI (GeoMean Fluorescence Intensity). All experiments were maintained on ice or at 4°C, except for incubation at 37°C.

[0318] The percentage reduction of cell surface molecules was calculated as follows: Cell surface molecule reduction % = (MFI 冰上 -MFI 37℃ ) / MFI 冰上 *100%.

[0319] The experimental results are shown in Table 4. The internalization capacity of antibody Hu 52H5D3B8-8 in Huh7 cells remained unchanged before and after conjugation with the cytotoxin, and the internalization capacity of each ADC molecule was similar. The results of internalization of each test substance in Huh7 cells (expressed as the percentage reduction of cell surface molecules) are shown in Table 4 and Figure 15.

[0320] Table 4: Internalization of GPC3 antibodies and ADCs in Huh7 cells

[0321] Example 5 In vitro pharmacodynamic study

[0322] The cell-killing activity of GPC3-targeting ADCs conjugated with various linker-payloads and varying DAR values ​​was compared. Non-binding indicates that the antibody is a non-binding human IgG1 isotype control that does not bind to the target on the tumor cell surface used in the experiment and serves as a negative control antibody.

[0323] Liver cancer cells growing in the logarithmic phase 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 ADC molecules 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 600 or 500 ng / mL. The cells were cultured in a cell culture incubator for 6 days, 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.

[0324] 1. Experimental reagents and sources:

[0325] Test drug

[0326] 2. Experimental results:

[0327] EC of each GPC3-ADC cell killing activity 50 The average values ​​are shown in Table 5. Figures 16-18 are representative graphs showing the killing effect of different GPC3-ADCs on Huh7 cells.

[0328] Table 5: EC values ​​of different GPC3-ADCs for cytotoxicity in Huh7 cells 50 value

[0329] In addition, other ADCs of the present invention, such as GPC3 (eg, Hu 52H5D3B8-8)-mcc-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)-Exatecan, also exhibited significant specific cell killing activity in Huh7 cells.

[0330] As can be seen from the above results, as shown in Table 5 and Figures 16-18, the various GPC3-ADCs of the present invention all exhibited significant specific cytotoxic activity in Huh7 cells. Furthermore, compared to GPC3-ADCs with a DAR of approximately 4, the corresponding GPC3-ADC molecules with a DAR of approximately 8 exhibited a more potent cytotoxic effect.

[0331] Example 6 In vivo pharmacodynamic study

[0332] The in vivo antitumor activity of GPC3-ADC was tested in the human hepatocellular carcinoma LIV#219 PDX mouse model.

[0333] The LIV219 PDX model has a high level of GPC3 RNA expression, and FACS analysis confirmed that it is GPC3 positive. The establishment process of the human liver cancer nude mouse LIV#219 PDX model (Jindi Biopharmaceutical Technology (Shanghai) Co., Ltd.) is as follows: a volume of about 15-30mm 3 The tumor tissue was transplanted subcutaneously on the right back of BALB / c nude mice. 3 The mice were randomly divided into groups according to the random block method, and the grouping day was designated as Day 0. There were 6 mice in each group, and the tumor volume and body weight were uniform among the groups. There were 15 groups in total, including the vehicle group, the Non-binding-MC-AAN-Exa(DAR8)(10 mg / kg) control group, the Non-binding-MC-AAQ-Exa(DAR8)(10 mg / kg) control group, the Non-binding-MCC-AAN-Exa(DAR8)(10 mg / kg) control group, the Non-binding-MCC-AAQ-Exa(DAR8)(10 mg / kg) control group, the Hu 52H5D3B8-8-MC-AAN-Exa(ADC-1, 10 mg / kg) group, the Hu 52H5D3B8-8-MCC-AAN-Exa(ADC-2, 10 and 3 mg / kg) group, and the Hu The 52H5D3B8-8-MC-AAQ-Exa (ADC-3, 10 and 3 mg / kg) and Hu 52H5D3B8-8-MCC-AAQ-Exa (ADC-4, 10 and 3 mg / kg) groups were administered with tail vein administration once on Day 0 and Day 7.

[0334] Data analysis: During the experiment, the body weight and tumor volume of mice were measured twice a week. The formula for calculating 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. Wherein, V0 is the tumor volume measured at the time of group administration (i.e., Day 0), V f The tumor volume was measured on the last day. Relative tumor growth rate (T / C) (%) = (RTV of the drug group / RTV of the vehicle group) × 100%. When T / C (%) ≤ 40% and P < 0.05, the test article was considered to have a significant inhibitory effect on tumor growth.

[0335] The experimental results are shown in Figures 19-20. On Day 29, the relative tumor proliferation rate T / C (%) of the Hu 52H5D3B8-8-MC-AAN-Exa (ADC-1, 10 mg / kg) group was 7.88% (P<0.05); the relative tumor proliferation rates T / C (%) of the Hu 52H5D3B8-8-MCC-AAN-Exa (ADC-2, 10 and 3 mg / kg) groups were 12.44% (P<0.05) and 77.71% (P>0.05), respectively; The relative tumor growth rates (T / C%) in the 52H5D3B8-8-MC-AAQ-Exa (ADC-3, 10, and 3 mg / kg) groups were 6.75% (P < 0.05) and 47.29% (P > 0.05), respectively. The relative tumor growth rates (T / C%) in the Hu52H5D3B8-8-MCC-AAQ-Exa (ADC-4, 10, and 3 mg / kg) groups were 7.37% (P < 0.05) and 53.71% (P > 0.05), respectively. Compared to Day 0, the mean body weight change in each group on Day 29 ranged from 0.87% to 4.88%.

[0336] Experimental results showed that at a dose of 10 mg / kg, all ADCs significantly inhibited tumor growth. At a dose of 3 mg / kg, Hu 52H5D3B8-8-MC-AAQ-Exa (ADC-3) and Hu 52H5D3B8-8-MCC-AAQ-Exa (ADC-4) exhibited lower relative tumor growth rates. All test products were well tolerated by tumor-bearing mice.

[0337] Example 7 In vivo pharmacodynamic study

[0338] The in vivo antitumor activity of Hu 52H5D3B8-8-MCC-AAN-Exa (ADC-2) and Hu 52H5D3B8-8-MCC-AAQ-Exa (ADC-4) was also tested in the human hepatocellular carcinoma LI6653 PDX mouse model.

[0339] The LI6653 PDX model (Crown Biotech (Taicang) Co., Ltd.) was confirmed to express GPC3 (++) by IHC. The establishment process of the human liver cancer nude mouse PDX model is as follows: tumor tissue was harvested from mice bearing human liver cancer xenografts, cut into 2-3 mm diameter tumor masses, and inoculated subcutaneously under the right anterior scapula of BALB / c nude mice. When the average tumor volume of the tumor-bearing mice reached approximately 170-200 mm, the tumor was inoculated with GPC3 (++). 3 Tumor-bearing mice were randomly divided into groups based on tumor size and dosed on Day 0. Six mice were included in each group to ensure uniform tumor volume and body weight across groups. Six groups were selected, including a vehicle group, a Non-binding-MCC-AAN-Exa (DAR8) (10 mg / kg) control group, a Hu 52H5D3B8-8-MCC-AAN-Exa (ADC-2, 10 and 3 mg / kg) group, and a Hu 52H5D3B8-8-MCC-AAQ-Exa (ADC-4, 10 and 3 mg / kg) group. Dosing was performed via the tail vein on Day 0 and Day 7.

[0340] Data analysis: During the experiment, the body weight and tumor volume of mice were measured twice a week. The formula for calculating 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. Wherein, V0 is the tumor volume measured at the time of group administration (i.e., Day 0), V f The tumor volume was measured on the last day. Relative tumor growth rate (T / C) (%) = (RTV of the drug group / RTV of the vehicle group) × 100%. When T / C (%) ≤ 40% and P < 0.05, the test article was considered to have a significant inhibitory effect on tumor growth.

[0341] The experimental results are shown in Figures 21-22. On Day 16, the relative tumor growth rates (T / C%) in the Hu 52H5D3B8-8-MCC-AAN-Exa (ADC-2, 10 and 3 mg / kg) treatment groups were 17.46% (P < 0.05) and 31.81% (P < 0.05), respectively; the relative tumor growth rates (T / C%) in the Hu 52H5D3B8-8-MCC-AAQ-Exa (ADC-4, 10 and 3 mg / kg) treatment groups were 13.05% (P < 0.05) and 21.10% (P < 0.05), respectively. Compared with Day 0, the average body weight change of mice in each group on Day 16 ranged from 4.38% to 11.97%.

[0342] Experimental results showed that both ADCs significantly inhibited tumor growth at a dose of 10 mg / kg. At a dose of 3 mg / kg, Hu 52H5D3B8-8-MCC-AAQ-Exa (ADC-4) exhibited a lower relative tumor growth rate than Hu 52H5D3B8-8-MCC-AAN-Exa (ADC-2). All test products were well tolerated by tumor-bearing mice.

Claims

1. An antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the antibody-drug conjugate has a structure as shown in formula I, Ab-(LD) p Formula I in: Ab is an anti-GPC3 antibody or an antigen-binding fragment thereof, wherein the anti-GPC3 antibody comprises a heavy chain variable region and a light chain variable region, wherein CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or mutants thereof, and CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the sequences shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, or mutants thereof; L is a linker selected from mc-AAN, mc-AAQ, mcc-AAN, mcc-AAQ, mcc-Ala-Ala-(3-cyano-alanine), mcc-Ala-Ala-(2-amino-4-cyanobutanoic acid), and mcc-Ala-Ala-Ala-Ala-Gln; D is a cytotoxin, which is Exatecan; p is any value between 1 and 10; Among them, the mcc or mc end of the L linker is covalently linked to Ab, and the other end of the L linker is covalently linked to D.

2. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: The structural formula of LD before conjugation with Ab is shown below: Each LD was coupled to the sulfhydryl group in Ab via the terminal succinimide.

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 antibody-drug conjugate has a structure as shown in formula (Ila), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh) or (IIi), 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: The anti-GPC3 antibody has one or more technical features selected from the following (1)-(2): (1) The heavy chain variable regions FR1, FR2, FR3, and FR4 of the anti-GPC3 antibody respectively comprise the residues shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or a mutant thereof; (2) The light chain variable region FR1, FR2, FR3, and FR4 of the anti-GPC3 antibody comprises the sequences shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, or a mutant thereof, respectively; or the light chain variable region FR1, FR2, FR3, and FR4 of the anti-GPC3 antibody comprises the sequences shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, and SEQ ID NO: 15, or a mutant thereof, respectively.

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 technical features selected from the following (1)-(2): (1) The heavy chain variable region of the anti-GPC3 antibody comprises the sequence shown in SEQ ID NO: 8 or a mutant thereof; (2) 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 mutant thereof.

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 technical features selected from the following (1)-(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 above 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 selected from a human lambda constant region, a kappa constant region or a mutant of the above constant region.

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 technical features selected from the following (1)-(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 a homology greater than 70%, preferably greater than 75%, 80%, 85%, 90%, 95%, or 99% 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 light chain constant region of the anti-GPC3 antibody comprises the sequence shown in SEQ ID NO: 20, or comprises a sequence having a homology greater than 70%, preferably greater than 75%, 80%, 85%, 90%, 95%, or 99% to the sequence shown in SEQ ID NO: 20; Preferably, the sequence of the light chain constant region of the anti-GPC3 antibody is as shown in SEQ ID NO:

20.

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: p is any value between 2 and 8; Preferably, p is any value between 4 and 8 (eg, 4.0, 7.9 or 8.0).

9. A pharmaceutical composition comprising 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; Optionally, the pharmaceutical composition further comprises at least one pharmaceutical excipient; Alternatively, the pharmaceutical composition further comprises at least one of a chemotherapeutic drug, an immunotherapeutic drug and an immunosuppressant for treating tumors.

10. Use of 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, or the pharmaceutical composition according to claim 9 in the preparation of a medicament for preventing and / or treating a disease associated with GPC3 positivity; Preferably, the disease associated with GPC3 positivity is selected from liver cancer, lung cancer, gastric cancer, head and neck cancer, esophageal cancer, Merkel cell carcinoma, liposarcoma, breast cancer, ovarian cancer, melanoma, squamous cell carcinoma, renal cell carcinoma, pancreatic cancer, prostate cancer, colorectal cancer, glioma, kidney cancer, bladder cancer, cervical cancer, gallbladder cancer, glioblastoma; More preferably, the disease associated with GPC3 positivity is liver cancer.

11. An in vitro non-therapeutic method for inhibiting tumor angiogenesis, delaying tumor progression, inhibiting tumor growth or inhibiting tumor cell proliferation, comprising: contacting tumor cells with 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, or the pharmaceutical composition according to claim 9, wherein the tumor is a tumor expressing GPC3; Preferably, the tumor expressing GPC3 is selected from liver cancer, lung cancer, gastric cancer, head and neck cancer, esophageal cancer, Merkel cell carcinoma, liposarcoma, breast cancer, ovarian cancer, melanoma, squamous cell carcinoma, renal cell carcinoma, pancreatic cancer, prostate cancer, colorectal cancer, glioma, kidney cancer, bladder cancer, cervical cancer, gallbladder cancer, glioblastoma; More preferably, the GPC3-expressing tumor is liver cancer.

12. A method for treating and / or preventing a disease associated with GPC3 positivity, comprising: Administering a therapeutically and / or prophylactically effective amount of 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, or the pharmaceutical composition according to claim 9 to a subject in need thereof; Preferably, the disease associated with GPC3 positivity is selected from liver cancer, lung cancer, gastric cancer, head and neck cancer, esophageal cancer, Merkel cell carcinoma, liposarcoma, breast cancer, ovarian cancer, melanoma, squamous cell carcinoma, renal cell carcinoma, pancreatic cancer, prostate cancer, colorectal cancer, glioma, kidney cancer, bladder cancer, cervical cancer, gallbladder cancer, glioblastoma; More preferably, the disease associated with GPC3 positivity is liver cancer.

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