5T4 antibody drug conjugate and application thereof

Abstract

Relates to a 5T4 antibody drug conjugate and application thereof, and particularly provides the antibody drug conjugate, or pharmaceutically acceptable salt, solvate or solvate of the salt, the antibody drug conjugate has a structure as shown in a formula I, and Ab is an anti-5T4 antibody. The antibody drug conjugate has good tumor cell growth inhibition activity in vivo and in vitro, and has a good application prospect. Ab-(L-D) p is represented by formula I.

Description

5T4 antibody-drug conjugate and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the application with CN application number 202311048671.0 and application date August 18, 2023, and claims its priority. The content of the CN application is hereby introduced as a whole into this application. Technical Field

[0003] The present invention relates to the field of biomedicine, and in particular to a 5T4 antibody-drug conjugate and applications thereof. Background Art

[0004] Oncofetal protein 5T4, also known as trophoblast glycoprotein (TPBG) and Wnt activation inhibitory factor 1 (WAIF1), is a highly N-terminally glycosylated transmembrane glycoprotein encoded by the TPBG gene. TPBG has a molecular weight of approximately 72 kDa and contains 420 amino acids. It consists of a 310-amino acid extracellular domain, a 20-amino acid transmembrane region, and a 44-amino acid cytoplasmic domain. It influences cytoskeletal remodeling and intercellular integrity by binding to PDZ-domain-containing proteins in the cytoplasm. Studies have shown that 5T4 is closely associated with various physiological and pathological processes, such as intercellular junctions, cell morphology and motility, cell adhesion, and cell membrane integrity. During embryonic development, 5T4 is relatively widely expressed, but in normal tissues, it is restricted to certain specialized epithelia, such as the basal stratified squamous epithelium, glandular and ductal epithelium, as well as retinal secondary neurons and the olfactory bulb.

[0005] In contrast, 5T4 is highly expressed in many malignant solid tumors, including pancreatic cancer, prostate cancer, ovarian cancer, mesothelioma, non-small cell lung cancer, breast cancer, head and neck cancer, cervical cancer, kidney cancer, gastric cancer, and colorectal cancer. Evidence suggests that 5T4 expression in colon, gastric, or ovarian cancer is associated with low cure rates. In tissues of non-small cell lung cancer, kidney cancer, or pancreatic cancer, 5T4 expression is as high as over 95%.

[0006] Existing tumor-related studies have shown that 5T4 may exert its effects through the following three mechanisms: 1) epithelial-mesenchymal transition (EMT); 2) regulation of the CXCL12 / CXCR4 biological axis; and 3) inhibition of Wnt signal transduction.

[0007] In summary, 5T4 is highly expressed in various solid tumor tissues, rarely found in normal mature tissues, and is associated with tumor metastasis and poor prognosis, making it an ideal target for oncology drugs. Targeting this target, clinical drug development involves a variety of immunotherapies, including monoclonal antibodies, bispecific antibodies, trispecific antibodies, ADCs, tumor vaccines, and CAR-raNK. ADCs currently in clinical development include PF-06263507 (A1-mcMMAF) developed by Pfizer, ASN-004 developed by Asana BioSciences, and SYD-1875 developed by Byondis BV. PF-06263507 (A1-mcMMAF) is composed of the 5T4-specific humanized antibody PF-06281192, a non-cleavable maleimidohexyl (mc) linker, and the tubulin inhibitor monomethyl auristatin F (MMAF). ASN-004 is composed of a 5T4-specific single-chain scFv-Fc antibody, a Dolaflexin drug linker (Mersana Therapeutics), and a tubulin inhibitor derivative (AF-HPA). SYD-1875 is composed of a 5T4-specific antibody, HCP41C, engineered with a cysteine ​​residue, and a linker payload, vc-seco-DUBA. According to reports, Pfizer terminated the clinical development of PF-06263507 due to the lack of objective responses observed with PF-06263507 and the observation of dose-limiting toxicities (DLTs), primarily ocular toxicity. Furthermore, there is no information regarding further clinical trials for SYD-1875 after the completion of Phase 1 clinical trials. Therefore, it is necessary to further develop new, safe, and more effective antibody-drug conjugates targeting 5T4.

[0008] Summary of the Invention

[0009] The inventors of the present application prepared a new anti-5T4 antibody-drug conjugate (ADC) through extensive experiments and creative work, and confirmed that it 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-5T4 antibody or an antigen-binding fragment thereof, wherein the anti-5T4 antibody comprises a heavy chain variable region and a light chain variable region,

[0015] The heavy chain variable region CDR1 comprises the sequence shown in SEQ ID NO: 3,

[0016] The heavy chain variable region CDR2 comprises the sequence shown in SEQ ID NO: 4,

[0017] The heavy chain variable region CDR3 comprises the sequence shown in SEQ ID NO: 5,

[0018] The light chain variable region CDR1 comprises the sequence shown in SEQ ID NO: 6,

[0019] the light chain variable region CDR2 comprises the sequence shown in SEQ ID NO: 7;

[0020] the light chain variable region CDR3 comprises the sequence shown in SEQ ID NO: 8;

[0021] L is the connector;

[0022] D is cytotoxin;

[0023] 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).

[0024] 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.

[0025] 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" 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.

[0026] The drug-antibody ratio (DAR) can be determined by conventional means such as mass spectrometry, ELISA assays, 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.

[0027] In some embodiments, the linker is selected from: MC-AAN, MCC-AAQ, 6-maleimidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), maleimidopropionyl (MP), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl (MCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB).

[0028] In some embodiments, the linker is selected from the group consisting of: MC-AAN, MCC-AAQ, 6-maleimidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB).

[0029] The structural formulas of MC, MCC, and MC-vc-PAB are as follows:

[0030] In some embodiments, the cytotoxin is selected from the group consisting of: topoisomerase inhibitors, Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), SN-38, gemcitabine, maytansinoids (e.g., Maytansine DM1, Maytansine DM4), calicheamicin, MGBA (e.g., duocarmycin), doxorubicin, ricin, diphtheria toxin, duocarmycin SA, I131, interleukins, tumor necrosis factor, chemokines, and nanoparticles.

[0031] In some embodiments, the topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor. Exemplary topoisomerase I inhibitors include camptothecin and its derivatives. Exemplary camptothecin derivatives include exatecan, belotecan, SN-38, topotecan, irinotecan (cpt-11), deruxtecan, etc.

[0032] In some embodiments, the cytotoxin is selected from the group consisting of: Exatecan (abbreviated as Exa), Monomethyl auristatin E (MMAE), and Monomethyl auristatin F (MMAF).

[0033] In some embodiments, LD is selected from the group consisting of: MC-AAN-Exa, MCC-AAQ-Exa, MC-vc-PAB-MMAE, MC-MMAF.

[0034] In some embodiments, the structural formula of LD is as follows:

[0035] When the four 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-Exa is covalently coupled to the Ab, the structure of the resulting ADC is as follows:

[0036] 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.

[0037] In some embodiments, the sequence of the heavy chain variable region CDR1 of the anti-5T4 antibody is shown as SEQ ID NO: 3, the sequence of the heavy chain variable region CDR2 is shown as SEQ ID NO: 4, the sequence of the heavy chain variable region CDR3 is shown as SEQ ID NO: 5, the sequence of the light chain variable region CDR1 is shown as SEQ ID NO: 6, the sequence of the light chain variable region CDR2 is shown as SEQ ID NO: 7, and the sequence of the light chain variable region CDR3 is shown as SEQ ID NO: 8.

[0038] In some embodiments, the sequence of the heavy chain variable region of the anti-5T4 antibody is shown in SEQ ID NO: 1, and the sequence of the light chain variable region of the anti-5T4 antibody is shown in SEQ ID NO: 2.

[0039] In some embodiments, the sequence of the heavy chain variable region of the anti-5T4 antibody is shown in SEQ ID NO: 9, and the sequence of the light chain variable region of the anti-5T4 antibody is shown in SEQ ID NO: 10.

[0040] In some embodiments, the sequence of the heavy chain variable region of the anti-5T4 antibody is shown in SEQ ID NO: 11, and the sequence of the light chain variable region of the anti-5T4 antibody is shown in SEQ ID NO: 10.

[0041] In some embodiments, the heavy chain constant region of the anti-5T4 antibody is selected from human IgG, IgM, IgA, IgD, IgE constant regions, or mutants of the aforementioned constant regions. In some embodiments, the IgG is selected from IgG1, IgG2, IgG3, and IgG4.

[0042] In some embodiments, the light chain constant region of the anti-5T4 antibody is selected from a human lambda constant region, a kappa constant region, or a mutant of the aforementioned constant region.

[0043] In some embodiments, p is any number between 2-8.

[0044] In some embodiments, p is any number between 3 and 8 (eg, 3.8, 4.1, 4.2, 7.9, or 8.0).

[0045] 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.

[0046] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0047] In some embodiments, the pharmaceutical composition further comprises at least one of a chemotherapeutic drug, an immunotherapeutic drug, and an immunosuppressant for treating tumors.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] In a third aspect, the present invention provides use of the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the aforementioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating diseases associated with 5T4.

[0052] In a fourth aspect, the present invention provides the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the aforementioned pharmaceutical composition, for use in preventing and / or treating diseases associated with 5T4.

[0053] In the fifth aspect of the present invention, the present invention provides a method for treating and / or preventing diseases associated with 5T4, comprising: administering to a subject in need thereof a therapeutically and / or preventatively effective amount of the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the aforementioned pharmaceutical composition.

[0054] In some embodiments, the disease associated with 5T4 is selected from pancreatic cancer, prostate cancer, ovarian cancer, mesothelioma, lung cancer (such as non-small cell lung cancer), breast cancer, head and neck cancer, cervical cancer, kidney cancer, gastric cancer, colorectal cancer (such as colon cancer), stomach cancer, bladder cancer, thyroid cancer, lymphoma, acute myeloid leukemia.

[0055] In some embodiments, the disease associated with 5T4 is breast cancer, non-small cell lung cancer, or colorectal cancer.

[0056] 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 5T4-expressing tumor.

[0057] In some embodiments, the 5T4-expressing tumor is selected from pancreatic cancer, prostate cancer, ovarian cancer, mesothelioma, lung cancer (such as non-small cell lung cancer), breast cancer, head and neck cancer, cervical cancer, kidney cancer, gastric cancer, colorectal cancer (such as colon cancer), stomach cancer, bladder cancer, thyroid cancer, lymphoma, acute myeloid leukemia.

[0058] In some embodiments, the 5T4-expressing tumor is selected from breast cancer, non-small cell lung cancer, and colorectal cancer. Beneficial effects

[0059] 1. The newly developed 5T4-ADC of the present invention exhibits a strong cell-killing effect in various tumor cells (such as breast cancer and non-small cell lung cancer).

[0060] 2. The newly developed 5T4-ADC of the present invention showed significant pharmacological effects in inhibiting tumor cell growth in multiple tumor models (such as the human colorectal cancer nude mouse CRC#047PDX model). BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1-1 shows the binding activity of chimeric antibody 14G12 to human 5T4-His protein and cynomolgus monkey 5T4-His protein;

[0062] Figure 1-2 shows the binding activity of chimeric antibody 14G12 to CHOK1-hu5T4 cells;

[0063] Figures 1-3 show the binding activity of humanized antibodies 14G12z28 and 14G12z43 to human 5T4-His protein and to CHOK1-hu5T4 cells, as well as the binding activity of humanized antibody 14G12z28 to MCF-7 cells;

[0064] FIG2 shows a hydrophobic interaction chromatogram of 14G12-vcMMAE;

[0065] FIG3 shows a hydrophobic interaction chromatogram of 14G12-MC-MMAF;

[0066] FIG4 shows a hydrophobic interaction chromatogram of 14G12z28-MC-MMAF;

[0067] FIG5 shows a hydrophobic interaction chromatogram of 14G12z43-MC-MMAF;

[0068] FIG6 shows a hydrophobic interaction chromatogram of 14G12z28-MC-AAN-Exa;

[0069] FIG7 shows a hydrophobic interaction chromatogram of 14G12z28-MCC-AAQ-Exa;

[0070] FIG8 shows the binding affinity of each test substance to tumor cells NCI-H1975;

[0071] FIG9 shows the binding affinity of each test substance to tumor cells HCT116;

[0072] FIG10 shows the internalization results of each test substance in HCT116 cells;

[0073] FIG11 is a representative graph showing the killing effect of different 5T4-ADCs on MCF7 tumor cells;

[0074] FIG12 is a representative graph showing the killing effect of different 5T4-ADCs on tumor cells NCI-H1975;

[0075] FIG13 is a representative graph showing the killing effect of different 5T4-ADCs on tumor cells MDA-MB-468;

[0076] FIG14 shows the in vivo anti-tumor activity of different 5T4-ADCs in the CRC#047PDX mouse model;

[0077] FIG15 shows the effects of different 5T4-ADCs on body weight in the CRC#047PDX mouse model. DETAILED DESCRIPTION

[0078] 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.

[0079] 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.

[0080] In the present invention, unless otherwise stated, any numerical range should be understood to include any value or any sub-range within the range.

[0081] In the present invention, the term "antibody" refers to an immunoglobulin molecule that is usually composed of two pairs of identical polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. The light chains of antibodies can be divided into two types: κ and λ. Heavy chains can be divided into five types: μ, δ, γ, α or ε. Depending on the heavy chain, antibodies can be divided into five types: IgM, IgD, IgG, IgA and IgE. Within the light chain and heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H 1. C H 2 and C H 3). Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the C1q component of the complement system. H and V L The V domain can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V LIt 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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)).

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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).

[0095] 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.

[0096] "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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] Example 1 Preparation, humanization and related performance testing of anti-5T4 chimeric antibodies

[0110] 1. Anti-human 5T4 monoclonal antibody immunoscreening

[0111] To obtain monoclonal antibodies targeting the human 5T4 protein, SJL, Balb / c, and C57BL / 6 mice were primed with human 5T4-His protein. Subsequently, these immunized mice were boosted with either human 5T4-His protein or CHO-K1 cells overexpressing human 5T4 (CHOK1-hu5T4). Antibody titers against human 5T4-His protein were measured in the sera of immunized mice by ELISA and FACS. Hybridoma cells were prepared from immunized mice with high serum antibody titers. Antibody binding activity in hybridoma supernatants was assessed by ELISA and FACS. Hybridomas were screened for binding to both human 5T4 and cynomolgus macaque 5T4-His protein (cyno5T4), specifically to CHOK1-hu5T4 cells. The resulting hybridoma clone, 14G12, had its variable region amino acid sequence determined by sequencing, as shown in Table 1-1.

[0112] Table 1-1: 14G12 antibody variable region sequences

[0113] 2. Binding activity of chimeric antibody 14G12

[0114] The chimeric antibody 14G12 was constructed by linking the variable region sequences of the 14G12 antibody to a human IgG1 / Cκ backbone.

[0115] The affinity of chimeric antibody 14G12 for human 5T4-His protein was determined using SPR. Chimeric antibody 14G12 was immobilized on a Protein A chip, and the binding affinity of chimeric antibody 14G12 for human 5T4-His protein was determined by fitting binding curves at different antigen concentrations, as shown in Tables 1-4.

[0116] The binding activity of chimeric antibody 14G12 to human 5T4-His and cynomolgus monkey 5T4-His proteins was tested by ELISA. After coating a 96-well plate with human 5T4-His or cynomolgus monkey 5T4-His protein, serially diluted chimeric antibody 14G12 was added and incubated at room temperature for 60 minutes. After washing with PBST, HRP-conjugated mouse anti-human IgG Fc antibody was added and incubated at room temperature for 30 minutes. After washing with PBST, TMB substrate was added and absorbance was analyzed at OD 450nm. The binding affinity of chimeric antibody 14G12 to human 5T4-His and cynomolgus monkey 5T4-His proteins is shown in Table 1-2 and Figure 1-1. Compared to the control antibody naptumomab, chimeric antibody 14G12 exhibited comparable human 5T4-His binding activity (Figure 1-1A) and significantly superior cynomolgus monkey 5T4-His binding activity (Figure 1-1B).

[0117] Table 1-2: Binding activity of chimeric antibody 14G12

[0118] The binding activity of chimeric antibody 14G12 to CHOK1-hu5T4 cells or MCF-7 cells was detected by FACS experiment. CHOK1-hu5T4 cells or MCF-7 cells in logarithmic phase were collected. After centrifugation, the cells were resuspended in FACS buffer and divided into 96-well U-bottom cell culture plates. Antibodies diluted in FACS buffer were added and incubated at 4°C for 30 minutes. After washing the cells in the wells with FACS buffer, PE Goat anti-Human IgG Fc Secondary Antibody (eBioscience) diluted in FACS buffer was added. TM , Invitrogen) and incubated at 4°C in the dark for 30 minutes. After washing the cells in the wells with FACS buffer, the cell samples were analyzed for fluorescence signals using a MACSQuant Analyzer 16 flow cytometer (Miltenyi). The results, as shown in Figures 1-2, demonstrate that chimeric antibody 14G12 has good binding affinity for CHOK1-hu5T4 cells.

[0119] 3. Humanization of chimeric antibody 14G12

[0120] By comparing the variable region framework sequence of the 14G12 antibody with existing human IgG variable region framework sequences, a highly compatible human sequence was screened. The 14G12 antibody CDR sequence was transplanted into the selected human IgG variable region framework sequence and backmutated to obtain a series of 14G12 humanized antibodies.

[0121] Through SPR method, ELISA experiment and FACS experiment (specific methods are the same as above), the humanized antibodies 14G12z28 (also known as Hu14G12-28) and 14G12z43 (also known as Hu14G12-43) with the best binding activity were screened, and their variable region amino acid sequences are shown in Tables 1-3.

[0122] Table 1-3: Humanized antibody variable region sequences

[0123] As shown in Tables 1-4, the affinity of humanized antibodies 14G12z28 and 14G12z43 for binding to human 5T4-His protein was slightly weaker than that of chimeric antibody 14G12.

[0124] Table 1-4: Affinity of chimeric and humanized antibodies

[0125] As shown in Figure 1-3, humanized antibodies 14G12z28 and 14G12z43 showed comparable binding activity to the chimeric antibody 14G12 for human 5T4-His protein (Figure 1-3A,B), and slightly better binding activity to CHOK1-hu5T4 cells than chimeric antibody 14G12 (Figure 1-3C,D). Furthermore, humanized antibody 14G12z28 exhibited good binding affinity to MCF-7 cells (Figure 1-3E).

[0126] Example 2 Preparation of Linker-Payload

[0127] 1. Preparation of MC-AAN-Exatecan

[0128] 1. Synthesis of Intermediate 1

[0129] 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:

[0130] 2. Synthesis of Intermediate 2

[0131] 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:

[0132] 3. Synthesis of Intermediate 3

[0133] 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:

[0134] 4. Synthesis of Intermediate 4

[0135] 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:

[0136] 5. Synthesis of Products

[0137] The intermediate 4 and isotectin mesylate (CAS No.: 169869-90-3) were subjected to a condensation reaction to obtain the product; the specific steps were as follows: at room temperature, the intermediate 4 (26 mg, 1.0 eq) was added to the reaction flask, DMF (1.5 mL) was added, and isotectin mesylate (29.6 mg, 1.0 eq), EEDQ (20.7 mg,

[0138] The reaction was carried out at room temperature. HPLC monitoring was performed after the reaction was completed, and the prepared solution was purified and concentrated to obtain the product. The reaction formula is as follows:

[0139] 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).

[0140] 2. Preparation of MCC-AAQ-Exatecan

[0141] 1. Synthesis of Intermediate 1: Fmoc-Gln-Exatecan

[0142] 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. DMF (1 ml), DIEA (29 mg 1.5 eq.), and TBTU (72.5 mg 1.1 eq.) were then added. 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.

[0143] The reaction formula is as follows:

[0144] 2. Synthesis of Intermediate 2 Gln-Exatecan

[0145] 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.

[0146] The reaction formula is as follows:

[0147] 3. Synthesis of Intermediate 3: Fmoc-Ala-Ala-Gln-Exatecan

[0148] 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.

[0149] The reaction formula is as follows:

[0150] 4. Synthesis of Intermediate 4: Ala-Ala-Gln-Exatecan

[0151] 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.

[0152] The reaction formula is as follows:

[0153] 5. Product Synthesis MCC-Ala-Ala-Gln-Exatecan

[0154] 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.

[0155] The reaction formula is as follows:

[0156] 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).

[0157] Example 3 Preparation of 5T4-ADC

[0158] 1. Preparation of Antibody Conjugates 5T4 Antibody-vcMMAE and 5T4 Antibody-MC-MMAF

[0159] a. Take a 5T4 antibody (such as 14G12, 14G12z28, or 14G12z43) 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 partially reduce the interchain disulfide bonds.

[0160] b. Add an excess of MC-vc-PAB-MMAE solution (produced by Shanghai Meiyake Biotechnology Co., Ltd. and commissioned by Hubei Huashitong Biopharmaceutical Technology Co., Ltd., dissolved in DMSO) or MC-MMAF solution (purchased from Borui Pharmaceutical Biology (Suzhou) Co., Ltd., 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, 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.

[0161] 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 conjugates 14G12-vcMMAE, 14G12-MC-MMAF, 14G12z28-MC-MMAF, and 14G12z43-MC-MMAF, which were then stored at 4°C.

[0162] d. The protein concentration of the antibody-drug conjugate was determined by UV / BCA. The DAR was determined by HIC (Figures 2 to 5). The DAR values ​​were 3.8, 3.8, 4.2, and 4.1, respectively. Purity was determined by SEC.

[0163] 2. Preparation of Antibody Conjugates 5T4 Antibody-MC-AAN-Exatecan and 5T4 Antibody-MCC-AAQ-Exatecan

[0164] a. Take a 5T4 antibody, such as 14G12z28, and adjust the pH of the antibody to approximately 7.5 with Tris-EDTA solution. Measure the protein concentration using a Nanodrop, weigh the antibody solution, and calculate the total protein content. Add TCEP solution to the antibody and place on a 3D shaker at room temperature for at least 120 minutes, mixing continuously to partially reduce the interchain disulfide bonds.

[0165] b. Add an excess of MC-AAN-Exatecan solution or MCC-AAQ-Exatecan (dissolved in DMSO) to the reduced antibody solution. Mix thoroughly, then 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. Incubate on a 3D shaker at room temperature for at least 30 minutes with continuous mixing.

[0166] 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 conjugates 14G12z28-MC-AAN-Exa and 14G12z28-MCC-AAQ-Exa. Store at 4°C.

[0167] 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 Figures 6 and 7). The DARs were 8.0 and 8.0, respectively. The purity was detected by SEC.

[0168] Example 4 Pharmacological and pharmacodynamic studies of 5T4-ADC

[0169] 1. Cell-binding activity of 5T4-ADC

[0170] FACS experiments confirmed that the binding activity of ADC to cells expressing the target antigen was basically unaffected before and after antibody conjugation.

[0171] Collect NCI-H1975 lung cancer cells or HCT116 colorectal cancer cells in logarithmic phase growth. After centrifugation, resuspend the cells in FACS buffer (PBS + 3% FBS), adjust the cell density, and plate the cells into 96-well U-bottom cell culture plates, with 200,000 to 500,000 cells per well. Add the antibody or ADC diluted fourfold in FACS buffer and mix thoroughly, achieving a final concentration of 10 μg / mL for each sample. Incubate the 96-well plate at 4°C for 45-90 minutes. Wash the cells thoroughly with FACS buffer to remove unbound antibody or ADC. Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 or 647 (Invitrogen, #A-11013 or #A-21445), diluted 1:1,000 in FACS buffer was added and incubated at 4°C in the dark for 30–60 min. The cells in the wells were washed thoroughly 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 Figures 8 and 9. The chimeric antibody 14G12 and the humanized antibody 14G12z28 showed no significant changes in binding affinity for 5T4-expressing NCI-H1975 or HCT116 cells before and after conjugation with the cytotoxin, indicating that conjugation of the antibody to the toxin has little effect on its cell-binding activity. The binding affinity of each test substance for tumor cells is shown in Table 2 and Figures 8 and 9.

[0172] Table 2: Binding of 5T4 antibody and ADC to tumor cells (EC 50 )

[0173] 2. Internalization of Antibodies in 5T4-ADC

[0174] Confirm the cellular internalization ability of 5T4-targeting mAbs and their ADCs.

[0175] HCT116 colorectal cells were harvested from logarithmically growing cells. After centrifugation, the cells were washed once and resuspended in pre-chilled cell culture medium containing 3% FBS. The cell density was adjusted and the cells were plated into 96-well U-bottom cell culture plates, with 500,000 to 800,000 cells per well. Antibodies or ADCs diluted in DMEM medium containing 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, centrifuged, and resuspended in cell culture medium. One 96-well plate was placed on ice, while the other three were incubated in a 37°C cell culture incubator. After 0.5, 1, and 2 hours, one of the 37°C plates was transferred to ice. After the third plate has been transferred to ice for 5–10 minutes, add diluted Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Invitrogen, #A-21445) and incubate on ice in the dark for 30–60 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 GeoMean Fluorescence Intensity (MFI). The entire experiment was maintained on ice or at 4°C, except for incubation at 37°C.

[0176] The percentage reduction of cell surface molecules was calculated as follows:

[0177] % reduction of cell surface molecules = (MFI 冰上 -MFI 37℃ ) / MFI 冰上 *100%.

[0178] The experimental results are shown in Table 3. The internalization ability of the humanized antibody 14G12z28 targeting 5T4 in HCT116 cells did not change before and after conjugation with the cytotoxin, indicating that the conjugated toxin has no significant effect on the cellular internalization of the antibody. The internalization results of each test substance in HCT116 cells (expressed as the percentage reduction of cell surface molecules) are shown in Table 3 and Figure 10.

[0179] Table 3: Internalization of 5T4 antibody and ADC on HCT116 cells

[0180] Example 5 In vitro pharmacodynamic study

[0181] The cell-killing activity of 5T4-targeting ADC molecules conjugated with various linker-payloads was evaluated.

[0182] Tumor 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 200 ng / mL or 10,000 ng / mL. The cells were cultured in a cell culture incubator for 4 days (MMAE or MMAF ADC) or 6 days (Exatecan ADC), 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.

[0183] 1. Experimental reagents and sources:

[0184] Table 4: Tested drugs

[0185] Table 5: Cell lines used in cell killing activity experiments

[0186] 2. Experimental results:

[0187] EC of each 5T4-ADC cell killing activity 50 The average values ​​are shown in Table 6. Figures 11 to 13 are representative graphs showing the killing of tumor cells by different 5T4-ADCs.

[0188] Table 6: EC values ​​of different 5T4-ADCs for cell killing activity in tumor cells 50 value

[0189] From the results in Table 6 and Figures 11 to 13 , it can be seen that the ADCs of the present invention formed by the toxin-conjugated 5T4 antibody all exhibited strong cell-killing activity.

[0190] Example 6 In vivo pharmacodynamic study

[0191] The in vivo antitumor activity of different 5T4-ADCs was tested in the CRC#047PDX mouse model.

[0192] The CRC#047PDX model was established by FACS analysis to confirm that the cells in the model were 5T4 positive. 3 The tumor tissue was transplanted subcutaneously on the right side of the back of BALB / c nude mice. 3 The mice were randomly divided into groups using the randomized block method, with grouping date designated Day 0. Each group consisted of six mice, ensuring uniform tumor volume and body weight across groups. Four groups were administered: vehicle, a non-binding MC-AAN-Exa (10 mg / kg) control, and 14G12z28-MC-AAN-Exa (10 and 3 mg / kg). The drug was administered via the tail vein on Day 0 and Day 7. The "non-binding" designation 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.

[0193] Data analysis: During the experiment, tumor volume was measured twice a week. The calculation formula of tumor volume (TV) is: TV = l × w 2 / 2. Where l and w represent the length and width of the tumor, respectively. The relative tumor volume (RTV) is calculated based on the measurement results. RTV = V f / V0. 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.

[0194] The experimental results are shown in Figures 14 and 15. On Day 28, the relative tumor growth rate (T / C%) in the 14G12z28-MC-AAN-Exa (10 mg / kg) group was 10.02% (P < 0.0001); the relative tumor growth rate (T / C%) in the 14G12z28-MC-AAN-Exa (3 mg / kg) group was 13.28% (P < 0.0001); and the relative tumor growth rate (T / C%) in the non-binding MC-AAN-Exa (10 mg / kg) control group was 55.54% (P > 0.05). Compared with Day 0, the average body weight change of mice in each group on Day 28 ranged from 3.74% to 5.21%.

[0195] The results showed that 14G12z28-MC-AAN-Exa significantly inhibited tumor growth at doses of 3 mg / kg and 10 mg / kg. Tumor-bearing mice showed good tolerance to all test products.

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-5T4 antibody or an antigen-binding fragment thereof, wherein the anti-5T4 antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region CDR1 comprises the sequence shown in SEQ ID NO: 3, The heavy chain variable region CDR2 comprises the sequence shown in SEQ ID NO: 4, The heavy chain variable region CDR3 comprises the sequence shown in SEQ ID NO: 5, The light chain variable region CDR1 comprises the sequence shown in SEQ ID NO: 6, The light chain variable region CDR2 comprises the sequence shown in SEQ ID NO: 7, The light chain variable region CDR3 comprises the sequence shown in SEQ ID NO: 8; L is the connector; D is cytotoxin; p is any value between 1 and 10.

2. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: The linker is selected from: MC-AAN, MCC-AAQ, 6-maleimidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), maleimidopropionyl (MP), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), 4-(N-maleimidomethyl)-cyclohexane-1-carboxyl (MCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB); Preferably, the linker is selected from: MC-AAN, MCC-AAQ, 6-maleimidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB).

3. The antibody drug conjugate according to any one of claims 1 to 2, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: The cytotoxin is selected from: Topoisomerase inhibitors, Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), SN-38, Gemcitabine, Maytansine alkaloids (e.g., Maytansine DM1, Maytansine DM4), calicheamicin, MGBA (e.g., duocarmycin), doxorubicin, ricin, diphtheria toxin, Duocarmycin SA, I131, interleukins, tumor necrosis factor, chemokines, and nanoparticles; Preferably, the cytotoxin is selected from: Exatecan, Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF).

4. The antibody drug conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: LD is selected from: MC-AAN-Exa, MCC-AAQ-Exa, MC-vc-PAB-MMAE, MC-MMAF.

5. The antibody drug conjugate according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: The anti-5T4 antibody has a technical feature selected from the following (i)-(ii): (i) the heavy chain variable region of the anti-5T4 antibody comprises the sequence shown in SEQ ID NO: 9, and the light chain variable region of the anti-5T4 antibody comprises the sequence shown in SEQ ID NO: 10; (ii) the heavy chain variable region of the anti-5T4 antibody comprises the sequence shown in SEQ ID NO: 11, and the light chain variable region of the anti-5T4 antibody comprises the sequence shown in SEQ ID NO:

10.

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-5T4 antibody has one or more technical features selected from the following (i)-(ii): (i) the heavy chain constant region of the anti-5T4 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; (ii) The light chain constant region of the anti-5T4 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: p is any value between 2 and 8; Preferably, p is any value between 3 and 8 (eg, 3.8, 4.1, 4.2, 7.9 or 8.0).

8. A pharmaceutical composition comprising 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; 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.

9. Use of 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, or the pharmaceutical composition according to claim 8 in the preparation of a medicament for preventing and / or treating a disease associated with 5T4; Preferably, the disease associated with 5T4 is selected from pancreatic cancer, prostate cancer, ovarian cancer, mesothelioma, lung cancer (such as non-small cell lung cancer), breast cancer, head and neck cancer, cervical cancer, kidney cancer, gastric cancer, colorectal cancer (such as colon cancer), gastric cancer, bladder cancer, thyroid cancer, lymphoma, acute myeloid leukemia.

10. 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 7, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the pharmaceutical composition according to claim 8, wherein the tumor is a tumor expressing 5T4; Preferably, the 5T4-expressing tumor is selected from pancreatic cancer, prostate cancer, ovarian cancer, mesothelioma, lung cancer (such as non-small cell lung cancer), breast cancer, head and neck cancer, cervical cancer, kidney cancer, gastric cancer, colorectal cancer (such as colon cancer), gastric cancer, bladder cancer, thyroid cancer, lymphoma, acute myeloid leukemia.

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