5T4 Antibody-Drug Conjugate and Its Application

Abstract

The invention relates to a 5T4 antibody-drug conjugate and its application, and specifically provides an antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the antibody-drug conjugate has a structure shown in Formula I, wherein Ab is an anti-5T4 antibody. The antibody-drug conjugate has good tumor cell growth inhibition activity both in vivo and in vitro, and has good application prospects. Ab-(L-D)p Formula I.

Description

[0001] Cross - reference to related applications

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

[0003] The present invention relates to the field of biomedicine, and specifically relates to a 5T4 antibody - drug conjugate and its applications. Background art

[0004] The carcino - embryonic protein 5T4, also known as trophoblast glycoprotein (TPBG), Wnt - activated inhibitory factor 1 (WAIF1), is a transmembrane glycoprotein encoded by the TPBG gene with highly glycosylated N - terminus. The molecular weight of the TPBG protein is approximately 72 kDa, containing 420 amino acids, consisting of an extracellular domain of 310 amino acids, a transmembrane region of 20 amino acids, and a cytoplasmic domain of 44 amino acids. It can affect the reconstruction of the cytoskeleton and the integrity between cells by binding to proteins containing PDZ domains in the cytoplasm. Studies have shown that 5T4 is closely related to different physiological and pathological processes, such as cell - cell junctions, cell morphology and motility, cell adhesion ability, and the integrity of the cell membrane. 5T4 is relatively widely expressed during embryonic development, while in normal tissues, it is only present in certain special epithelia, such as the basal - layer stratified squamous epithelium, glands and duct epithelia, as well as secondary neurons in the retina 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, etc. There is evidence that the expression level of 5T4 is related to the low cure rate of cancer in colon cancer, gastric cancer, or ovarian cancer. In tissues of non - small cell lung cancer, kidney cancer, or pancreatic cancer, the expression of 5T4 is as high as over 95%.

[0006] Existing tumor - related research shows that 5T4 may play a role through the following three mechanisms: 1) epithelial - mesenchymal transition (EMT); 2) regulation of the CXCL12 / CXCR4 biological axis; 3) inhibition of Wnt signal transduction.

[0007] In summary, the high expression of 5T4 in various solid tumor tissues, its rarity in normal mature tissues, and its correlation with tumor metastasis and poor prognosis make it an ideal target for tumor drugs. For this target, the clinical drugs under research involve various immunotherapies including monoclonal antibodies, bispecific antibodies, trispecific antibodies, ADCs, tumor vaccines, CAR-raNK, etc. Currently, the ADC drugs in clinical research 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) consists of a 5T4-specific humanized antibody PF-06281192, an uncleavable maleimide hexyl (mc) linker, and a tubulin inhibitor monomethyl auristatin F (MMAF). ASN-004 consists of a 5T4-specific single-chain scFv-Fc antibody, a Dolaflexin drug linker (Mersana Therapeutics), and a tubulin inhibitor auristatin derivative (AF-HPA). SYD-1875 consists of an antibody HCP41C with 5T4-specific engineered cysteine residues and a linker payload vc-seco-DUBA. According to reports, Pfizer terminated the clinical development of PF-06263507 because no objective remission was observed and dose-limiting toxicity (DLT) mainly manifested as ocular toxicity was observed. In addition, there is no information on further clinical trials after SYD-1875 completed a Phase 1 clinical trial. Therefore, it is necessary to further develop new, safe, and more effective antibody-drug conjugates targeting 5T4. Summary of the Invention

[0008] Through a large number of experiments and creative work, the inventors of the present application prepared a new anti-5T4 antibody-drug conjugate (ADC) and confirmed its good biological activity, thus completing the present invention.

[0009] Therefore, 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, and the antibody-drug conjugate has the structure shown in Formula I,

[0010] Ab-(L-D) p

[0011] Formula I

[0012] Wherein:

[0013] Ab is an anti-5T4 antibody or an antigen-binding fragment thereof, and the anti-5T4 antibody comprises a heavy-chain variable region and a light-chain variable region.

[0014] The heavy-chain variable region CDR1 comprises the sequence shown in SEQ ID NO: 3.

[0015] The heavy-chain variable region CDR2 comprises the sequence shown in SEQ ID NO: 4.

[0016] The heavy-chain variable region CDR3 comprises the sequence shown in SEQ ID NO: 5.

[0017] The light-chain variable region CDR1 comprises the sequence shown in SEQ ID NO: 6.

[0018] The light-chain variable region CDR2 comprises the sequence shown in SEQ ID NO: 7.

[0019] The light-chain variable region CDR3 comprises the sequence shown in SEQ ID NO: 8.

[0020] L is a linker.

[0021] D is a cytotoxin.

[0022] p is any value between 1 and 10 (such as 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).

[0023] In formula I, L-D represents that the linker and the cytotoxin are covalently linked to form an L-D molecule; Ab-(L-D) p represents that p L-D molecules are covalently coupled to Ab.

[0024] In the present invention, the drug antibody ratio (DAR) refers to the number of drug molecules conjugated to the antibody (for example, p in Formula I). The number of drug molecules contained in the antibody-drug conjugate 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 conjugated to each antibody. "p is any value between 1 and 10" means that p can be any integer selected from 1 to 10 (including the endpoints 1 and 10), or any decimal selected from 1 to 10. At the same time, those skilled in the art can understand that even if the same preparation method is used, the DAR values of the antibody-drug conjugates prepared in different batches may not be exactly the same. For example, it can fluctuate within a range of not more than 0.5 up and down.

[0025] The drug antibody ratio (DAR) can be determined by conventional means, such as mass spectrometry, ELISA assay, HIC, and HPLC. The quantitative distribution of the ADC in terms of p can also be determined. In some cases, the isolation, purification, and verification of the homogeneous ADC with a certain value of p from the ADCs with other drug payloads can be achieved by means such as HIC, reverse-phase HPLC, or electrophoresis.

[0026] In some embodiments, the linker is selected from: MC-AAN, MCC-AAQ, 6-maleimidohexanoyl (MC), 6-maleimidohexanoyl-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).

[0027] In some embodiments, the linker is selected from: MC-AAN, MCC-AAQ, 6-maleimidohexanoyl (MC), 6-maleimidohexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB).

[0028] Among them, the structural formulas of MC, MCC, and MC-vc-PAB are shown as follows:

[0029]

[0030]

[0031] In some embodiments, the cytotoxin is selected from: topoisomerase inhibitors, Monomethylauristatin E (MMAE), Monomethyl auristatin F (MMAF), SN-38, Gemcitabine, maytansine alkaloids (such as Maytansine DM1, Maytansine DM4), calicheamicin, MGBA (such as duocarmycin), doxorubicin, ricin, diphtheria toxin, Duocarmycin SA, I131, interleukins, tumor necrosis factors, chemokines, and nanoparticles.

[0032] 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.

[0033] In some embodiments, the cytotoxin is selected from: Exatecan (abbreviated as Exa), Monomethylauristatin E (MMAE), Monomethyl auristatin F (MMAF).

[0034] In some embodiments, L-D is selected from: MC-AAN-Exa, MCC-AAQ-Exa, MC-vc-PAB-MMAE, MC-MMAF.

[0035] In some embodiments, the structural formula of L-D is as follows:

[0036]

[0037]

[0038] When the above four L-Ds are covalently coupled to Ab, they are formed by coupling the succinimide at the end of L-D with the thiol group in the antibody. For example, when MC-AAN-Exa is covalently coupled to Ab, the structural formula of the formed ADC is as follows:

[0039]

[0040] Among them, in the ADC formed above, the antibody Ab is connected to the carbon atom of the succinimide at the L-D end through -S-. This -S- is not a mercapto group introduced into Ab additionally, but a mercapto group contained in the antibody itself after the disulfide bond of the antibody Ab is reduced and opened.

[0041] In some embodiments, the sequence of CDR1 of the heavy chain variable region of the anti-5T4 antibody is as shown in SEQ ID NO: 3, the sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO: 4, the sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO: 5, the sequence of CDR1 of the light chain variable region is as shown in SEQ ID NO: 6, the sequence of CDR2 of the light chain variable region is as shown in SEQ ID NO: 7, and the sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO: 8.

[0042] In some embodiments, the sequence of the heavy chain variable region of the anti-5T4 antibody is as shown in SEQ ID NO: 1, and the sequence of the light chain variable region of the anti-5T4 antibody is as shown in SEQ ID NO: 2.

[0043] In some embodiments, the sequence of the heavy chain variable region of the anti-5T4 antibody is as shown in SEQ ID NO: 9, and the sequence of the light chain variable region of the anti-5T4 antibody is as shown in SEQ ID NO: 10.

[0044] In some embodiments, the sequence of the heavy chain variable region of the anti-5T4 antibody is as shown in SEQ ID NO: 11, and the sequence of the light chain variable region of the anti-5T4 antibody is as shown in SEQ ID NO: 10.

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

[0046] In some embodiments, the light chain constant region of the anti-5T4 antibody is selected from human lambda constant region, kappa constant region or mutants of the above constant regions.

[0047] In some embodiments, p is any value between 2 and 8.

[0048] In some embodiments, p is any value between 3 and 8 (such as 3.8, 4.1, 4.2, 7.9 or 8.0).

[0049] In a 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 thereof, or a solvate of said salt.

[0050] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutical excipient.

[0051] In some embodiments, the pharmaceutical composition further comprises at least one of a chemotherapeutic drug, an immunotherapeutic drug, and an immunosuppressant for treating tumors.

[0052] In some embodiments, the chemotherapeutic drug is, for example, Adriamycin, cyclophosphamide, taxanes [such as paclitaxel (Taxol), docetaxel (Taxotere)], capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (Arimidex, Femara, Aromasin), 5-FU plus leucovorin, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, etc., or a combination thereof.

[0053] In some embodiments, the immunotherapeutic drug is, for example, a PD-1 monoclonal antibody (such as pembrolizumab, nivolumab), a PD-L1 monoclonal antibody (such as Atezolizumab), a TIGIT monoclonal antibody, a 4-1BB monoclonal antibody, a VEGFR2 monoclonal antibody (such as Ramucirumab, apatinib), a HER2 monoclonal antibody (such as trastuzumab, Trastuzumab biosimilar, Trastuzumab-dkst), etc., or a combination thereof.

[0054] In some embodiments, the immunosuppressant is selected from: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporine and fostriecin, etc.; (3) antimetabolites, such as azathioprine and 6-mercaptopurine, etc.; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as antilymphocyte globulin and OKT3, etc.; (5) alkylating agents, such as cyclophosphamide. In some specific embodiments, the immunosuppressant is, for example, methylprednisolone, prednisone, azathioprine, tacrolimus, daclizumab, basiliximab, cyclosporine, tacrolimus, sirolimus, mycophenolate mofetil, mizoribine, cyclophosphamide, fingolimod, etc.

[0055] In a third aspect of the present invention, the present invention provides the use of the foregoing antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the foregoing pharmaceutical composition in the preparation of a drug for preventing and / or treating 5T4-related diseases.

[0056] In a fourth aspect of the present invention, the present invention provides the foregoing antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the foregoing pharmaceutical composition for preventing and / or treating 5T4-related diseases.

[0057] In a fifth aspect of the present invention, the present invention provides a method for treating and / or preventing 5T4-related diseases, which includes: administering to a subject in need a therapeutically and / or prophylactically effective amount of the foregoing antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the foregoing pharmaceutical composition.

[0058] In some embodiments, the 5T4-related diseases are 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.

[0059] In some embodiments, the 5T4-related diseases are breast cancer, non-small cell lung cancer, and colorectal cancer.

[0060] In a sixth aspect of the present invention, the present invention provides a method for non-therapeutically inhibiting tumor angiogenesis, delaying tumor progression, inhibiting tumor growth or inhibiting tumor cell proliferation in vitro, which includes: contacting tumor cells with the foregoing antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the foregoing pharmaceutical composition, wherein the tumor is a 5T4-expressing tumor.

[0061] In some embodiments, the 5T4-expressing tumors are 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.

[0062] In some embodiments, the 5T4-expressing tumors are selected from breast cancer, non-small cell lung cancer, and colorectal cancer.

[0063] Beneficial effects

[0064] 1. The newly developed 5T4-ADC of the present invention exhibits strong cytotoxic effects in various tumor cells (such as breast cancer and non-small cell lung cancer).

[0065] 2. The newly developed 5T4-ADC of the present invention exhibits significant anti-tumor cell growth efficacy in various tumor models (such as the human colorectal cancer nude mouse CRC#047PDX model). BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1-1 Shows the binding activity of the chimeric antibody 14G12 to human 5T4-His protein and cynomolgus monkey 5T4-His protein;

[0067] Figure 1-2 Shows the binding activity of the chimeric antibody 14G12 to CHOK1-hu5T4 cells;

[0068] Figure 1-3 Shows the activity of the humanized antibodies 14G12z28 and 14G12z43 in binding to human 5T4-His protein and CHOK1-hu5T4 cells, and the activity of the humanized antibody 14G12z28 in binding to MCF-7 cells;

[0069] Figure 2 Shows the hydrophobic interaction chromatogram of 14G12-vcMMAE;

[0070] Figure 3 Shows the hydrophobic interaction chromatogram of 14G12-MC-MMAF;

[0071] Figure 4 Shows the hydrophobic interaction chromatogram of 14G12z28-MC-MMAF;

[0072] Figure 5 Shows the hydrophobic interaction chromatogram of 14G12z43-MC-MMAF;

[0073] Figure 6 Shows the hydrophobic interaction chromatogram of 14G12z28-MC-AAN-Exa;

[0074] Figure 7 Shows the hydrophobic interaction chromatogram of 14G12z28-MCC-AAQ-Exa;

[0075] Figure 8 Shows the binding affinity of each test substance to the tumor cell NCI-H1975;

[0076] Figure 9 Shows the binding affinity of each test substance to the tumor cell HCT116;

[0077] Figure 10 Shows the internalization results of each test substance on HCT116 cells;

[0078] Figure 11Representative figures showing the killing of tumor cells MCF7 by different 5T4-ADCs;

[0079] Figure 12 Representative figures showing the killing of tumor cells NCI-H1975 by different 5T4-ADCs;

[0080] Figure 13 Representative figures showing the killing of tumor cells MDA-MB-468 by different 5T4-ADCs;

[0081] Figure 14 Representative figures showing the in vivo anti-tumor activity of different 5T4-ADCs in the CRC#047PDX mouse model;

[0082] Figure 15 Representative figures showing the effect of different 5T4-ADCs on the body weight of the CRC#047PDX mouse model. Detailed implementation mode

[0083] The following will describe the implementation schemes of the present invention in detail in combination with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0084] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. And the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation steps used herein are all widely used terms and conventional steps in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0085] In the present invention, unless otherwise specified, any numerical range should be understood to include any value or any sub-range within the range.

[0086] In the present invention, the term "antibody" refers to an immunoglobulin molecule usually composed of two pairs of identical polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). The light chains of antibodies can be divided into two classes, κ and λ. The heavy chains can be divided into five types, μ, δ, γ, α or ε. According to the different heavy chains, antibodies can be divided into five classes, IgM, IgD, IgG, IgA and IgE. Within the light and heavy chains, 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 a heavy chain constant region (C H) consists of. The heavy chain constant region consists of 3 domains (C H 1, C H 2, and C H 3). Each light chain consists of a light chain variable region (V L ) and a light chain constant region (C L ). The light chain constant region consists of one domain C L . The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system such as C1q. V H and V L regions can also be further divided into regions with high variability (called complementarity-determining regions (CDRs)), which are interspersed with relatively conserved regions called framework regions (FRs). Each V H and V L consists of 3 CDRs and 4 FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (V H and V L ) of each heavy chain / light chain pair form the antibody binding site respectively. The assignment of amino acids to each region or domain follows the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0087] In the present invention, a "humanized" antibody refers to a non-human (e.g., murine) antibody form that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody), containing the minimal sequence derived from a non-human immunoglobulin. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody), wherein the residues of the complementarity-determining regions (CDRs) of the recipient antibody are replaced by the CDR residues from a non-human species (donor antibody) such as a mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0088] In addition, in humanization, amino acid residues within the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may also be mutated, thereby improving one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, by PCR-mediated mutagenesis, and the effects of such mutations on antibody binding or other functional properties can be evaluated using the in vitro or in vivo assays described herein. Generally, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, mutations within the CDRs typically do not exceed one or two.

[0089] In the present invention, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as the "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated 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, diabody, single-domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), probody, and such polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0090] In the present invention, the term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "Fab fragment" means an antibody fragment consisting of the 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 in the hinge region; the term "Fab’ fragment" means the fragment obtained by reducing the disulfide bond linking the two heavy-chain fragments in the F(ab’)2 fragment, and consists of a complete light chain and the Fd fragment of the heavy chain (consisting of the VH and CH1 domains).

[0091] In the present invention, the term "Fv" means 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 capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for the antigen) can recognize and bind an antigen, although its affinity may be lower than that of the complete binding site.

[0092] In the present invention, the term "scFv" refers to a single polypeptide chain comprising the VL and VH domains, wherein the VL and VH are linked by a linker (see, e.g., 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 can 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 can also exist between the VH and VL of the scFv. In certain embodiments of the present invention, the scFv can form a di-scFv, which refers to two or more individual scFvs linked in series to form an antibody. In certain embodiments of the present invention, the scFv can form a (scFv)2, which refers to two or more individual scFvs linked in parallel to form an antibody.

[0093] 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 of the same chain, thus forcing the domains to pair with the complementary domains of the other chain and generating two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak R.J. et al., Structure 2:1121-1123 (1994)).

[0094] In the present invention, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, which refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy-chain variable region), which retains the ability to specifically bind the same antigen as the full-length antibody binds. Single-domain antibodies are also referred to as nanobodies.

[0095] 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 murine antibody and the constant region sequence is derived from a human antibody.

[0096] Each of the above antibody fragments retains the ability to specifically bind the same antigen as the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen.

[0097] Antigen-binding fragments of an antibody (e.g., the above antibody fragments) can be obtained from a given antibody (e.g., the antibodies provided by the present invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as for the intact antibody.

[0098] 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)). Additionally, these antigen-binding fragments can also 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 directly obtained from host cells; the Fab' fragments can be chemically conjugated to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). Additionally, Fv, Fab, or F(ab')2 fragments can also be directly isolated from the culture broth of recombinant host cells. Those of ordinary skill in the art are fully aware of other techniques for preparing these antigen-binding fragments.

[0099] In the present invention, the algorithms for determining percent sequence homology and percent sequence similarity are, for example, the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acid. Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Using, for example, the parameters described in the literature or the default parameters, BLAST and BLAST 2.0 can be used to determine the percent amino acid sequence homology of the present invention. The software for performing BLAST analysis is available to the public through the National Center for Biotechnology Information (NCBI) of the United States.

[0100] In the present invention, mutants of the amino acid sequence refer to sequences having a homology greater than 70% with the amino acid sequence, such as greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence, such as sequences having 3, 2, or 1 amino acid substitutions, deletions, or additions. Preferably, the substituted, added, or deleted amino acids do not exceed 3 amino acids. More preferably, the substituted, added, or deleted amino acids do not exceed 2 amino acids. Most preferably, the substituted, added, or deleted amino acids do not exceed 1 amino acid.

[0101] "Substituted" variants are variants in which at least one amino acid residue in the native sequence has been removed and a different amino acid inserted in its place. The substitution can be single, where only one amino acid in the molecule is replaced; or multiple, where two or more amino acids in the same molecule are replaced. Multiple substitutions can be located at contiguous sites. Also, one amino acid can be replaced by multiple residues, where such variants include both substitution and insertion. "Inserted" (or "added") variants are variants in which one or more amino acids have been inserted adjacent to an amino acid at a particular position in a stretch of native sequence. Adjacent amino acid means linked to the α-carboxyl or α-amino functional group of the amino acid. "Deleted" variants are variants in which one or more amino acids have been removed from the native amino acid sequence. Typically, deleted variants have one or two amino acids deleted in a particular region of their molecule.

[0102] In certain embodiments, fewer than the theoretical maximum of drug modules are conjugated to the antibody in the conjugation reaction. Generally, antibodies do not contain many free and reactive cysteine thiol groups that can link drug modules; in fact, most cysteine thiol groups in antibodies are present as disulfide bridges. In certain embodiments, the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarboxyethylphosphine (TCEP) under partial or complete reducing conditions to generate reactive cysteine thiol groups.

[0103] In the present invention, the term "pharmaceutically acceptable salt" refers to: (i) salts formed by acidic functional groups present in the conjugates provided by the present invention with appropriate inorganic or organic cations (bases), 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, tris(hydroxymethyl)aminomethane salts. And, (ii) salts formed by basic functional groups present in the conjugates provided by the present invention with appropriate inorganic or organic anions (acids), and includes but is not limited to, hydrohalic acid salts such as hydrofluoric acid salts, hydrochloric acid salts, hydrobromic acid salts, hydroiodic acid salts, etc.; inorganic acid salts such as nitrate salts, perchlorate salts, sulfate salts, phosphate salts, etc.; lower alkanesulfonate salts such as methanesulfonate salts, trifluoromethanesulfonate salts, ethanesulfonate salts, etc.; arylsulfonate salts such as benzenesulfonate salts, p-benzenesulfonate salts, etc.; organic acid salts such as acetate salts, malate salts, fumarate salts, succinate salts, citrate salts, tartrate salts, oxalate salts, maleate salts, etc.; amino acid salts such as glycine salts, trimethylglycine salts, arginine salts, ornithine salts, glutamate salts, aspartate salts, etc.

[0104] 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 that provides a pharmaceutically acceptable anion, or by reacting a sufficient amount of an acidic substance with a suitable base that provides a pharmaceutically acceptable cation.

[0105] In the present invention, solvates refer to those forms of the antibody-drug conjugates of the present invention: complexes in solid or liquid form formed by the antibody-drug conjugates coordinating with solvent molecules. Hydrates are a specific form of solvates and have coordinated water molecules. In the present invention, hydrates are the preferred solvates.

[0106] Methods for preparing various pharmaceutical compositions containing a certain amount of the active ingredient are known or will be apparent to those skilled in the art from the disclosure of the present invention. As described in REMINGTON’S PHARMACEUTICAL SCIENCES, Martin, E.W., ed., Mack Publishing Company, 19th ed. (1995), the methods for preparing the pharmaceutical compositions include incorporating appropriate pharmaceutical excipients, carriers, diluents, etc., which are non-toxic to the cells or mammals exposed to them at the doses and concentrations employed.

[0107] In the present invention, the pharmaceutical excipients refer to excipients and additives used in the production of drugs and the preparation of prescriptions. They are substances that have been reasonably evaluated in terms of safety except for the active ingredients and are included in pharmaceutical preparations. In addition to shaping, serving as carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, solubilization assistance, sustained and controlled release, etc. They are important components that may affect the quality, safety, and effectiveness of drugs. According to their sources, they can be divided into natural products, semi-synthetic products, and fully synthetic products. According to their functions and uses, they can be divided into: solvents, propellants, solubilizers, solubilization aids, emulsifiers, coloring agents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, permeation promoters, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc.; according to their routes of administration, they can be divided into oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration, and ophthalmic administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations with different routes of administration and has different functions and uses.

[0108] In the present invention, the pharmaceutical composition can be made into various suitable dosage forms according to the route of administration. For example, tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder aerosols, sprays, etc. Among them, the pharmaceutical composition or the suitable dosage form can contain 0.01 mg to 1000 mg of the antibody-drug conjugate of the present invention, or its pharmaceutically acceptable salt, solvate, or solvate of the salt.

[0109] As used herein, the term "treatment" generally refers to obtaining the desired pharmacological and / or physiological effect. This effect can be prophylactic according to completely or partially preventing a disease or its symptoms; and / or can be therapeutic according to partially or completely stabilizing or curing a disease and / or side effects caused by the disease. "Treatment" as used herein encompasses any treatment of a patient's disease, including: (a) preventing a disease or symptoms from occurring in a patient who is susceptible to the disease or symptoms but has not been diagnosed with the disease; (b) inhibiting the symptoms of a disease, i.e., preventing its development; or (c) relieving the symptoms of a disease, i.e., causing the disease or symptoms to regress.

[0110] 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.

[0111] In the present invention, "effective amount" refers to the amount that effectively achieves the desired therapeutic or prophylactic effect at the required dosage and time. The "therapeutically effective amount" of the substance / molecule of the present invention may vary depending on factors such as the disease state, age, sex, and body weight of the individual, and the ability of the substance / molecule to elicit the desired response in the individual. The therapeutically effective amount also encompasses the amount where the therapeutic beneficial effects of the substance / molecule outweigh any toxic or harmful consequences. "Prophylactically effective amount" refers to the amount that effectively achieves the desired prophylactic effect at the required dosage and time. Generally but not necessarily, since the prophylactic dose is administered to a subject before the onset of the disease or in the early stage of the disease, the prophylactically effective amount will be lower than the therapeutically effective amount. In the case of cancer, the therapeutically effective amount of a drug may reduce the number of cancer cells; shrink the tumor volume; inhibit (i.e., slow down to a certain extent, preferably stop) the infiltration of cancer cells 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.

[0112] In the present invention, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0113] The present invention will be further explained below with reference to specific embodiments, but these embodiments do not limit the scope of the present invention.

[0114] Example 1 Preparation, Humanization and Related Performance Detection of Anti-5T4 Chimeric Antibody

[0115] 1. Immunoscreening of Anti-Human 5T4 Monoclonal Antibody

[0116] To obtain monoclonal antibodies targeting human 5T4 protein, initial immunizations were first carried out in SJL mice, Balb / c mice, and C57BL / 6 mice using human 5T4-His protein, and then booster immunizations were performed in the immunized mice using human 5T4-His protein or CHO-K1 cells overexpressing human 5T4 (CHOK1-hu5T4). Through ELISA experiments and FACS experiments, the antibody titers against human 5T4-his protein in the sera of immunized mice were detected. Immunized mice with high serum antibody titers were selected to prepare hybridoma cells. The binding activities of the antibodies in the hybridoma supernatants were detected through ELISA experiments and FACS experiments, and hybridoma cells that could bind both human 5T4-His protein (human 5T4) and cynomolgus monkey 5T4-His protein (cyno5T4), and specifically bind to CHOK1-hu5T4 cells were screened. The screened hybridoma clone 14G12 antibody was sequenced to analyze its variable region amino acid sequence, as shown in Table 1-1.

[0117] Table 1-1: Variable region sequence of 14G12 antibody

[0118]

[0119]

[0120] 2. Binding activity of chimeric antibody 14G12

[0121] The chimeric antibody 14G12 was constructed by linking the variable region sequence of the 14G12 antibody to the human IgG1 / Cκ backbone.

[0122] The affinity characteristics of the chimeric antibody 14G12 binding to human 5T4-His protein were detected by SPR method. The chimeric antibody 14G12 was immobilized on a Protein A chip, and the affinity of the chimeric antibody 14G12 binding to human 5T4-His protein was detected by fitting the binding curves under different antigen concentration conditions, as shown in Table 1-4.

[0123] The binding activities of the chimeric antibody 14G12 to human 5T4-His protein and cynomolgus monkey 5T4-His protein were detected by ELISA experiment. After coating a 96-well plate with human 5T4-His protein or cynomolgus monkey 5T4-His protein, serially diluted chimeric antibody 14G12 was added and incubated at room temperature for 60 min. After washing with PBST, mouse-anti-human IgG Fc antibody conjugated with HRP was added and incubated at room temperature for 30 min. After washing with PBST, TMB substrate was added and the absorbance at OD 450 nm was analyzed. The binding affinities of the chimeric antibody 14G12 to human 5T4-His protein and cynomolgus monkey 5T4-His protein are shown in Table 1-2 and Figure 1-1As shown, compared with the control antibody naptumomab, the chimeric antibody 14G12 has comparable human 5T4-His binding activity ( Figure 1-1 A) and significantly superior cynomolgus monkey 5T4-His binding activity ( Figure 1-1 B).

[0124] Table 1-2: Binding activity of chimeric antibody 14G12

[0125]

[0126] The binding activity of the chimeric antibody 14G12 to CHOK1-hu5T4 cells or MCF-7 cells was detected by FACS experiment. CHOK1-hu5T4 cells or MCF-7 cells in the logarithmic growth phase were collected. After centrifugation, the cells were resuspended in FACS buffer and dispensed into a 96-well U-bottom cell culture plate. Antibodies serially diluted with FACS buffer were added, and the cells were incubated at 4°C for 30 min. After washing the cells in the wells with FACS buffer, PE Goat anti-Human IgG Fc Secondary Antibody (eBioscience TM , Invitrogen) diluted with FACS buffer was added, and the cells were incubated in the dark at 4°C for 30 min. After washing the cells in the wells with FACS buffer, the fluorescence signals of the cell samples were analyzed using a MACSQuant Analyzer 16 flow cytometer (Miltenyi). The experimental results are as Figure 1-2 shown, and the chimeric antibody 14G12 has good binding affinity for CHOK1-hu5T4 cells.

[0127] 3. Humanization of chimeric antibody 14G12

[0128] By comparing the variable region framework sequences of the 14G12 antibody with existing human IgG variable region framework sequences, human sequences with high matching degrees were screened. The CDR sequences of the 14G12 antibody were transplanted into the selected human IgG variable region framework sequences, and after back-mutation modification, a series of humanized 14G12 antibodies were obtained.

[0129] By SPR method, ELISA experiment and FACS experiment (the 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 Table 1-3.

[0130] Table 1-3: Variable region sequences of humanized antibodies

[0131]

[0132]

[0133] As shown in Table 1-4, the affinities of the humanized antibodies 14G12z28 and 14G12z43 for binding to the human 5T4-His protein are slightly weaker than those of the chimeric antibody 14G12.

[0134] Table 1-4: Affinities of chimeric and humanized antibodies

[0135]

[0136] As Figure 1-3 shown, the activities of the humanized antibodies 14G12z28 and 14G12z43 for binding to the human 5T4-His protein are comparable to those of the chimeric antibody 14G12 ( Figure 1-3 A,B), and their activities for binding to CHOK1-hu5T4 cells are slightly better than those of the chimeric antibody 14G12 ( Figure 1-3 C,D). In addition, the humanized antibody 14G12z28 has good binding affinity for MCF-7 cells ( Figure 1-3 E).

[0137] Example 2 Preparation of linker-payload

[0138] I. Preparation of MC-AAN-Exatecan

[0139]

[0140] 1. Synthesis of Intermediate 1

[0141] After activating Fmoc-Ala-OH (N-Fluorenylmethoxycarbonyl-L-alanine, CAS No.: 35661-39-3) with HOSu (N-Hydroxysuccinimide, CAS No.: 6066-82-6), it reacts with L-Ala (L-alanine, CAS No.: 56-41-7) to obtain Intermediate 1; specifically, the steps are as follows: Add Fmoc-Ala-OH (3 g, 1.0 eq), HOSu (1.45 g, 1.3 eq) into the reaction flask, add 21 mL of THF, control the temperature at room temperature, and slowly add DCC (2.59 g, 1.3 eq) under stirring conditions, then carry out the reaction at room temperature, monitor with HPLC. After the reaction is completed, filter the reaction solution, and wash the filter cake with THF (6 mL). Add purified water (15 mL) to the filtrate, then add L-Ala (1.12 g, 1.3 eq), solid sodium bicarbonate (0.81 g, 1.0 eq), stir and react at room temperature, monitor with HPLC. After the reaction is completed, add citric acid (2.02 g, 1.0 eq) and stir, then extract the reaction solution with ethyl acetate, concentrate the organic phase, add DMF (12 mL) to dissolve the product, filter, and then pass through prep-HPLC (Preparative High Performance Liquid Chromatography). After concentrating the preparation solution until no obvious droplets flow out, extract with ethyl acetate, and concentrate the ethyl acetate phase to dryness to obtain Intermediate 1. The reaction formula is as follows:

[0142]

[0143] 2. Synthesis of Intermediate 2

[0144] After activating Intermediate 1 (N-[Fluorenylmethoxycarbonyl]-L-alanyl-L-alanine, CAS No.: 87512-31-0) with HOSu (N-Hydroxysuccinimide, CAS No.: 6066-82-6), it reacts with L-Asn (L-asparagine, CAS No.: 70-47-3) to obtain Intermediate 2; specifically, the steps are as follows: Add Intermediate 1 (0.87 g, 1.0 eq), HOSu (0.34 g, 1.3 eq), and THF (9 mL) into the reaction flask, control the temperature at room temperature, and slowly add DCC (0.61 g, 1.3 eq) under stirring conditions, then carry out the reaction at room temperature, monitor with HPLC. After the reaction is completed, filter the reaction solution, and wash the filter cake with THF (2 mL). Add purified water (10 mL) to the filtrate, add L-Asn (0.34 g, 1.1 eq), solid sodium bicarbonate (0.19 g, 1.0 eq), stir and react at room temperature, monitor with HPLC. After the reaction is completed, add citric acid monohydrate (0.48 g, 1.0 eq) and stir, concentrate the reaction solution to remove most of the solvent, prepare the purified residue. After concentrating the preparation solution until no obvious droplets flow out, extract with ethyl acetate, and concentrate the organic phase to dryness to obtain Intermediate 2. The reaction formula is as follows:

[0145]

[0146] 3. Synthesis of Intermediate III

[0147] After removing Fmoc from Intermediate II by adding DEA (diethylamine, CAS No.: 109 - 89 - 7), Intermediate III is obtained. The specific steps are as follows: Add Intermediate II (100 mg) and DMF (1.5 mL) to a reaction flask, control the temperature at room temperature, dropwise add DEA (300 μL), carry out the reaction at room temperature, monitor by HPLC until there is no remaining Intermediate II, concentrate to remove DMF, add DCM (4 mL) and purified water (4 mL), stir and then separate the layers. Concentrate the aqueous phase to dryness to obtain Intermediate III. The reaction formula is as follows:

[0148]

[0149] 4. Synthesis of Intermediate IV

[0150] Intermediate III reacts with N - succinimidyl 6 - (maleimido)hexanoate (CAS No.: 55750 - 63 - 5) to obtain Intermediate IV. The specific steps are as follows: Add Intermediate III (92 mg, 1.0 eq), N - succinimidyl 6 - (maleimido)hexanoate (135 mg, 1.3 eq), DMF (1.5 mL), and DIPEA (0.059 mL, 1.0 eq) to a reaction flask, monitor by HPLC. After the reaction is completed, carry out preparative purification and concentrate the preparation solution to obtain Intermediate IV. The reaction formula is as follows:

[0151]

[0152] 5. Synthesis of the Product

[0153] Intermediate IV and irinotecan mesylate (CAS No.: 169869 - 90 - 3) undergo a condensation reaction to obtain the product. The specific steps are as follows: Under room - temperature conditions, add Intermediate IV (26 mg, 1.0 eq) to a reaction flask, add DMF (1.5 mL), and successively add irinotecan mesylate (29.6 mg, 1.0 eq), EEDQ (20.7 mg,

[0154] 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), and carry out the reaction at room temperature. Monitor by HPLC. After the reaction is completed, carry out preparative purification and concentrate the preparation solution to obtain the product. The reaction formula is as follows:

[0155]

[0156] 11H NMR (400 MHz, DMSO-d6) δ 8.22 - 8.29 (d, J = 2.0 Hz, 1H), 7.94 - 8.05 (d, J = 2.0 Hz, 3H), 7.83 - 7.88 (d, J = 2.0 Hz, 1H), 7.75 - 7.82 (d, J = 2.0 Hz, 1H), 7.34 - 7.40 (m, 1H), 7.28 - 7.33 (m, 1H), 6.96 - 7.02 (d, J = 8.0 Hz, 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).

[0157] II. Preparation of MCC-AAQ-Exatecan

[0158]

[0159] 1. Synthesis of Intermediate 1: Fmoc-Gln-Exatecan

[0160] Add Fmoc-Gln-OH (N-Fluorenylmethoxycarbonyl-L-glutamine) (76.3 mg, 1.1 eq.), Exatecan (100 mg, 1.0 eq.) into a reaction flask, add DMF (1 ml), DIEA (29 mg, 1.5 eq.), TBTU (72.5 mg, 1.1 eq.), react at room temperature, monitor by HPLC until no raw materials remain. After the reaction is completed, purify by medium-pressure column chromatography (DCM / MeOH). Collect the product, concentrate to dryness to obtain Intermediate 1.

[0161] The reaction equation is as follows:

[0162]

[0163] 2. Synthesis of Intermediate 2: Gln-Exatecan

[0164] Add intermediate I Fmoc-Gln-Exatecan (130 mg, 1.0 eq.) to a reaction flask, add DCM (2 ml), and stir at room temperature. Add DEA (0.5 ml), react at room temperature, monitor by HPLC until no raw materials remain. Stop the reaction, slowly add MTBE (10 ml), a large amount of solid precipitates, stir for 30 min. Filter, wash with MTBE to obtain an off-white solid, dry to obtain intermediate II.

[0165] The reaction formula is as follows:

[0166]

[0167] 3. Synthesis of intermediate III Fmoc-Ala-Ala-Gln-Exatecan

[0168] Add intermediate II Gln-Exatecan (100 mg, 1.0 eq.) to a reaction flask, DMF (1 mL), Fmoc-Ala-Ala-OH (67.8 mg, 1.0 eq), TBTU (68.4 mg, 1.2 eq.), DIEA (34.4 mg, 1.5 eq.), react at room temperature, monitor by HPLC until the reaction ends. Purify by medium-pressure column chromatography (DCM / MeOH), collect the product to obtain intermediate III.

[0169] The reaction formula is as follows:

[0170]

[0171] 4. Synthesis of intermediate IV Ala-Ala-Gln-Exatecan

[0172] Add intermediate III Fmoc-Ala-Ala-Gln-Exatecan (140 mg, 1.0 eq.) to a reaction flask, add DCM (2 ml), and stir at room temperature. Add DEA (0.5 ml), react at room temperature, monitor by HPLC until the reaction ends. Slowly add MTBE (10 ml), a large amount of solid precipitates, stir for 30 min. Filter, wash with MTBE to obtain an off-white solid, dry to obtain intermediate IV.

[0173] The reaction formula is as follows:

[0174]

[0175] 5. Synthesis of product MCC-Ala-Ala-Gln-Exatecan

[0176] Add the intermediate tetra-Ala-Ala-Gln-Exatecan (50 mg, 1.0 eq.) into a reaction flask, add DMF (1 mL), then add MCC (18 mg, 1.1 eq.) and DIEA (12.1 mg, 1.5 eq.). React at room temperature and monitor by HPLC until the intermediate tetra is completely consumed, then stop the reaction. Purify by Prep-HPLC, collect the product, and concentrate to dryness to obtain the product.

[0177] The reaction formula is as follows:

[0178]

[0179] 1 H NMR (400 MHz, DMSO-d6) δ 8.17 - 8.26 (d, J = 2.0 Hz, 1H), 7.83 - 7.91 (d, J = 2.0 Hz, 2H), 7.65 - 7.74 (d, J = 2.0 Hz, 2H), 7.21 - 7.28 (d, J = 8.0 Hz, 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).

[0180] Example 3 Preparation of 5T4-ADC

[0181] 1. Preparation of antibody conjugates 5T4 antibody-vcMMAE and 5T4 antibody-MC-MMAF

[0182] a. Take 5T4 antibody (such as 14G12, 14G12z28 or 14G12z43), adjust the pH of the antibody to about 7.5 with Tris-EDTA solution; detect the protein concentration with Nanodrop, weigh the net weight of the antibody solution, and calculate the total amount of protein. Add TCEP solution to the antibody, place it on a 3D shaker, and react at room temperature for more than 120 min, continuously mixing to partially reduce the inter-chain disulfide bonds of the antibody.

[0183] b. Add an excessive amount of MC-vc-PAB-MMAE solution (produced by Hubei Huashitong Biomedical Technology Co., Ltd. commissioned by Shanghai Medyac Biotechnology Co., Ltd., dissolved in DMSO) or MC-MMAF solution (purchased from Borui Pharmaceutical Co., Ltd., dissolved in DMSO) to the reduced antibody solution. After mixing evenly, place it on a 3D shaker and react for more than 30 minutes at room temperature with continuous mixing. After the reaction, add an excessive amount of N-acetylcysteine solution to the reaction solution, place it on a 3D shaker, and react for more than 30 minutes at room temperature with continuous mixing.

[0184] c. Purify the conjugate using a 30KD ultrafiltration centrifugal tube and replace it into the storage solution (10 mM Histidine, pH around 5.5), with a replacement multiple greater than 1000 times. Then filter it through a 0.22 μm sterilizing filter to obtain the antibody-drug conjugates 14G12-vcMMAE, 14G12-MC-MMAF, 14G12z28-MC-MMAF, and 14G12z43-MC-MMAF, which are stored at 4°C.

[0185] d. Detect the protein concentration of the antibody-drug conjugate using the UV / BCA method, and perform DAR detection using HIC (as shown in Figures 2 to 5 ), with DAR values of 3.8, 3.8, 4.2, and 4.1 respectively, and perform purity detection using SEC.

[0186] 2. Preparation of antibody conjugates 5T4 antibody-MC-AAN-Exatecan and 5T4 antibody-MCC-AAQ-Exatecan

[0187] a. Take the 5T4 antibody such as 14G12z28, adjust the pH of the antibody to about 7.5 using Tris-EDTA solution; detect the protein concentration using Nanodrop, weigh the net weight of the antibody solution, and calculate the total protein amount. Add TCEP solution to the antibody, place it on a 3D shaker, and react for more than 120 minutes at room temperature with continuous mixing to partially reduce the inter-chain disulfide bonds of the antibody.

[0188] b. Add an excessive amount of MC-AAN-Exatecan solution or MCC-AAQ-Exatecan (dissolved in DMSO) to the reduced antibody solution. After mixing evenly, place it on a 3D shaker and react for more than 30 minutes at room temperature with continuous mixing. After the reaction, add an excessive amount of N-acetylcysteine solution to the reaction solution, place it on a 3D shaker, and react for more than 30 minutes at room temperature with continuous mixing.

[0189] c. The conjugate product was purified using a 30KD ultrafiltration centrifugal tube and replaced into the storage solution (10 mM Histidine, pH around 5.5), with a replacement multiple greater than 1000-fold. Then it was filtered through a 0.22 μm sterilizing filter to obtain the antibody-drug conjugates 14G12z28-MC-AAN-Exa and 14G12z28-MCC-AAQ-Exa, which were stored at 4°C.

[0190] d. The protein concentration of the antibody-drug conjugate was detected by the UV / BCA method, and the DAR was detected by HIC (as Figures 6 to 7 shown), with DAR values of 8.0 and 8.0 respectively, and the purity was detected by SEC.

[0191] Example 4 Pharmacology and pharmacodynamics study of 5T4-ADC

[0192] 1. Cellular binding activity of 5T4-ADC

[0193] It was confirmed by FACS experiment that the cellular binding activity of the ADC to the cells expressing the target antigen was basically unaffected before and after antibody conjugation.

[0194] NCI-H1975 lung cancer cells or HCT116 colorectal cancer cells in the logarithmic growth phase were collected. After centrifugation, the cells were resuspended in FACS buffer (PBS + 3% FBS), the cell density was adjusted, and the cells were dispensed into a 96-well U-bottom cell culture plate so that each well contained 200,000 - 500,000 cells. Antibody or ADC diluted 4-fold with FACS buffer was added and mixed well, so that the final starting concentration of each sample was 10 μg / mL. The 96-well plate was incubated at 4°C for 45 - 90 min. The cells in the wells were washed 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 with 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 as Figures 8 to 9 shown. The binding affinities of the chimeric antibody 14G12 and the humanized antibody 14G12z28 to NCI-H1975 or HCT116 cells expressing 5T4 did not show significant changes before and after conjugation with the cytotoxin, indicating that the antibody-cytotoxin conjugation basically did not affect its cellular binding activity. The binding affinities of each test substance to tumor cells are shown in Table 2 andFigures 8 to 9 as shown

[0195] Table 2: Binding of 5T4 antibody and ADC to tumor cells (EC 50 )

[0196]

[0197] 2. Internalization of the antibody in 5T4-ADC

[0198] Confirm the cellular internalization ability of the monoclonal antibody targeting 5T4 and its ADC.

[0199] Collect HCT116 colorectal cells in the logarithmic growth phase. After centrifugation, wash the cells once, resuspend the cells in pre-cooled cell culture medium containing 3% FBS, adjust the cell density, and aliquot the cells into a 96-well U-bottom cell culture plate so that each well contains 500,000 - 800,000 cells. Add the antibody or ADC diluted with DMEM medium containing 3% FBS and mix well to make the final concentration of the sample 10 μg / mL. Incubate the 96-well plate on ice for 60 min. Wash the cells in the wells thoroughly with pre-cooled FACS buffer (PBS + 3% FBS) to remove unbound antibody or ADC. Aliquot the cells in the wells into 4 96-well U-bottom plates. After centrifugation, resuspend the cells with cell culture medium. Keep 1 96-well plate on ice and incubate the other 3 plates in a 37 °C cell culture incubator. After 0.5 h, 1 h, and 2 h, transfer 1 plate incubated at 37 °C to ice. After the 3rd plate is transferred to ice for 5 - 10 min, add diluted Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Invitrogen, #A-21445) and incubate in the dark on ice for 30 - 60 min. Wash the cells in the wells with pre-cooled FACS buffer to remove unbound secondary antibody. Analyze the fluorescence signal of the cell samples using a CytoFLEX flow cytometer (Beckman Coulter), and express it as MFI (GeoMean fluorescence intensity). Keep the whole experimental process on ice or at 4 °C except for the incubation at 37 °C.

[0200] The calculation method for the percentage reduction of cell surface molecules is as follows:

[0201] Percentage reduction of cell surface molecules = (MFI 冰上 - MFI 37℃ ) / MFI 冰上 * 100%.

[0202] The experimental results are shown in Table 3. The internalization ability of the humanized antibody 14G12z28 targeting 5T4 towards HCT116 cells remained unchanged before and after conjugation with the cytotoxin, indicating that the conjugated toxin had no significant effect on the cellular internalization of the antibody. The internalization results (expressed as the percentage reduction of cell surface molecules) of each test substance on HCT116 cells are shown in Table 3 and Figure 10 as follows.

[0203] Table 3: Internalization of 5T4 antibody and ADC on HCT116 cells

[0204]

[0205] Example 5 In vitro Pharmacodynamic Study

[0206] The cytotoxic activities of ADC molecules targeting 5T4 conjugated with various linker-payloads were evaluated.

[0207] Tumor cells in the logarithmic growth phase were collected. After centrifugation, the cells were resuspended in fresh medium and counted. The cells were seeded in 96-well clear-bottom black cell culture plates (Costar) and cultured overnight in a cell incubator. The next day, the ADC molecules were serially diluted 4-fold with medium, and the diluted solutions were carefully transferred to the black culture plates such that the final starting concentrations of each sample were 200 ng / mL or 10,000 ng / mL. They were cultured in a cell 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, followed by incubation in the cell incubator for 1 h. The fluorescence signals were read using a SpectraMax M5 microplate reader, with the excitation and emission wavelengths of the instrument set at 560 nm and 590 nm, respectively. The obtained fluorescence signal data were analyzed using SoftMax Pro 6.5 software.

[0208] 1. Experimental reagents and sources:

[0209] Table 4: Test drugs

[0210]

[0211] Table 5: Cell lines used in the cytotoxic activity experiment

[0212]

[0213] 2. Experimental results:

[0214] The average values of the EC 50 of the cytotoxic activities of each 5T4-ADC are shown in Table 6. Figures 11 to 13Representative graphs of the killing of tumor cells by different 5T4-ADCs.

[0215] Table 6: EC of the cell killing activity of different 5T4-ADCs in tumor cells 50 Value

[0216]

[0217] As can be seen from Table 6 and Figures 11 to 13 the results, all the ADCs of the present invention formed by coupling the 5T4 antibody with the toxin showed strong cell killing activity.

[0218] Example 6 In vivo pharmacodynamic study

[0219] The in vivo antitumor activity of different 5T4-ADCs was tested in the CRC#047PDX mouse model.

[0220] The CRC#047PDX model was confirmed by FACS analysis to have 5T4 positive expression in its cells. The establishment process of the human colorectal cancer nude mouse CRC#047PDX model was as follows: Tumor tissue with a volume of approximately 30 mm 3 was transplanted subcutaneously into the right back of BALB / c nude mice. When the tumor volume reached 200 - 300 mm 3 , it was grouped by the randomized block method, and the day of grouping was recorded as Day 0. There were 6 mice in each group, ensuring that the tumor volumes among groups were uniform and taking into account the body weights. There were a total of 4 groups, including the vehicle group, the Non-binding-MC-AAN-Exa (10 mg / kg) control drug administration group, and the 14G12z28-MC-AAN-Exa (10 and 3 mg / kg) drug administration groups. Tail vein injection was performed once on Day 0 and Day 7. Among them, Non-binding represents the non-binding human IgG1 isotype control antibody, which has no binding to the target on the surface of the tumor cells used in the experiment and is used as a negative control antibody.

[0221] Data analysis: During the experiment, the tumor volume was measured twice a week. The calculation formula for the tumor volume (Tumor Volume, TV) was: TV = l × w 2 / 2. Where l and w represent the measured length and width of the tumor respectively. The relative tumor volume (relative tumor volume, RTV) was calculated based on the measurement results, and RTV = V f / V0. Where V0 is the tumor volume measured at the time of grouped drug administration (i.e., Day 0), and V fIt is the tumor volume measured on the last day. The relative tumor proliferation rate T / C(%) = (RTV of the dosing group / RTV of the Vehicle group) × 100%. When T / C(%) ≤ 40% and P < 0.05, the test article is considered to have a significant inhibitory effect on tumor growth.

[0222] The experimental results are as Figures 14 to 15 shown. At Day 28, the relative tumor proliferation rate T / C(%) of the 14G12z28-MC-AAN-Exa (10 mg / kg) dosing group was 10.02% (P < 0.0001); the relative tumor proliferation rate T / C(%) of the 14G12z28-MC-AAN-Exa (3 mg / kg) dosing group was 13.28% (P < 0.0001); the relative tumor proliferation rate T / C(%) of the Non-binding-MC-AAN-Exa (10 mg / kg) control dosing group was 55.54% (P > 0.05). Compared with Day 0, the average body weight change of each group of mice at Day 28 was in the range of 3.74% - 5.21%.

[0223] The experimental results show that 14G12z28-MC-AAN-Exa has a significant inhibitory effect on tumor growth at dosing doses of 3 mg / kg and 10 mg / kg. The tumor-bearing mice have good tolerance to all the test articles.

Claims

1. An antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the antibody-drug conjugate has the structure shown in Formula I, Ab-(LD) p Formula I in: Ab is an anti-5T4 antibody or an antigen-binding fragment thereof, wherein the anti-5T4 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, The sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO: 3, The sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO: 4, The sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 5, The sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 6, The sequence of the light chain variable region CDR2 is shown in SEQ ID NO: 7, The sequence of the light chain variable region CDR3 is shown in SEQ ID NO: 8; LD is selected from: MC-AAN-Exa, MCC-AAQ-Exa, MC-vc-PAB-MMAE, MC-MMAF; 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 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.

3. The antibody drug conjugate according to claim 1, 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, and IgE constant regions; (ii) The light chain constant region of the anti-5T4 antibody is selected from a human lambda constant region and a kappa constant region.

4. The antibody-drug conjugate according to claim 3, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: The IgG is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

5. The antibody drug conjugate according to claim 1, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: p is any value between 2 and 8.

6. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: p is any value between 3 and 8.

7. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: p is 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.

9. The pharmaceutical composition according to claim 8, wherein The pharmaceutical composition further comprises at least one pharmaceutical excipient.

10. The pharmaceutical composition according to claim 8, wherein The pharmaceutical composition also contains at least one of a chemotherapeutic drug, an immunotherapeutic drug and an immunosuppressant for treating tumors.

11. 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 any one of claims 8 to 10 in the preparation of a medicament for preventing and / or treating a disease associated with 5T4; The disease associated with 5T4 is selected from non-small cell lung cancer, breast cancer, and colorectal cancer.

12. The use according to claim 11, wherein The colorectal cancer is colon cancer.

13. An in vitro non-therapeutic method for 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 any one of claims 8 to 10, wherein the tumor is a tumor expressing 5T4; The 5T4-expressing tumor is selected from non-small cell lung cancer, breast cancer, and colorectal cancer.

14. The method of claim 13, wherein: The colorectal cancer is colon cancer.

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