WO2025040000A1 - 5t4 antibody-drug conjugate and use thereof - Google Patents

Abstract

The present application relates to a 5T4 antibody-drug conjugate and a use thereof, and specifically provides an antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof or a solvate of the salt. The antibody-drug conjugate has a structure as 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 a good application prospect. Ab-(L-D)p Formula I

Description

5T4 antibody-drug conjugate and its application

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on and claims priority to an application with CN application number 202311048671.0 and filing date August 18, 2023. The contents of the CN application are hereby introduced as a whole into this application.

Technical Field

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

Background Art

Oncofetal protein 5T4, also known as Trophoblast glycoprotein (TPBG) and Wnt activation inhibitory factor 1 (WAIF1), is a highly glycosylated transmembrane glycoprotein encoded by the TPBG gene. The molecular weight of TPBG protein is about 72kDa, containing 420 amino acids, composed 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 of 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-to-cell connections, cell morphology and movement, cell adhesion ability, and cell membrane integrity. 5T4 is relatively widely expressed during embryonic development, while normal tissues only exist in certain special epithelia, such as basal stratified squamous epithelium, glandular and ductal epithelium, as well as retinal secondary neurons and olfactory bulbs.

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. In colon cancer, gastric cancer or ovarian cancer, there is evidence that the expression of 5T4 is associated with a low cure rate of cancer. In tissues of non-small cell lung cancer, kidney cancer or pancreatic cancer, the expression of 5T4 is as high as more than 95%.

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.

In summary, 5T4 is highly expressed in a variety of solid tumor tissues, but rarely exists in normal mature tissues, and its correlation with tumor metastasis and poor prognosis makes it one of the ideal targets for tumor drugs. For this target, the clinical drugs under development involve a variety of immunotherapies including monoclonal antibodies, bispecific antibodies, trispecific antibodies, ADCs, tumor vaccines, CAR-raNK, etc. At present, the ADC drugs entering 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) is composed of 5T4-specific humanized antibody PF-06281192, non-cleavable maleimidohexyl (mc) linker and microtubule 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 auristatin derivative (AF-HPA). SYD-1875 is composed of a 5T4-specific engineered cysteine residue antibody HCP41C and a linker payload vc-seco-DUBA. According to reports, Pfizer terminated the clinical development of PF-06263507 because no objective response was observed with PF-06263507 and dose-limiting toxicity (DLT) mainly in the form of ocular toxicity was observed. In addition, there is no information on further clinical trials after SYD-1875 completed Phase 1 clinical trials. Therefore, it is necessary to further develop new, safe, and more effective antibody-drug conjugates targeting 5T4.

Summary of the invention

The inventors of the present application prepared a new anti-5T4 antibody-drug conjugate (ADC) through a large number of experiments and creative work, and confirmed that it has good biological activity, thereby completing the present invention.

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 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 (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).

In Formula I, LD indicates that the linker and the cytotoxin are connected by a covalent bond to form an LD molecule; Ab-(LD) _p indicates that p LD molecules are coupled to Ab by a covalent bond.

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 conjugate described herein may be an integer or a decimal. Whether it is an integer or a decimal, it refers to the average number of drug molecules coupled to each antibody. "p is any value between 1-10", which means that p can be any integer selected from 1-10 (including endpoints 1 and 10), or any decimal selected from 1-10. At the same time, those skilled in the art will understand that even if the same preparation method is used, the DAR values of antibody-drug conjugates prepared in different batches may not be exactly the same, for example, they may fluctuate within a range of no more than 0.5.

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

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).

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).

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

In some embodiments, the cytotoxin is selected from: topoisomerase inhibitors, Monomethyl auristatin 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 factor, chemokines and nanoparticles.

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.

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

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

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

When the above four LDs are coupled to Ab via covalent bonds, the succinimide at the end of the LD is coupled to the thiol group in the antibody. For example, when MC-AAN-Exa is coupled to Ab via covalent bonds, the structural formula of the formed ADC is as follows:

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

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

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.

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.

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.

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.

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

In some embodiments, p is any value between 2-8.

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

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.

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

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

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, Furlong, Aromasin), 5-FU plus folinic acid, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, etc., or a combination thereof.

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.

In some embodiments, the immunosuppressant is selected from: (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, sulelac, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, mizoribine, cyclophosphamide, fingolimod, etc.

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

In the fourth aspect of the present invention, 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.

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

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), gastric cancer, bladder cancer, thyroid cancer, lymphoma, acute myeloid leukemia.

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

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 tumor expressing 5T4.

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), gastric cancer, bladder cancer, thyroid cancer, lymphoma, acute myeloid leukemia.

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

Beneficial Effects

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).

2. The newly developed 5T4-ADC of the present invention showed significant pharmacological effects in inhibiting tumor cell growth in various tumor models (such as the human colorectal cancer nude mouse CRC#047PDX model).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1-1 shows the binding activity of chimeric antibody 14G12 to human 5T4-His protein and cynomolgus monkey 5T4-His protein;

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

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

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

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

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

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

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

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

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

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

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

FIG11 is a representative graph showing the killing of tumor cells MCF7 by different 5T4-ADCs;

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

FIG13 is a representative graph showing the killing of tumor cells MDA-MB-468 by different 5T4-ADCs;

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

FIG. 15 shows the effects of different 5T4-ADCs on body weight in CRC#047PDX mouse model.

DETAILED DESCRIPTION

The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

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. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

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

In the present invention, the term "antibody" refers to an immunoglobulin molecule generally composed of two pairs of identical polypeptide chains (each pair having a "light" (L) chain and a "heavy" (H) chain). The light chain of an antibody can be divided into two types: κ and λ. The heavy chain 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. In 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 ( _VH ) and a heavy chain constant region ( _CH ). The heavy chain constant region consists of three domains ( _CH1 , _CH2 and _CH3 ). Each light chain consists of a light chain variable region ( _VL ) and a light chain constant region ( _CL ). The light chain constant region consists of one domain _{, CL} . 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. The _VH and _VL regions can be further subdivided into regions of high variability called complementarity determining regions (CDRs). The variable regions of each heavy chain/light chain pair ( _VH and _VL ) 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.

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) containing minimal sequences derived from non-human immunoglobulins. Preferably, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues in the complementary determining regions (CDRs) of the recipient antibodies are replaced by CDR residues from non-human species (donor antibodies) such as mice, rats, or rabbits having the desired specificity, affinity, and capacity.

In addition, in humanization, it is also possible to mutate the amino acid residues in 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. For example, PCR-mediated mutations can be used to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions. In addition, the mutations in the CDRs are usually no more than one or two.

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, which 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 contain 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.

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 connected by a disulfide bridge on the hinge region; the term "Fab'fragment" means a fragment obtained after reducing the disulfide bonds connecting 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.

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 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., a 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 a complete binding site.

In the present invention, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, 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 useful 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, the scFv There may also be a disulfide bond between VH and VL. In certain embodiments of the present invention, scFv may form a di-scFv, which refers to two or more single scFvs in series to form an antibody. In certain embodiments of the present invention, scFv may form a (scFv) ₂ , which refers to two or more single scFvs in parallel to form an antibody.

In the present invention, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains of the same chain is used, 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 R.J. et al., Structure 2:1121-1123 (1994)).

In the present invention, the term "single-domain antibody (sdAb)" has the meaning generally 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) that retains the ability to specifically bind to the same antigen as the full-length antibody. Single-domain antibodies are also called nanobodies.

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.

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.

Antibody antigen-binding fragments (e.g., the antibody fragments 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 antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.

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)). In addition, 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; 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 the culture medium of recombinant host cells. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

In the present invention, algorithms used to determine the percentage of 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 the percentage of amino acid sequence homology of the present invention, using, for example, the parameters described in the literature or the default parameters. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI).

In the present invention, the mutant of the amino acid sequence refers to a sequence having a homology with the amino acid sequence of greater than 70%, such as greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, such as a sequence having 3, 2 or 1 substituted, deleted or added amino acids. 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.

A "substitution" variant is a variant in which at least one amino acid residue in the native sequence is removed and inserted into the same position by a different amino acid. The substitution may 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 may be located at consecutive sites. Similarly, an amino acid may be substituted by multiple residues, in which such variants include both substitutions and insertions. An "insertion" (or "addition") variant is a variant in which one or more amino acids are inserted into an amino acid at a specific position adjacent to a native sequence. Adjacent amino acids means connected to the α-carboxyl or α-amino functional group of the amino acid. A "deletion" variant is a variant in which one or more amino acids in the native amino acid sequence are removed. Typically, a deletion variant has one or two amino acids deleted in a specific region of the molecule.

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 full reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine thiol groups.

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, tris(hydroxymethyl)aminomethane salts. And, (ii) salts formed by 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, hydroiodides, etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkane sulfonates, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; aryl sulfonates, such as benzenesulfonates, p-toluenesulfonates, etc.; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; amino acid salts, such as glycine, trimethylglycine, arginine, ornithine, glutamate, aspartate, etc.

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.

In the present invention, solvate refers to these forms of the antibody drug conjugate of the present invention: the antibody drug conjugate forms a complex in a solid or liquid form by coordination with solvent molecules. Hydrate is a specific form of solvate, which has coordinated water molecules. In the present invention, hydrate is a preferred solvate.

Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredients are known or will be apparent to those skilled in the art based on the disclosure of the present invention. As described in REMINGTON’S PHARMACEUTICAL SCIENCES, Martin, E.W., ed., Mack Publishing Company, 19th ed. (1995), the method for preparing the pharmaceutical composition includes incorporating appropriate pharmaceutical excipients, carriers, diluents, etc., which are non-toxic to cells or mammals exposed thereto at the doses and concentrations used.

In the present invention, the pharmaceutical excipients refer to excipients and additives used in the production of drugs and the preparation of prescriptions. They refer to substances that have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations in addition to active ingredients. In addition to excipients, carriers, and stability improvement, pharmaceutical excipients also have important functions such as solubilization, solubilization, and sustained and controlled release. 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, 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, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc.; according to their route of administration, they can be divided into oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration and ocular administration, etc. The same pharmaceutical excipients can be used in drug preparations with different routes of administration and have different functions and uses.

In the present invention, the pharmaceutical composition can be prepared into various suitable dosage forms according to the administration route, 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, sprays, etc. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the antibody-drug conjugate of the present invention, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof.

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 in terms of partially or completely stabilizing or curing a disease and/or As a side effect of a disease, it can be therapeutic. As used herein, "treatment" encompasses any treatment of a patient's disease, including: (a) preventing the disease or symptoms from occurring in a patient who is susceptible to the disease or symptoms but has not yet been diagnosed with the disease; (b) inhibiting the symptoms of the disease, i.e., preventing its development; or (c) alleviating the symptoms of the disease, i.e., causing the disease or symptoms to regress.

In the present invention, "subject" refers to a vertebrate. In certain embodiments, a 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, a mammal refers to a human.

In the present invention, "effective amount" refers to an amount that is effective in achieving the desired therapeutic or preventive effect at the necessary dose and time. 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 induce the desired response in the individual. The therapeutically effective amount also encompasses the amount in which the therapeutic beneficial effects of the substance/molecule outweigh any toxic or harmful consequences. "Preventively effective amount" refers to an amount that is effective in achieving the desired preventive effect at the necessary dose and time. Usually, but not necessarily, since the preventive dose is used for the subject before the onset of the disease or in the early stages of the disease, the preventively effective amount will be lower than the therapeutically effective amount. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the size of the tumor; 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.

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.

The present invention is further explained below in conjunction with specific embodiments, but these embodiments are not intended to limit the scope of the present invention.

Example 1 Preparation of anti-5T4 chimeric antibody, humanization transformation and related performance testing

1. Anti-human 5T4 monoclonal antibody immunoscreening

In order to obtain monoclonal antibodies targeting human 5T4 protein, human 5T4-His protein was first used for primary immunization in SJL mice, Balb/c mice, and C57BL/6 mice, and then human 5T4-His protein or CHO-K1 cells overexpressing human 5T4 (CHOK1-hu5T4) were used for enhanced immunization in immunized mice. The antibody titer against human 5T4-his protein in the sera of immunized mice was detected by ELISA and FACS experiments. Hybridoma cells were prepared from immunized mice with high serum antibody titers. The binding activity of antibodies in hybridoma supernatants was detected by ELISA and FACS experiments, and hybridoma cells that could simultaneously bind to human 5T4-His protein (human 5T4) and cynomolgus monkey 5T4-His protein (cyno5T4) and specifically bind to CHOK1-hu5T4 cells were screened. The hybridoma clone 14G12 antibody obtained by screening was sequenced to obtain its variable region amino acid sequence, as shown in Table 1-1.

Table 1-1: 14G12 antibody variable region sequence

2. Binding activity of chimeric antibody 14G12

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

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

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

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

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 growing 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 ^TM , Invitrogen) diluted in FACS buffer was added and incubated at 4°C for 30 minutes in the dark. After washing the cells in the wells with FACS buffer, the cell samples were analyzed for fluorescence signals using MACSQuant Analyzer 16 flow cytometer (Miltenyi). The experimental results are shown in Figures 1-2. The chimeric antibody 14G12 has good binding affinity to CHOK1-hu5T4 cells.

3. Humanization of chimeric antibody 14G12

By comparing the variable region framework sequence of 14G12 antibody with the existing human IgG variable region framework sequence, a human sequence with a high degree of match was screened. The 14G12 antibody CDR sequence was transplanted into the selected human IgG variable region framework sequence, and after back mutation, a series of 14G12 humanized antibodies were obtained.

Through the SPR method, ELISA experiment and FACS experiment (the specific method is the same as before), 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.

Table 1-3: Humanized antibody variable region sequences

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

Table 1-4: Affinity of chimeric and humanized antibodies

As shown in Figure 1-3, the humanized antibodies 14G12z28 and 14G12z43 have comparable binding activity to the chimeric antibody 14G12 in terms of human 5T4-His protein (Figure 1-3A, B), and slightly better binding activity to CHOK1-hu5T4 cells than the chimeric antibody 14G12 (Figure 1-3C, D). In addition, the humanized antibody 14G12z28 has good binding affinity to MCF-7 cells (Figure 1-3E).

Example 2 Preparation of Linker-Payload

1. Preparation of MC-AAN-Exatecan

1. Synthesis of Intermediate 1

Fmoc-Ala-OH (N-fluorenylmethoxycarbonyl-L-alanine, CAS No.: 35661-39-3) was activated by 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 are: Fmoc-Ala-OH (3 g, 1.0 eq) and HOSu (1.45 g, 1.3 eq) were added to the reaction bottle, 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 then the reaction was carried out at room temperature, monitored by HPLC, and 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, DMF (12 mL) was added to dissolve the product, filtered, and passed through prep-HPLC (preparative high performance liquid chromatography). After the preparation solution was concentrated until no obvious droplets flowed out, it was extracted with ethyl acetate, and the ethyl acetate phase was concentrated to dryness to obtain intermediate 1. The reaction formula is as follows:

2. Synthesis of Intermediate 2

The intermediate 1 (N-[fluorenylmethoxycarbonyl]-L-alanyl-L-alanine, CAS No.: 87512-31-0) is activated by HOSu (N-hydroxysuccinimide, CAS No.: 6066-82-6), and then reacted with L-Asn (L-asparagine, CAS No.: 70-47-3) to obtain the intermediate 2; the specific steps are: adding the intermediate 1 (0.87 g, 1.0 eq), HOSu (0.34 g, 1.3 eq), and THF (9 mL) into the reaction flask, controlling the temperature at room temperature, slowly adding DCC (0.61 g, 1.3 eq) under stirring conditions, reacting at room temperature, and monitoring by HPLC. After the reaction is completed, the reaction solution is filtered, and the filter cake is rinsed with THF (2 mL). Purified water (10 mL) was added to the filtrate, and L-Asn (0.34 g, 1.1 eq) and solid sodium bicarbonate (0.19 g, 1.0 eq) were added. 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:

3. Synthesis of Intermediate 3

After the intermediate 2 is added with DEA (diethylamine, CAS No.: 109-89-7) to remove Fmoc, the intermediate 3 is obtained; the specific steps are: add the intermediate 2 (100 mg) and DMF (1.5 mL) into the reaction bottle, control the temperature at room temperature, add DEA (300 μL) dropwise, react at room temperature, monitor by HPLC, until there is no residue of the intermediate 2, concentrate to remove DMF, add DCM (4 mL), purified water (4 mL), stir and separate, concentrate the aqueous phase to dryness to obtain the intermediate 3, the reaction formula is as follows:

4. Synthesis of Intermediate 4

Intermediate three reacts with 6-(maleimido)hexanoic acid succinimidyl ester (CAS No.: 55750-63-5) to obtain intermediate four; the specific steps are: adding intermediate three (92 mg, 1.0 eq), 6-(maleimido)hexanoic acid succinimidyl ester (135 mg, 1.3 eq), DMF (1.5 mL), DIPEA (0.059 mL, 1.0 eq) into a reaction flask, HPLC monitoring, after the reaction is completed, preparing and purifying, concentrating the preparation liquid, and obtaining intermediate four, the reaction formula is as follows:

5. Synthesis of Products

The intermediate 4 and exotecan mesylate (CAS No.: 169869-90-3) are subjected to condensation reaction to obtain the product; the specific steps are: under room temperature, the intermediate 4 (26 mg, 1.0 eq) is added to the reaction bottle, DMF (1.5 mL) is added, and exotecan mesylate (29.6 mg, 1.0 eq), EEDQ (20.7 mg,

1.5eq), HATU (31.8mg, 1.5eq), DMAP (0.7mg, 0.1eq), DIPEA (29.2μL, 3.0eq), react at room temperature. HPLC monitoring, after the reaction is completed, prepare and purify, concentrate the preparation solution to obtain the product, the reaction formula is as follows:

¹ H NMR (400MHz, DMSO-d ₆ )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),7.34-7.4 0(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).

2. Preparation of MCC-AAQ-Exatecan

1. Synthesis of Intermediate 1: Fmoc-Gln-Exatecan

Fmoc-Gln-OH (N-fluorenylmethoxycarbonyl-L-glutamine) (76.3 mg 1.1 eq.) and Exatecan (100 mg 1.0 eq.) were added to the reaction bottle, and DMF (1 ml), DIEA (29 mg 1.5 eq.), and TBTU (72.5 mg 1.1 eq.) were added. The reaction was carried out at room temperature and monitored by HPLC. No raw material remained. After the reaction was completed, it was purified by medium pressure column (DCM/MeoH). The product was collected and concentrated to obtain intermediate 1.

The reaction formula is as follows:

2. Synthesis of Intermediate 2 Gln-Exatecan

Add intermediate 1 Fmoc-Gln-Exatecan (130 mg 1.0 eq.) to the reaction bottle, add DCM (2 ml), and stir at room temperature. Add DEA (0.5 ml), react at room temperature, HPLC monitoring, no raw material remaining. Stop the reaction, slowly add MTBE (10 ml), a large amount of solid precipitates, stir for 30 min. Filter, wash with MTBE, obtain an off-white solid, and dry to obtain intermediate 2.

The reaction formula is as follows:

3. Synthesis of Intermediate 3: Fmoc-Ala-Ala-Gln-Exatecan

Add intermediate II Gln-Exatecan (100 mg 1.0 eq.) into a reaction bottle, 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, and monitor by HPLC until the reaction is complete. Purify by medium pressure column (DCM/MeOH), and collect the product to obtain intermediate III.

The reaction formula is as follows:

4. Synthesis of Intermediate 4: Ala-Ala-Gln-Exatecan

Add intermediate three Fmoc-Ala-Ala-Gln-Exatecan (140mg 1.0eq.) to the reaction bottle, add DCM (2ml), and stir at room temperature. Add DEA (0.5ml), react at room temperature, and monitor by HPLC until the reaction is completed. Slowly add MTBE (10ml), a large amount of solid precipitates, and stir for 30min. Filter, wash with MTBE, and obtain an off-white solid, which is dried to obtain intermediate four.

The reaction formula is as follows:

5. Product Synthesis MCC-Ala-Ala-Gln-Exatecan

Add intermediate 4 Ala-Ala-Gln-Exatecan (50mg 1.0eq.) to the reaction bottle, DMF (1mL), then add MCC (18mg 1.1eq.), DIEA (12.1mg 1.5eq.), react at room temperature, monitor by HPLC until there is no residue of intermediate 4, then stop the reaction. Purify by Prep-HPLC, collect the product, and concentrate to dryness to obtain the product.

The reaction formula is as follows:

¹ H NMR (400MHz, DMSO-d ₆ )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).

Example 3 Preparation of 5T4-ADC

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

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

b. Add an excess of MC-vc-PAB-MMAE solution (Shanghai Meiyake Biotechnology Co., Ltd. commissioned Hubei Huashitong Biopharmaceutical Technology Co., Ltd. to produce, dissolved in DMSO) or MC-MMAF solution (purchased from Borei Pharmaceutical Biology (Suzhou) Co., Ltd., dissolved in DMSO) to the reduced antibody solution, mix well, place on a 3D shaker, react at room temperature for more than 30 minutes, and mix continuously. After the reaction is completed, add an excess of N-acetylcysteine solution to the reaction solution, place on a 3D shaker, react at room temperature for more than 30 minutes, and mix continuously.

c. Use a 30KD ultrafiltration centrifuge tube to purify the conjugated product and replace it in the storage solution (10mM Histidine, pH 5.5 or so), with the replacement multiple greater than 1000 times. Then filter it with a 0.22um sterilizing filter to obtain antibody-drug conjugates 14G12-vcMMAE, 14G12-MC-MMAF, 14G12z28-MC-MMAF, and 14G12z43-MC-MMAF, which are stored at 4°C.

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 2 to 5). The DARs were 3.8, 3.8, 4.2 and 4.1, respectively. The purity was detected by SEC.

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

a. Take 5T4 antibody such as 14G12z28, adjust the pH of the antibody to about 7.5 with Tris-EDTA solution; use Nanodrop to detect protein concentration, weigh the net weight of the antibody solution, and calculate the total amount of protein. Add TCEP solution to the antibody, place on a 3D shaker, react for more than 120 minutes at room temperature, and continuously mix to partially reduce the disulfide bonds between the antibody chains.

b. Add excess MC-AAN-Exatecan solution or MCC-AAQ-Exatecan (dissolved in DMSO) to the reduced antibody solution, mix well, place on a 3D shaker, react at room temperature for more than 30 minutes, and continue to mix. After the reaction is completed, add excess N-acetylcysteine solution to the reaction solution, place on a 3D shaker, react at room temperature for more than 30 minutes, and continue to mix.

c. Use a 30KD ultrafiltration centrifuge tube to purify the coupling product and replace it in the storage solution (10mM Histidine, pH 5.5 or so), the replacement multiple is greater than 1000 times. Then filter it with a 0.22um sterilizing filter. The antibody drug conjugates 14G12z28-MC-AAN-Exa and 14G12z28-MCC-AAQ-Exa were obtained and stored at 4°C.

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.

Example 4 5T4-ADC pharmacology and pharmacodynamics study

1. Cell binding activity of 5T4-ADC

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

Collect NCI-H1975 lung cancer cells or HCT116 colorectal cancer cells growing in the logarithmic phase. After centrifugation, resuspend the cells in FACS buffer (PBS + 3% FBS), adjust the cell density, and divide the cells into 96-well U-bottom cell culture plates so that each well contains 200,000 to 500,000 cells. Add antibodies or ADCs diluted 4 times with FACS buffer and mix well so that the final concentration of each sample is 10μg/mL. Incubate the 96-well plate at 4℃ for 45 to 90 minutes. Wash the cells in the wells thoroughly with FACS buffer to remove unbound antibodies or ADCs. 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 fully washed with FACS buffer to remove unbound secondary antibodies. The cell samples were analyzed for fluorescence signals 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 did not show significant changes in their binding affinity to NCI-H1975 or HCT116 cells expressing 5T4 before and after coupling with cytotoxins, indicating that the antibody and toxin coupling basically did not affect their cell binding activity. The binding affinity of each test substance to tumor cells is shown in Table 2 and Figures 8 and 9 .

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

2. Internalization of Antibodies in 5T4-ADC

Confirm the cellular internalization ability of mAbs targeting 5T4 and their ADCs.

Collect HCT116 colorectal cells growing in the logarithmic phase. After centrifugation, wash the cells once, resuspend the cells in pre-cooled cell culture medium containing 3% FBS, adjust the cell density, and divide the cells into 96-well U-bottom cell culture plates so that each well contains 500,000 to 800,000 cells. Add antibodies or ADCs diluted in DMEM medium containing 3% FBS and mix well to make the final concentration of the sample 10μg/mL. Incubate the 96-well plate on ice for 60min. Wash the cells in the wells thoroughly with pre-cooled FACS buffer (PBS+3% FBS) to remove unbound antibodies or ADCs. Divide the cells in the wells equally into 4 96-well U-bottom plates, centrifuge, and resuspend the cells in cell culture medium. Place one 96-well plate on ice, and incubate the other three in a cell culture incubator at 37°C. After 0.5h, 1h, and 2h, transfer one culture plate at 37°C to ice. After the third culture plate was transferred to ice for 5-10 minutes, diluted Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Invitrogen, #A-21445) was added and incubated on ice in the dark for 30-60 minutes. The cells in the wells were washed with pre-cooled FACS buffer to remove unbound secondary antibody. The fluorescence signal of the cell samples was analyzed using CytoFLEX flow cytometer (Beckman Coulter), and MFI (GeoMean fluorescence intensity) was used to represent the fluorescence signal. Except for incubation, the whole experiment was kept on ice or at 4°C.

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

% reduction of cell surface molecules = (MFI _{on ice} - MFI _{at 37°C} ) / MFI _{on ice} * 100%.

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 coupling with cytotoxins, indicating that the coupling with toxins had no significant effect on the cell internalization of the antibody. The results of the internalization of each test substance on HCT116 cells (expressed as the percentage of cell surface molecule reduction) are shown in Table 3 and Figure 10.

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

Example 5 In vitro pharmacodynamic study

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

Collect tumor cells growing in the logarithmic phase. After centrifugation, resuspend the cells in new culture medium and count the cells. The cells were inoculated in a 96-well bottom transparent black cell culture plate (Costar) and cultured overnight in a cell culture incubator. On the second day, the ADC molecule was diluted 4-fold in a gradient with culture medium, and the dilution was carefully transferred to the black culture plate so that the final concentration of each sample was 200ng/mL or 10,000ng/mL. Cultured in a cell culture incubator for 4 days (MMAE or MMAF ADC) or 6 days (Exatecan ADC), then added 1/10 of the well volume of PrestoBlue reagent (Invitrogen) and incubated in a cell culture incubator for 1h. The fluorescence signal was read using a SpectraMax M5 plate reader, and the excitation and emission wavelengths of the instrument were set to 560nm and 590nm, respectively. The obtained fluorescence signal data was analyzed using SoftMax Pro 6.5 software.

1. Experimental reagents and sources:

Table 4: Tested drugs

Table 5: Cell lines used in cell killing activity experiments

2. Experimental results:

The average values of EC ₅₀ of each 5T4-ADC cell killing activity are shown in Table 6. Representative graphs of tumor cell killing.

Table 6: _EC50 values of cell killing activity of different 5T4-ADCs in tumor cells

It can be seen from the results in Table 6 and Figures 11 to 13 that the ADCs of the present invention formed by the 5T4 antibody coupled to toxin all exhibited strong cell killing activity.

Example 6 In vivo pharmacodynamic study

The in vivo antitumor activities of different 5T4-ADCs were tested in the CRC#047PDX mouse model.

FACS analysis confirmed that the cells of the CRC#047PDX model had positive expression of 5T4. The establishment process of the human colorectal cancer nude mouse CRC#047PDX model was as follows: tumor tissue with a volume of about 30 mm ³ was transplanted subcutaneously on the right side of the back of BALB/c nude mice. When the tumor volume reached 200-300 mm ³ , the mice were grouped by random block method, and the day of grouping was Day 0. There were 6 mice in each group to ensure that the tumor volume between groups was uniform and the body weight was taken into account. There were 4 groups in total, including the solvent group, the Non-binding-MC-AAN-Exa (10 mg/kg) control administration group and the 14G12z28-MC-AAN-Exa (10 and 3 mg/kg) administration group. On Day 0 and Day 7, the tail vein was administered once. Among them, Non-binding means that the antibody is a non-binding human IgG1 isotype control, 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.

Data analysis: During the experiment, the 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 measured length and width of the tumor, respectively. The relative tumor volume (RTV) was calculated based on the measurement results, RTV = V _f /V _0. Where V ₀ is the tumor volume measured at the time of group administration (i.e. Day 0), and V _f is the tumor volume measured on the last day. Relative tumor proliferation rate T/C (%) = (RTV of drug administration group/RTV of 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.

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

The experimental results showed that 14G12z28-MC-AAN-Exa had a significant inhibitory effect on tumor growth at a dose of 3 mg/kg and 10 mg/kg. Tumor-bearing mice had good tolerance to all the test products.

Claims (10)

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. 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). 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). 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 L-D is selected from: MC-AAN-Exa, MCC-AAQ-Exa, MC-vc-PAB-MMAE, MC-MMAF. 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): Sign: (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. 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. 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). 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. 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. 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.