An anti-trop2 antibody-drug conjugate and preparation method and use thereof

Abstract

The application provides an anti-TROP2 antibody-drug conjugate or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the heavy chain of the anti-TROP2 antibody comprises at least one non-natural amino acid residue as shown in formula (I). The application also provides a preparation method and uses of the antibody-drug conjugate. The anti-TROP2 antibody and auristatin toxin are conjugated at a specific site in the application, so that a brand new antibody-drug conjugate is obtained, which has very excellent biological activity and is obviously superior to existing similar products, and the preparation process is simple and easy to control.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to an anti-TROP2 antibody-drug conjugate, a preparation method and uses thereof, and further relates to a pharmaceutical composition comprising the anti-TROP2 antibody-drug conjugate and uses thereof. BACKGROUND

[0002] Malignant tumor is one of the major health problems in the world, and is also a major public health problem that seriously endangers human health and social development. In 2022, the global number of new cancer cases reached 20 million, and the number of deaths reached 9.7 million. For patients with advanced tumors with more serious disease progression, the traditional cytotoxic chemotherapy with poor selectivity and strong side effects is still the main treatment choice. Therefore, the development of targeted therapeutic drugs is of great significance to meet the treatment needs of such patients.

[0003] Molecular targeted therapy of tumor is to combine target drugs with corresponding targets, directly affect the function of target molecules, or carry effector molecules through the targeting of target molecules to kill or inhibit target cells. Due to the existence of a clear target, targeted drugs usually have high selectivity, and can effectively kill or inhibit target cells without or with only small toxic side effects on normal cells. As a new treatment model after traditional surgery, radiotherapy and chemotherapy, molecular targeted drugs have become one of the hotspots in clinical research of tumors.

[0004] Antibody-drug conjugate (ADC) is a kind of antibody or antibody fragment that can specifically recognize specific target points on the surface of tumor cells, connected with bioactive effector molecules through a linker. ADC reaches the surface of tumor cells through the guidance of antibody or antibody fragment, enters tumor cells through endocytosis, releases effector molecules in tumor cells, or directly releases effector molecules on the surface of tumor cells, achieving the effect of specifically killing tumor cells without damaging normal tissue cells.

[0005] ADC is usually composed of four parts: 1) antibody or antibody fragment with targeting function, used to recognize target cells and bring effector molecules to the surface or inside of cells; 2) cytotoxic or other effector small molecules, which play a role in killing cells or regulating cell function; 3) linker connecting antibody and effector molecule, which plays a connecting and supporting role; 4) linker and antibody conjugation linker with special active groups. The structure of each part of ADC affects the overall function of the molecule.

[0006] TROP2 is a human trophoblast cell surface glycoprotein antigen 2, also known as tumor-associated calcium signal transducer 2 (TACSTD2), epithelial glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA733-1), surface marker 1 (M1S1), which is a type I cell surface glycoprotein expressed by the TACSTD2 gene in the chromosome 1p32 region [CUBAS R et al., Biochim Biophys Acta, 1796(2):309-14, 2009]. The TROP2 protein is a 36 kD polypeptide composed of 323 amino acids, consisting of a hydrophobic leader peptide (AA 1-26), an extracellular domain (AA 27-274), a transmembrane domain (AA 275-297), and a cytoplasmic tail (AA 298-323). TROP2 and EpCAM (epithelial cell adhesion molecule, also known as TROP-1, KSA or GA733-2) have about 49% amino acid homology, and human and mouse TROP2 have 87% homology. The primary structure is modified by N-glycosylation to form a type I cell membrane glycoprotein different from EpCAM, i.e., TROP2 protein [VIDMAR T et al., Protein Expr Purif, 91(1):69-76, 2013]. TROP2 has important significance in the process of embryonic development and tumor cell proliferation and metastasis, and is overexpressed in various malignant tumors, such as pancreatic cancer, triple-negative breast cancer, non-small cell lung cancer, thyroid cancer, colon cancer, gastric cancer, ovarian cancer, and cervical cancer, etc., and can promote tumor cell proliferation, metastasis, and invasion, etc. Its high expression is closely related to the short survival period and poor prognosis of tumor patients, but the expression of TROP2 in adult normal tissues is very low, so the research of anti-tumor drugs targeting TROP2 has important significance.

[0007] At present, the ADC targeting TROP2, Sacituzumab Govitecan (Trodelvy) has been approved for marketing in many countries around the world, and is used for treating unresectable locally advanced or metastatic TNBC adult patients who have received at least two systemic treatments. In addition, SKB264 of Kolon Biotech and Datopotamab deruxtecan developed by Dailchi Sankyo have submitted marketing applications in China. The above three ADCs targeting TROP2 all use a DNA topoisomerase inhibitor, Camptothecin (CPT) derivative, as the effector small molecule. Compared with microtubule inhibitors, the IC50 value of Camptothecin and its derivatives is high, and in order to achieve the same tumor killing effect, as many effector small molecules as possible need to be coupled to each antibody molecule, but Camptothecin derivatives are highly hydrophobic, and high drug loading will lead to easy aggregation of ADC, thereby reducing drug stability, increasing toxicity and increasing immunogenicity. The above three ADCs targeting TROP2 all use a cleavable linker, which to some extent increases the risk of off-target toxicity. Moreover, the above three ADCs targeting TROP2 all use thiol coupling, and since there are multiple potentially coupled sulfhydryl groups in IgG antibodies, the coupling process control complexity is high, and the product uniformity is low. Therefore, there is a great space for further optimization of the ADC targeting TROP2. SUMMARY

[0008] In order to make up for the deficiencies in the prior art, one object of the present application is to provide an anti-TROP2 antibody-drug conjugate formed by site-specific coupling of an anti-TROP2 antibody and an Auristatin toxin, which can achieve very excellent biological activity, and the product is uniform, the preparation process is simple and easy to control.

[0009] Another object of the present application is to provide a preparation method and use of the anti-TROP2 antibody-drug conjugate.

[0010] The first aspect of the present application provides an anti-TROP2 antibody-drug conjugate or a stereoisomer, a pharmaceutically acceptable salt thereof, wherein the heavy chain of the anti-TROP2 antibody comprises at least one non-natural amino acid residue represented by formula (I),

[0011]

[0012] wherein X represents a C1-C6 linear or branched alkylene group, one or more -CH2- of the alkylene group is optionally replaced by one or more of -O-, -S-, -NH-;

[0013] L represents a C1-C6 linear or branched alkylene group;

[0014] D represents an auristatin toxin;

[0015] m represents an integer from 2 to 10;

[0016] represents a linking point in the amino acid sequence.

[0017] The anti-TROP2 antibody used in the anti-TROP2 antibody-drug conjugate provided by the present application can be any antibody and analog thereof commonly used in the art, as long as it has the desired biological activity.

[0018] The number of non-natural amino acid residues as shown in formula (I) in the anti-TROP2 antibody-drug conjugate provided by the present application can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In some preferred embodiments, the number of non-natural amino acid residues as shown in formula (I) can be 2, 4, 6 or 8, and the number on each heavy chain is the same. In some more preferred embodiments, each heavy chain contains 1 or 2 of the non-natural amino acid residues.

[0019] The anti-TROP2 antibody-drug conjugate provided by the present application can also be a mixture of various conjugates, so that the drug-antibody ratio (DAR) value in it can be a non-integer. In some preferred embodiments, the DAR value can be an integer or a fraction in the range of 1-10, such as 1-6.0, 1.2-5.5, 1.4-5.0, 1.5-4.5, 1.6-4.0, 1.7-3.5, 1.8-3.0, 1.8-2.5, 1.8-2.4, 1.8-2.3, 1.9-2.2, 1.9-2.1, 1.9-2.0, etc.

[0020] In some preferred embodiments, the amino acid sequences of the heavy chain and the light chain of the anti-TROP2 antibody provided by the present application are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, wherein one or more of the positions 122, 212 and 278 of the heavy chain is the non-natural amino acid residue. In some more preferred embodiments, the position 122 of each heavy chain of the anti-TROP2 antibody is the non-natural amino acid residue.

[0021] In the anti-TROP2 antibody-drug conjugate provided by the present application, D can represent any auristatin toxin commonly used in the art, which is an artificial synthetic derivative of the natural product dolastatin 10, belongs to an antimitotic agent, blocks tubulin polymerization by acting on the β-subunit of α-β tubulin dimer, promotes cell cycle arrest and apoptosis, and the auristatin toxin includes but is not limited to MMAE, MMAF, MMAD, etc. In some preferred embodiments, D can represent one of the following structures:

[0022]

[0023] wherein, represents the connection point with L.

[0024] In the anti-TROP2 antibody-drug conjugate provided by the present application, X can represent a C1-C4 straight chain alkylene, and one -CH2- of the alkylene can be optionally replaced by -O- or -NH-.

[0025] In the anti-TROP2 antibody-drug conjugate provided by the present application, X can further represent -CH2-O-, -O-CH2-, -CH2-, -CH2-O-CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-O-CH2-CH2-O-, -CH2-O-CH2-CH2-O-CH2-CH2-O-, -O-, -CH2-NH-CH2-, -NH-CH2- or -CH2-NH-.

[0026] In the anti-TROP2 antibody-drug conjugate provided by the present application, m can further represent 3, 4, 5 or 6.

[0027] In the anti-TROP2 antibody-drug conjugate provided by the present application, L can further represent a C1-C3 straight chain alkylene, for example, methylene (-CH2-), ethylene (-CH2-CH2-), propylene (-CH2-CH2-CH2-). In some preferred embodiments, L can represent methylene or ethylene.

[0028] The heavy chain of the anti-TROP2 antibody-drug conjugate provided by the present application preferably has the following structure:

[0029]

[0030] wherein, the direction of R1 to R2 is the N-terminal to C-terminal direction of the amino acid sequence, and R1 and R2 each independently represent a polypeptide, which together constitute the heavy chain of the anti-TROP2 antibody.

[0031] The second aspect of the present application provides a toxin derivative as shown in formula (I') or a stereoisomer, a pharmaceutically acceptable salt thereof,

[0032]

[0033] wherein X, L, D, m are each independently defined in the first aspect of the present application.

[0034] The first aspect of the present application provides an anti-TROP2 antibody-drug conjugate which is decomposed after being targeted into cells for endocytosis to form a toxin derivative carrying unnatural amino acids as shown in formula (I'), the toxin derivative has good physiological activity, for example, tumor cell inhibition or killing activity, and thus can be used for preparing a drug, for example, a drug for treating cancer.

[0035] The third aspect of the present application provides a preparation method of the anti-TROP2 antibody-drug conjugate according to any one of the above technical solutions, comprising the following steps:

[0036] S1: at least one unnatural amino acid as shown in formula (II) is inserted into the heavy chain of the anti-TROP2 antibody, to obtain a recombinant antibody;

[0037]

[0038] S2: the recombinant antibody obtained in step S1 is subjected to an oximation reaction with a toxin as shown in formula (III), to obtain the anti-TROP2 antibody-drug conjugate;

[0039] H2NO(CH2CH2O)m—L—D

[0040] Formula (III)

[0041] wherein X, L, D, m are each independently defined in the first aspect of the present application.

[0042] In the preparation method provided by the present application, the unnatural amino acid as shown in formula (II) contains a terminal carbonyl group, and the toxin as shown in formula (III) contains a hydroxylamine group, and the terminal carbonyl group reacts with the hydroxylamine group to form an oxime bond, thereby forming the antibody-drug conjugate.

[0043] In some preferred embodiments, the unnatural amino acid as shown in formula (II) can be further an unnatural amino acid as shown in formula (II'),

[0044]

[0045] wherein X is defined in the first aspect of the present application.

[0046] In some more preferred embodiments, the unnatural amino acid represented by formula (II) can be those described in Chinese patents ZL202110863816.7 and ZL 202110865364.6, for example, can be a compound having the following structure:

[0047]

[0048] In some preferred embodiments, the toxin as represented by formula (III) can be:

[0049]

[0050] In the preparation method provided by the present application, step S1 can be achieved by codon expansion technology or by chemical synthesis to insert at least one unnatural amino acid as represented by formula (II) into the heavy chain of the anti-TROP2 antibody to obtain a recombinant antibody. In some preferred embodiments, the codon expansion technology is achieved in mammalian engineering cells.

[0051] The fourth aspect of the present application provides a pharmaceutical composition comprising the anti-TROP2 antibody-drug conjugate or a stereoisomer, a pharmaceutically acceptable salt thereof according to the first aspect of the present application, and optionally one or more pharmaceutically acceptable excipients.

[0052] The fifth aspect of the present application provides a pharmaceutical composition comprising the toxin derivative or a stereoisomer, a pharmaceutically acceptable salt thereof according to the second aspect of the present application, and optionally one or more pharmaceutically acceptable excipients.

[0053] The sixth aspect of the present application provides the use of the anti-TROP2 antibody-drug conjugate or a stereoisomer, a pharmaceutically acceptable salt thereof according to the first aspect of the present application, the toxin derivative or a stereoisomer, a pharmaceutically acceptable salt thereof according to the second aspect of the present application, and the pharmaceutical composition according to the fourth aspect and the fifth aspect of the present application in the preparation of a medicament for treating cancer.

[0054] The seventh aspect of the present application provides a method for treating cancer, comprising administering a therapeutically effective dose of the anti-TROP2 antibody-drug conjugate or a stereoisomer, a pharmaceutically acceptable salt thereof according to the first aspect of the present application, the toxin derivative or a stereoisomer, a pharmaceutically acceptable salt thereof according to the second aspect of the present application, or the pharmaceutical composition according to the fourth aspect or the fifth aspect of the present application to a patient in need thereof.

[0055] The cancer described in the present application can be a common solid tumor and hematoma. In some preferred embodiments, the cancer can be pancreatic cancer, breast cancer (e.g., triple negative breast cancer), lung cancer (e.g., non-small cell lung cancer), urothelial carcinoma, thyroid cancer, colon cancer, gastric cancer, endometrial cancer, prostate cancer, ovarian cancer or cervical cancer.

[0056] The present application firstly site-couples an anti-TROP2 antibody and an auristatin toxin, thereby obtaining a brand-new antibody-drug conjugate, which at least has the following advantages:

[0057] (1) The anti-TROP2 antibody and the auristatin toxin are site-coupled through a non-natural amino acid, a fixed number of effector small molecules can be coupled on each antibody, the product is uniform, which is conducive to stable drug efficacy, and the preparation process is simple and easy to control.

[0058] (2) The antibody-drug conjugate provided by the present application has excellent biological activity, and the cancer cell inhibition effect in vivo and in vitro is obviously better than that of existing similar products, which has a very good application prospect as an anticancer drug. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 A schematic diagram of the expression plasmid pCDNA3.1-TROP2Mab-UAG122 prepared in Example 1.

[0060] Figure 2 A hydrophobic HPLC chromatogram of the anti-TROP2 monoclonal antibody-122 (NPAK)-P4AE conjugation reaction product prepared in Example 1.

[0061] Figure 3 A hydrophobic HPLC chromatogram of the anti-TROP2 monoclonal antibody-212 (NPAK)-P4AE conjugation reaction product prepared in Example 1.

[0062] Figure 4 A hydrophobic HPLC chromatogram of the anti-TROP2 monoclonal antibody-278 (NPAK)-P4AE conjugation reaction product prepared in Example 1.

[0063] Figure 5 A hydrophobic HPLC chromatogram of the anti-TROP2 monoclonal antibody-122 (NBGK)-P4AE conjugation reaction product prepared in Example 1.

[0064] Figure 6 A hydrophobic HPLC chromatogram of the anti-TROP2 monoclonal antibody-122 (NBOK)-P4AE conjugation reaction product prepared in Example 1.

[0065] Figure 7Figure 1 shows the hydrophobic HPLC chromatogram of the anti-TROP2 mAb-122 (NPAK)-P4AF conjugation reaction product prepared for Example 1. DETAILED DESCRIPTION

[0066] In the present application, "C1-Cn" includes C1-C2,... C1-Cn. For example, the "C1-C6" group means that the moiety has 1 to 6 carbon atoms, i.e. the group contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.

[0067] The conjugates of the present application can contain asymmetric or chiral centers, and as such can exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the conjugates of the present application, including but not limited to, diastereomeric, enantiomeric, atropisomeric and geometric (conformational) isomers and mixtures thereof, such as racemic mixtures, are within the scope of the present application. Unless otherwise stated, structures depicted herein are also meant to include all isomeric (such as diastereomeric, enantiomeric, atropisomeric and geometric (conformational) isomeric) forms of the structure described. For example, the R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, atropisomers (see Basic Organic Chemistry, 2nd Ed., Hehng Qi Yi et al., p104-105), PAC, 1996, 68, 2193. (Basic terminology of stereochemistry (IUPAC Recommendations 1996, on page 2201)), (Z) and (E) conformational isomers of the conjugates of the present application are within the scope of the application. Thus, individual stereoisomers of the conjugates of the present application, as well as mixtures of enantiomers, mixtures of diastereomers, mixtures of atropisomers and mixtures of geometric (conformational) isomers are within the scope of the present application.

[0068] The term "antibody", used alone or in combination, is construed in its broadest sense to include intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies), as long as they exhibit the desired biological activity. Herein, "antibody" and "immunoglobulin" are used interchangeably.

[0069] The term "pharmaceutically acceptable salt" as used herein alone or in combination refers to a salt of the conjugate of the present application which retains the biological effectiveness and properties of the free acids or free bases and is obtained by reacting the free acid with a non-toxic inorganic or organic base or the free base with a non-toxic inorganic or organic acid. Standard procedures used by those skilled in the art can be used. Suitable salts are listed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977).

[0070] The term "pharmaceutical composition" as used herein alone or in combination refers to a composition comprising one or more conjugates of the present application or stereoisomers, pharmaceutically acceptable salts thereof, and other components such as pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism and absorption of the active ingredient to exert a biological activity. The pharmaceutical composition can be formulated into various suitable dosage forms according to the route of administration, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, eye preparations, pills, implants, aerosols, powder aerosols, spray, etc.

[0071] The term "excipient" as used herein alone or in combination refers to a substance other than the active ingredients of a drug, which has been reasonably evaluated in terms of safety and is included in a pharmaceutical preparation. The pharmaceutical excipient has important functions such as solubilization, solubilization aid, sustained release, and the like, in addition to the functions of excipient and carrier, and improvement of stability, and is an important ingredient that can affect the quality, safety, and effectiveness of a drug. The pharmaceutical excipient can be classified into natural products, semi-synthetic products, and synthetic products according to its origin, and can be classified into solvents, propellants, solubilizers, solubilization aids, emulsifiers, coloring agents, binders, excipients, disintegrants, fillers, lubricants, humectants, osmotic pressure adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusting agents, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filtration aids, release retardants, and the like according to its function and use. The same pharmaceutical excipient can be used for pharmaceutical preparations for different administration routes, and has different functions and uses.

[0072] The terms "treat" and other similar synonyms used herein, alone or in combination, include alleviating, abating, or ameliorating a disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a symptom caused by the disease or condition, or stopping the symptoms of the disease or condition, in addition, the term encompasses prophylactic purposes. The term also includes obtaining a therapeutic effect and / or a prophylactic effect.

[0073] The technical solutions of the present application are further described in detail below in combination with specific embodiments.

[0074] In the embodiments of the present application, the non-natural amino acids NBGK, NPAK and NBOK used are prepared according to Chinese Patent ZL202110863816.7, the toxins P4AE and P4AF used are prepared according to Chinese Patent ZL201810957127.0, and other raw materials or reagents are commercially available products unless otherwise specified.

[0075] In the embodiments of the present application, the eluent proportions are all volume ratios, and other percentages involved are mass percentages unless otherwise specified.

[0076] Preparation of anti-TROP2 ADC in Example 1

[0077] The anti-TROP2 monoclonal antibodies containing non-natural amino acids at different sites are expressed in a eukaryotic expression system using non-natural amino acids NBGK, NPAK and NBOK, and conjugates with toxins are prepared.

[0078] (1) Obtaining of the helper plasmid

[0079] The helper plasmid pCMV-MbPylRS is purchased from the plasmid depository organization addgene (item #91706), which encodes an aminoacyl tRNA synthetase that specifically recognizes pyrrolysine-derived non-natural amino acids in mammalian cells and the corresponding tRNA (recognizing the amber codon UAG).

[0080] (2) Construction of anti-TROP2 monoclonal antibody expression vector containing an amber codon in the internal gene reading frame

[0081] The nucleic acid fragments encoding the heavy chain and light chain DNA of the anti-TROP2 monoclonal antibody (the corresponding amino acid sequences are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively) are synthesized by full gene synthesis, and are subcloned into the eukaryotic expression vector pCDNA3.1+, and then the obtained expression vector is subjected to point mutation to obtain the expression plasmid pCDNA3.1-TROP2Mab-UAG122 in which the amino acid codon at position 122 of the heavy chain reading frame is mutated to an amber codon, and the map thereof is as shown in Figure 1The complete sequence is shown as SEQ ID NO: 3.

[0082] The heavy chain amino acid sequence of the anti-TROP2 mAb (SEQ ID NO: 1) is as follows:

[0083]

[0084] The light chain amino acid sequence of the anti-TROP2 mAb (SEQ ID NO: 2) is as follows:

[0085]

[0086] The gene sequence of the expression plasmid pCDNA3.1-TROP2Mab-UAG122 (SEQ ID NO: 3) is as follows:

[0087]

[0088]

[0089]

[0090]

[0091]

[0092] According to the same method as described above, the expression plasmid pCDNA3.1-TROP2Mab-UAG212 in which the amino acid codon at position 212 of the heavy chain reading frame is mutated to an amber codon, and the expression plasmid pCDNA3.1-TROP2Mab-UAG278 in which the amino acid codon at position 278 of the heavy chain reading frame is mutated to an amber codon, are obtained. The maps of both are consistent with Figure 1 the map of the expression plasmid pCDNA3.1-TROP2Mab, and their gene sequences are also substantially consistent with SEQ ID NO: 3 (wherein, relative to SEQ ID NO: 3, the DNA sequence of the expression plasmid pCDNA3.1-TROP2Mab-UAG212 is TAG at positions 1610-1612 and GCC at positions 1340-1342; the DNA sequence of the expression plasmid pCDNA3.1-TROP2Mab-UAG278 is TAG at positions 1808-1810 and GCC at positions 1340-1342).

[0093] (3) Insertion of a non-natural amino acid

[0094] Suspension-adapted HEK293 cells (ATCC, Cat. No. CRL-1573) were used to inoculate Wayne293 5 at a density of 0.3 x 10 TMCulture medium (Quacell Biotechnology, catalog number A21501), using 1L shake flasks, 240mL of liquid, cultured with shaking at 120rpm, 5% CO2, and 80% humidity, until the cell density reaches approximately 1×10⁻⁶. 6 Transfection was performed at a concentration of / mL. The helper plasmid pCMV-MbPylRS described in steps (1) and (2) and the expression plasmid pCDNA3.1-TROP2Mab-UAG122 (or expression plasmid pCDNA3.1-TROP2Mab-UAG212 or expression plasmid pCDNA3.1-TROP2Mab-UAG278) were extracted and endotoxins were removed for later use. 120 μg of expression plasmid and helper plasmid were used for each shake flask transfection. The plasmid was added to a centrifuge tube and diluted to 7.2 mL with 1×PBS buffer. PEI transfection reagent (polyethyleneimine, P) was added to another centrifuge tube. 720 μg of olyethyleneimine was added and diluted to 7.2 mL with 1×PBS buffer. After mixing, the two centrifuge tubes were allowed to stand for 5 minutes each. The liquid in the two centrifuge tubes was then gently mixed and allowed to stand for 10 minutes. A total of 14.4 mL was slowly added dropwise to 240 mL of cell suspension in a 1 L shake flask, while gently shaking continuously during the addition. The cells were cultured at 120 rpm, 5% CO2, and 80% humidity for 4 hours. Then, non-natural amino acids were added to a final concentration of 1 mM. The cells were then cultured at 120 rpm, 5% CO2, and 80% humidity for another 5 days. The cell culture supernatant was harvested for antibody purification.

[0095] (4) Purification

[0096] Cell culture supernatant was purified using HiTrap Protein A in a 1 mL pre-packed column. The elution buffer consisted of 100 mmol / L glycine and 200 mmol / L acetate at pH 3.5, yielding purified anti-TROP2 monoclonal antibodies with inserted non-natural amino acids. Anti-TROP2 monoclonal antibodies with NPAK inserted at positions 122, 212, and 278 of the heavy chain were named anti-TROP2 monoclonal antibody-122 (NPAK), anti-TROP2 monoclonal antibody-212 (NPAK), and anti-TROP2 monoclonal antibody-278 (NPAK), respectively. Anti-TROP2 monoclonal antibodies with NBGK and NBOK inserted at position 122 of the heavy chain were named anti-TROP2 monoclonal antibody-122 (NBGK) and anti-TROP2 monoclonal antibody-122 (NBOK), respectively.

[0097] (5) Coupling

[0098] The synthetic route is shown below (taking the coupling reaction of an anti-TROP2 monoclonal antibody with inserted NPAK and P4AE as an example):

[0099]

[0100] Toxins (P4AE) containing aminoxy end groups were site-specifically coupled to purified anti-TROP2 mAbs inserted with unnatural amino acids (wherein the direction of R1 to R2 is the N-terminal to C-terminal direction of the amino acid sequence) by oximation reaction to obtain mAb-toxin conjugates: i.e. anti-TROP2 mAb-122(NPAK)-P4AE (anti-TROP2 mAb-122(NPAK) modified by toxin P4AE), anti-TROP2 mAb-212(NPAK)-P4AE (anti-TROP2 mAb-212(NPAK) modified by toxin P4AE), anti-TROP2 mAb-278(NPAK)-P4AE (anti-TROP2 mAb-278(NPAK) modified by toxin P4AE), TROP2 mAb-122(NBGK)-P4AE (anti-TROP2 mAb-122(NBGK) modified by toxin P4AE), TROP2 mAb-122(NBOK)-P4AE (anti-TROP2 mAb-122(NBOK) modified by toxin P4AE).

[0101] Similarly, toxins (P4AF) containing aminoxy end groups were site-specifically coupled to purified anti-TROP2 mAb-122(NPAK) by oximation reaction to obtain anti-TROP2 mAb-122(NPAK)-P4AF (anti-TROP2 mAb-122(NPAK) modified by toxin P4AF).

[0102] The specific operation of the coupling process is as follows: P4AE or P4AF was added to the solution of purified anti-TROP2 mAbs inserted with unnatural amino acids at a molar ratio of 12:1, mixed and dissolved, and then the pH was adjusted to 4.0 using 10M acetic acid, and the mixture was shaken on a shaker (25°C, 200 rpm), sampled after 48h, and the reaction of the anti-TROP2 mAb with the toxin was detected by HPLC based on the principle of hydrophobic chromatography (HIC-HPLC), which was displayed in the form of drug-to-antibody ratio (DAR value), as shown in Table 1, and the chromatograms are shown in Figures 2-7 The results showed that P4AE or P4AF was combined to the unnatural amino acids NBGK, NPAK or NBOK of the anti-TROP2 mAb in the form of site-specific coupling. Since each heavy chain of the anti-TROP2 mAb inserted 1 unnatural amino acid available for coupling reaction, 2 molecules of toxin could be coupled to each mAb molecule under ideal conditions, and the DAR value was 2.

[0103] Table 1 Coupling effect of anti-TROP2 mAb with toxin

[0104] Sample Name DAR Value Anti-TROP2 mAb-122 (NPAK)-P4AE 1.95 Anti-TROP2 mAb-212 (NPAK)-P4AE 1.95 Anti-TROP2 mAb-278 (NPAK)-P4AE 1.96 Anti-TROP2 mAb-122 (NBGK)-P4AE 1.95 Anti-TROP2 mAb-122 (NBOK)-P4AE 1.92 Anti-TROP2 mAb-122 (NPAK)-P4AF 1.95

[0105] The conjugated mAb-toxin conjugate was buffer exchanged by 50 kDa ultrafiltration centrifuge tubes to remove unreacted toxin starting material and replace the buffer with 20 mM histidine buffer, pH 6.5.

[0106] The HIC-HPLC analysis conditions are as follows:

[0107] Mobile phase A (2 M ammonium sulfate, 75 mM K2HPO4, pH 7.2 ± 0.2);

[0108] Mobile phase B (75 mM K2HPO4, 25% isopropanol, pH 7.2 ± 0.2).

[0109]

[0110]

[0111] The peak area of conjugated 1 toxin (DRUG1), the peak area of conjugated 2 toxins (DRUG2), and the peak area of conjugated 3 toxins (DRUG3) were calculated by area normalization method, and the DAR value was calculated. The DAR value calculation formula: DAR value = (DRUG1 + DRUG2 x 2 + DRUG3 x 3) / (DRUG1 + DRUG2 + DRUG3).

[0112] In vitro activity analysis of Example 2

[0113] The NCI-N87 (ATCC, Cat. No. CRL-5822), HCC1954 (ATCC, Cat. No. CRL-2338) cell lines were used to detect the inhibitory effect of the ADC sample on cell proliferation.

[0114] The specific process is as follows: NCI-N87, HCC1954 cells are cultured in RPMI-1640 Medium containing 10% fetal bovine serum under the condition of 37°C, 5% carbon dioxide, and cultured to a sufficient amount, and the cell density is adjusted to an appropriate cell density with a cell culture solution, and the cells are seeded in a cell plate so that 4000 cells are added to each well, 90 μL / well, and the solvent control wells are added with culture medium without cells. The cell culture plate is incubated in a CO2 incubator (37±1°C, 5±0.5% CO2) overnight. All samples (Trodelvy, anti-TROP2 mAb-122 (NPAK)-P4AE, anti-TROP2 mAb-212 (NPAK)-P4AE, anti-TROP2 mAb-278 (NPAK)-P4AE, anti-TROP2 mAb-122 (NBGK)-P4AE, anti-TROP2 mAb-122 (NBOK)-P4AE, anti-TROP2 mAb-122 (NPAK), anti-TROP2 mAb-212 (NPAK), anti-TROP2 mAb-278 (NPAK), anti-TROP2 mAb-122 (NPAK)-P4AF) are diluted from 100 nM to 0.05 nM in a gradient, a total of 9 concentrations, and 2 replicate wells for each dilution. The diluted samples are transferred to the culture plate inoculated with NCI-N87, HCC1954 cells, 10 μL per well, and 10 μL of medium is added to the solvent control wells and blank control wells, and incubated at 37°C, 5% carbon dioxide for 96 h±2 h, then 50 μL of Promega Cell Titer-Glo detection reagent is added to each well, shaken for 2 min, and incubated at room temperature for 10 min before reading the plate. The inhibition rate (Growth inhibition) of the test sample is calculated using the following formula: Inhibition rate (Emax, %) = (1-(RLU compound-RLU blank) / (RLU control-RU blank)) x 100%. The inhibition rates of different concentrations of compounds are calculated in Excel, and the IC50 is calculated using GraphPad Prism 7 software, and the results are shown in Table 2.

[0115] Table 2 Cell proliferation inhibition activity test results

[0116]

[0117] The results show that, compared with the control drug Trodelvy, the tumor killing effect (inhibition rate) of the P4AE or P4AF modified anti-TROP2 mAb containing non-natural amino acids is significantly higher in the NCI-N87 cell line and the HCC1954 cell line, and the P4AE or P4AF modification at position 122 of the anti-TROP2 mAb has the best inhibition activity.

[0118] The IC50 value of the P4AE-modified anti-TROP2 monoclonal antibody containing unnatural amino acids was significantly reduced compared to the anti-TROP2 monoclonal antibody containing only unnatural amino acids, indicating that the latter successfully linked the small toxin molecule, and thus the cell inhibition activity was greatly improved.

[0119] Example 3 Analysis of in vivo activity

[0120] The anti-tumor effect of the test sample (histidine buffer at pH 6.5 as a drug solvent) was evaluated in a female BALB / c nude mouse animal model of subcutaneous xenotransplantation of TROP2-positive human gastric cancer NCI-N87 and human breast cancer HCC1954 cell lines.

[0121] The specific process is as follows:

[0122] Cell culture: NCI-N87 or HCC1954 cells were cultured in RPMI1640 culture medium containing 10% fetal bovine serum. Exponentially growing tumor cells were collected and resuspended in PBS to an appropriate concentration for inoculation.

[0123] Animal modeling: BALB / c nude experimental mice (Jiangsu Jizhuangkang Biotechnology Co., Ltd. Changzhou Branch) were inoculated subcutaneously on the right anterior back with 10 x 10 6 cells in 1:1 PBS and Matrigel (0.1 mL per mouse), and tumor growth was observed regularly. When the tumor grew to an average volume of 100-200 mm 3 According to the tumor size and mouse body weight, the mice were randomly divided into groups for drug administration. On the day of grouping (day 0), drug administration was started according to the experimental design (as shown in Table 3).

[0124] Table 3 Experimental design table

[0125]

[0126] After the start of drug administration, the body weight and tumor size of the mice were measured twice a week.

[0127] Tumor volume calculation formula: Tumor volume (mm 3 ) = 1 / 2 x (a x b 2 ) (where a represents the long diameter and b represents the short diameter)

[0128] The tumor inhibition efficacy TGI (%) of the test sample was calculated after the end of the experiment.

[0129] TGI (%) calculation: TGI (%) = 1 - (average tumor volume at the end of administration in a certain treatment group / average tumor volume at the end of treatment in the control group x 100.

[0130] The results are shown in Tables 4-1 and 4-2.

[0131] Table 4-1 Anti-tumor efficacy evaluation of the test samples on NCI-N87 inoculated tumor model (calculated based on tumor volume on day 28 after grouping administration)

[0132]

[0133] Table 4-2 Anti-tumor efficacy evaluation of the test samples on HCC1954 inoculated tumor model (calculated based on tumor volume on day 28 after grouping administration)

[0134]

[0135] The results show that in the NCI-N87 model, anti-TROP2 monoclonal antibody-122 (NPAK)-P4AE and anti-TROP2 monoclonal antibody-122 (NPAK)-P4AF high and low dose groups, and Trodelvy all have tumor growth inhibition effect (TGI > 60%), and have dose dependence, and the low dose groups of anti-TROP2 monoclonal antibody-122 (NPAK)-P4AE and anti-TROP2 monoclonal antibody-122 (NPAK)-P4AF have better tumor inhibition effect in the NCI-N87 model than Trodelvy; in the HCC1954 model, the high dose groups of anti-TROP2 monoclonal antibody-122 (NPAK)-P4AE and anti-TROP2 monoclonal antibody-122 (NPAK)-P4AF have tumor growth inhibition effect (TGI > 60%), and the tumor inhibition effect is comparable to Trodelvy, and the low dose groups have no obvious tumor growth inhibition effect (TGI < 60%).

[0136] Unless specifically limited, the terms used in the present application are of the meanings commonly understood by those skilled in the art.

[0137] The embodiments described in the present application are only for illustrative purposes, and are not intended to limit the protection scope of the present application, and those skilled in the art can make various other replacements, changes and improvements within the scope of the present application, therefore, the present application is not limited to the above embodiments, but is limited only by the claims.

Claims

1. An anti-TROP2 antibody-drug conjugate or a pharmaceutically acceptable salt thereof, characterized in that, The amino acid sequences of the heavy chain and the light chain of the anti-TROP2 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, wherein one of positions 122, 212 and 278 of the heavy chain comprises a non-natural amino acid residue shown in formula (I), Formula (I) wherein X represents a C1-C6 linear or branched alkylene, one or more -CH2- of the alkylene is optionally replaced by one or more of -O-, -S-, -NH-; L represents a C1-C6 linear or branched alkylene; D represents an auristatin toxin; m represents an integer of 2-6; represents the point of attachment in the heavy chain.

2. The anti-TROP2 antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, characterized in that, D represents an auristatin toxin MMAE, MMAF or MMAD.

3. The anti-TROP2 antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 2, characterized in that, D represents one of the following structures: wherein represents the point of attachment to L.

4. The anti-TROP2 antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein X represents a C1-C4 linear alkylene, one -CH2- of the alkylene is optionally replaced by -O- or -NH-.

5. The anti-TROP2 antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein X represents -CH2-O-, -CH2-, -CH2-O-CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-O-CH2-CH2-O-, -O-, -CH2-NH-CH2- or -CH2-NH-.

6. The anti-TROP2 antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein m represents 3, 4, 5 or 6.

7. The anti-TROP2 antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-6, characterized in that, L represents a C1-C3 linear alkylene.

8. The anti-TROP2 antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 7, characterized in that, L represents methylene or ethylene.

9. A method of preparing an anti-TROP2 antibody-drug conjugate according to any one of claims 1-8, characterized in that, comprising the following steps: S1: site-specifically inserting a non-natural amino acid shown in formula (II) into the heavy chain of the anti-TROP2 antibody to obtain a recombinant antibody; Formula (II) S2: subjecting the recombinant antibody obtained in step S1 to an oxime reaction with a toxin shown in formula (III) to obtain the anti-TROP2 antibody-drug conjugate; Formula (III) wherein X, L, D and m are each independently defined in any one of claims 1-8.

10. The method of claim 9, wherein, In step S1, the non-natural amino acid shown in formula (II) is site-specifically inserted into the heavy chain of the anti-TROP2 antibody by codon expansion technology or by chemical synthesis to obtain a recombinant antibody.

11. The method of claim 10, wherein, The codon expansion technology is implemented in a mammalian engineered cell.

12. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the anti-TROP2 antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-8, and optionally one or more pharmaceutically acceptable excipients.

13. Use of the anti-TROP2 antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-8, or the pharmaceutical composition according to claim 12, in the preparation of a medicament for treating cancer.

14. Use according to claim 13, characterized in that, The cancer is pancreatic cancer, breast cancer, lung cancer, urothelial cancer, thyroid cancer, colon cancer, gastric cancer, endometrial cancer, prostate cancer, ovarian cancer or cervical cancer.

15. Use according to claim 14, characterized in that, The breast cancer is triple-negative breast cancer.

16. Use according to claim 14, characterized in that, The lung cancer is non-small cell lung cancer.

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