CN115845080A - Eribulin derivative-anti-folate receptor antibody conjugate - Google Patents

Abstract

The present disclosure relates to eribulin derivative-anti-folate receptor antibody conjugates. Drug conjugates of eribulin derivatives Specifically, antibody conjugates of eribulin derivatives-anti-folate receptor α (FRA) antibodies or antigen-binding fragments thereof, their preparation methods and their use in Medical applications. The present disclosure further relates to methods and compositions for treating cancer by administering the antibody-drug conjugates provided herein.

Description

Eribulin derivative-anti-folate receptor antibody conjugate

Technical Field

The present disclosure relates to an eribulin derivative-anti-folate receptor antibody conjugate.

Background Art

Antibody drug conjugates (ADCs) connect monoclonal antibodies or antibody fragments to biologically active drugs through stable chemical linker compounds, making full use of the specificity of antibodies binding to surface antigens of normal cells and tumor cells and the high efficiency of drugs, while avoiding the defects of the former's low efficacy and the latter's excessive toxic side effects. This means that compared with traditional chemotherapy drugs, antibody drug conjugates can accurately bind to tumor cells and reduce the impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12: 329-332; DiJoseph JF, Armellino DC, (2004) Blood, 103: 1807-1814).

Since the first antibody-drug conjugate Mylotarg (gemtuzumab ozogamicin, Wyeth Pharmaceuticals Co., Ltd.) was approved by the U.S. FDA for the treatment of acute myeloid leukemia in 2000, there have been 164 ADC drugs in clinical trials, most of which (n=100) are in Phase I clinical trials, 46 are in Phase II clinical trials, 7 are in Phase III clinical trials, and 3 have applied for BLA. The representative drugs of the third generation are Polatuzumab vedotin (trade name, Polivy, approved for marketing in June 2019) jointly developed by Genentech and Genetics, Enfortumab vedotin (trade name, Padcev, December 2019) jointly developed by Agensys (a subsidiary of Astellas) and Seattle Genetics, and Fam-trastuzumab deruxtecan (trade name, Enhertu) developed by Daiichi Sankyo.

Microtubules are powerful, filamentous cytoskeletal proteins associated with a variety of cellular functions including intracellular migration and transport, cell signaling, and maintenance of cell shape. Microtubules also play a key role in mitotic cell division by forming the mitotic spindle required for the separation of chromosomes into two daughter cells. The biological functions of microtubules in all cells are largely regulated by their polymerization dynamics, which is reversibly and non-covalently attached to the ends of microtubules by α and β tubulin dimers. This dynamic behavior and the resulting control of microtubule length are indispensable for the proper function of the mitotic spindle. Even minor changes in microtubule dynamics can involve the axis checkpoint, inhibit cell cycle progression during mitosis, and subsequently cause cell death (Mukhtar et al. (2014) Mol. Cancer Ther. 13: 275-84). Due to the rapid cell division of cancer cells, they are generally more sensitive to compounds that bind to tubulin and disrupt its normal function than normal cells. Therefore, tubulin inhibitors and other agents targeting microtubules are expected to become a class of drugs for treating cancer (Dumontet and Jordan (2010) Nat. Rev. Drug Discov. 9: 790-803).

On the other hand, folate receptor alpha (FRA) is a glycerophosphatidylinositol (GPI)-linked membrane protein that binds folate. Although the role of FRA in the biology of normal and cancerous tissues is not fully understood, it is overexpressed in a high percentage of ovarian cancers of epithelial origin (O'Shannessy et al., Int. J. Gynecol. Pathol. 2003, 32(3): 258-68), and in a certain percentage of non-small cell lung cancers (Christoph et al. Clin. Lung Cancer 2014 15(5): 320-30). FRA also has limited expression in normal tissues. These properties make FRA an attractive target for cancer immunotherapy.

Summary of the invention

The present disclosure provides an antibody-drug conjugate (ADC) having a structure as shown in Formula I or a pharmaceutically acceptable salt or solvate thereof:

Ab-(LD) _k (I)

Wherein, Ab is an anti-folate receptor α (FRA) antibody or an antigen-binding fragment thereof,

L is a linker that covalently links Ab to D, and k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any number between any two numbers),

-D is as follows:

In some embodiments, k in the antibody-drug conjugate Ab-(LD) _k is selected from 1 to 10 and can be an integer or a decimal.

In some embodiments, the linker is stable outside of the cell, so that the ADC remains intact when present in the extracellular environment, but is capable of cleavage when internalized in a cell, such as a cancer cell. In some embodiments, when the ADC enters a cell expressing an antigen specific for the antibody portion of the ADC, the eribulin derivative drug portion is cleaved from the antibody portion, and the cleavage releases the unmodified form of the eribulin derivative. In some embodiments, the linker comprises a cleavable portion positioned so that no portion of the linker or the antibody portion remains bound to the eribulin derivative after cleavage.

In some embodiments, the cleavable portion in the linker is a cleavable peptide portion. In some embodiments, relative to an ADC comprising an alternative cleavable portion, an ADC comprising a cleavable peptide portion shows a lower aggregation level, an improved antibody: drug ratio, an increased targeted killing of cancer cells, a reduced off-target killing of non-cancerous cells, and/or a higher drug load. In some embodiments, relative to a non-cleavable linker, the addition of a cleavable portion increases cytotoxicity and/or efficacy. In some embodiments, the increased efficacy and/or cytotoxicity is in cancers that express a moderate level of an antigen targeted by the antibody portion of the ADC (e.g., moderate FRA expression). In some embodiments, the cleavable peptide portion can be cleaved by an enzyme, and the linker is a linker that the enzyme can cleave. In some embodiments, the enzyme is a cathepsin, and the linker is a linker that the cathepsin can cleave. In certain embodiments, compared to alternative cleavage mechanisms, linkers that the enzyme can cleave (e.g., linkers that the cathepsin can cleave) show one or more of the above-mentioned improved properties.

In some embodiments, the linker comprises an amino acid unit, which preferably comprises a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, more preferably valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe) or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

In some embodiments, the disclosure includes a linker of an amino acid unit selected from the group consisting of:

In some embodiments, the amino acid unit comprises valine-citrulline (Val-Cit). In some embodiments, the ADC comprising Val-Cit exhibits increased stability, reduced off-target cell killing, increased on-target cell killing, lower aggregation levels, and/or higher drug loading relative to ADCs comprising alternative amino acid units or alternative cleavable moieties.

In another aspect, some embodiments provide a linker comprising a cleavable sulfonamide moiety, wherein the linker is capable of being cleaved under reducing conditions.

In some embodiments, the linker comprises a cleavable disulfide moiety, and the linker is capable of being cleaved under reducing conditions.

In another aspect, in the antibody conjugates of the present disclosure, the linker comprises at least one spacer unit that attaches the eribulin derivative D to the cleavable moiety. In some embodiments, the linker comprises a spacer unit attached to D.

In some embodiments, the spacer unit comprises p-aminobenzyloxycarbonyl (pAB),

In some embodiments, the spacer unit comprises:

Wherein, Z ₁ to Z ₄ are selected from carbon atoms or nitrogen atoms; R ⁴ is selected from alkyl, cycloalkyl, aryl and heteroaryl, and the alkyl, cycloalkyl, aryl and heteroaryl are each independently substituted by one or more substituents selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R ¹ and R ² are each independently selected from hydrogen, C _1-6 alkyl, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen; or, R ¹ and R ² together with the carbon atom to which they are connected form a C _3-6 cycloalkyl; X is selected from -O- or -NH-; L is selected from an integer between 1 and ₄ ;

其中R⁵选自-COOH或CH₂OH。在一些实施方案中，V选自-COOH。Q is VE, VE provides a glycosidic bond that can be cleaved by intracellular glycosidases, E is selected from -O-, -S- or -NR ³ -, R ³ is selected from hydrogen or methyl, and further, V is selected from

wherein R ⁵ is selected from -COOH or CH ₂ OH. In some embodiments, V is selected from -COOH.

In some embodiments, the spacer unit comprises:

In another aspect, in the antibody conjugate (ADC) of the present disclosure, L-D is a chemical moiety represented by the following formula:

-Str-(Pep)-Sp-D

Str is a stretching unit covalently linked to Ab.

Sp is a spacer unit,

Pep is selected from amino acid units.

In another aspect, the ADC wherein Str is selected from a chemical moiety represented by the following formula:

其中R⁶选自-W-C(O)-、-C(O)-W-C(O)-、(CH₂CH₂O)_p1C(O)-、(CH₂CH₂O)_p1CH₂C(O)-、(CH₂CH₂O)_p1CH₂CH₂C(O)-，其中W选自C_1-8亚烷基、C_1-8亚烷基-环烷基或1至8个原子的直链杂亚烷基，所述杂亚烷基包含1至3个选自N、O或S的杂原子，其中所述的C_1-8亚烷基、C_1-8亚烷基-环烷基和直链杂亚烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、烷氧基和环烷基的一个或多个取代基所取代；

wherein R ⁶ is selected from -WC(O)-, -C(O)-WC(O)-, (CH ₂ CH ₂ O) _p1 C(O)-, (CH ₂ CH ₂ O) _p1 CH ₂ C(O)-, (CH ₂ CH ₂ O) _p1 CH ₂ CH ₂ C(O)-, wherein W is selected from C _1-8 alkylene, C _1-8 alkylene-cycloalkyl or a straight-chain heteroalkylene of 1 to 8 atoms, wherein the heteroalkylene contains 1 to 3 heteroatoms selected from N, O or S, wherein the C _1-8 alkylene, C _1-8 alkylene-cycloalkyl and straight-chain heteroalkylene are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, alkoxy and cycloalkyl;

L ¹ is selected from -NR ⁷ (CH ₂ CH ₂ O) _p1 CH ₂ CH ₂ C(O)-, -NR ⁷ (CH ₂ CH ₂ O) _p1 CH ₂ C(O)-, -S(CH ₂ ) _p1 C(O)-, -(CH ₂ ) _p1 C(O)- or a chemical bond, wherein p1 is an integer from 1 to 20, preferably a chemical bond; p1 is an integer from 1 to 20, and R ⁷ is selected from a hydrogen atom, an alkyl group, a halogenated alkyl group and a hydroxyalkyl group.

其中p1为整数1至20，例如

(PEG)₂；

(PEG)₄；In some embodiments, the linker can include at least one polyethylene glycol (PEG) moiety. The PEG moiety can, for example, include -(PEG) _p1 -,

Where p1 is an integer from 1 to 20, for example

(PEG) ₂ ;

(PEG) ₄ ;

(PEG)₅。

(PEG) ₅ .

In some embodiments, the spacer unit in the linker comprises (PEG) _2. In some embodiments, despite the shorter linker length, ADCs comprising shorter spacer units (e.g., (PEG) ₂ ) exhibit lower aggregation levels and/or higher drug loading relative to ADCs comprising longer spacer units (e.g., (PEG) ₈ ).

In some embodiments, ^R7 in the antibody-drug conjugate is selected from _C1-6 alkylene C(O)-, -( _{CH2 - CH2O} ) _2C (O)-, -(CH2-CH2O) _2CH2C ( _O )-, -( _CH2 -CH2O) _2CH2CH2C (O)- _{, -} (CH2-CH2O) _2CH2CH2C (O)-, -( _CH2 - _CH2O ) _3C (O ₎ -, and -( _CH2 - _CH2O ) _4C (O)-.

In some embodiments, the linker L in the antibody-drug conjugate comprises: maleimide-(PEG) ₂ -Val-Cit, maleimide-(PEG) ₆ -Val-Cit, maleimide-(PEG) ₈ -Val-Cit, maleimide-(PEG) ₄ -CH ₂ CH ₂ C(O)-Val-lys, maleimide-(CH ₂ ) ₅ -Val-Cit, maleimide-(CH ₂ ) ₅ -Val-lys, maleimide-(CH ₂ ) ₅ -Gly-Gly-Phe-Gly, maleimide-(PEG) ₂ -Ala-Ala-Asn, maleimide-(PEG) ₆ -Ala-Ala-Asn, maleimide-(PEG) ₈ -Val-Cit, -Ala-Ala-Asn, maleimide-(PEG) ₄ -triazole-(PEG) ₃ -sulfonamide, maleimide-(PEG) _2- CH ₂ CH ₂ C(O)-Val-lys, maleimide-(PEG) ₄ -triazole-(PEG) ₃ -sulfonamide or Mal-(PEG) ₄ -triazole-(PEG) ₃ -disulfide.

On the other hand, some embodiments provide the antibody-drug conjugate wherein Str is selected from a chemical moiety represented by the following formula:

其中R⁸选自C_1-10亚烷基、C_2-10烯基、(C_1-10亚烷基)O-、N(R^d)-(C_2-6亚烷基)-N(R^d)和N(R^d)-(C_2-6亚烷基)；且每个R^d独立为H或C₁-C₆烷基。

wherein R ⁸ is selected from C _1-10 alkylene, C _2-10 alkenyl, (C _1-10 alkylene)O—, N(R ^d )-(C _2-6 alkylene)-N(R ^d ) and N(R ^d )-(C _2-6 alkylene); and each R ^d is independently H or C ₁ -C ₆ alkyl.

In some embodiments, the antibody-drug conjugate is represented by the following formula:

k选自1至10，可以为整数，也可以为小数；P1选自2、4、6或8；P2选自0、1或2；

k is selected from 1 to 10, and can be an integer or a decimal; P1 is selected from 2, 4, 6 or 8; P2 is selected from 0, 1 or 2;

k选自1至10，可以为整数，也可以为小数；P1选自2、4、6或8；P2选自0、1或2；

k is selected from 1 to 10, and can be an integer or a decimal; P1 is selected from 2, 4, 6 or 8; P2 is selected from 0, 1 or 2;

k选自1至10，可以为整数，也可以为小数；P1选自2、4、6或8；P2选自0、1或2；

k is selected from 1 to 10, and can be an integer or a decimal; P1 is selected from 2, 4, 6 or 8; P2 is selected from 0, 1 or 2;

k选自1至10，可以为整数，也可以为小数；P2选自1-6之间整数；

k is selected from 1 to 10, and can be an integer or a decimal; P2 is selected from an integer between 1 and 6;

k选自1至10，可以为整数，也可以为小数；P2选自1-6之间整数；

k is selected from 1 to 10, and can be an integer or a decimal; P2 is selected from an integer between 1 and 6;

k选自1至10，可以为整数，也可以为小数；P2选自1-6之间整数。

k is selected from 1 to 10 and can be an integer or a decimal; P2 is selected from an integer between 1 and 6.

Further, the antibody-drug conjugate (ADC) of the present disclosure is selected from:

其中，k选自1至10，可以为整数，也可以为小数。

Wherein, k is selected from 1 to 10 and can be an integer or a decimal.

In some embodiments, the antibody or antigen-binding fragment thereof is an anti-folate receptor alpha (FRA) antibody or antigen-binding fragment thereof.

In some embodiments, the anti-FRA antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO:7, and LCDR1, LCDR2 and LCDR3 having the same sequence as the light chain variable region shown in SEQ ID NO:8.

In some embodiments, the anti-FRA antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

The CDR sequences of the anti-FRA antibody or antigen-binding fragment thereof encoded according to Kabat are shown in the following table:

name sequence serial number HCDR1 GYGLS SEQ ID NO: 1 HCDR2 MISSGGSYTYYADSVKG SEQ ID NO: 2 HCDR3 HGDDPAWFAY SEQ ID NO: 3 LCDR1 SVSSSISSNNLH SEQ ID NO: 4 LCDR2 GTSNLAS SEQ ID NO: 5 LCDR3 QQWSSYPYMYT SEQ ID NO: 6

In some embodiments, the anti-folate receptor alpha (FRA) antibody is a humanized antibody or a fragment thereof.

In an alternative embodiment, the anti-folate receptor alpha (FRA) antibody or antigen-binding fragment thereof described in the present disclosure is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment comprises a human IgG1 heavy chain constant domain and an Igκ light chain constant domain.

Further, in some embodiments, the anti-folate receptor alpha (FRA) antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8.

Heavy chain variable region:

EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTVSS

SEQ ID NO: 7

Light chain variable region:

DIQLTQSPSSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSYPYMYTFGQGTKVEIK

SEQ ID NO: 8

In an embodiment of the present disclosure, the anti-folate receptor alpha (FRA) antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO:9 and a light chain having an amino acid sequence of SEQ ID NO:10.

Heavy Chain:

EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 9

Light chain:

DIQLTQSPSSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSYPYMYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

SEQ ID NO: 10

Further, the aforementioned antibody-conjugate uses heterogeneous connection technology, i.e., amine-based lysine conjugation and thiol-based cysteine conjugation. In some embodiments, the stretching unit Str covalently linked to Ab in the aforementioned antibody-conjugate is linked to a cysteine amino acid on an anti-folate receptor α (FRA) antibody.

On the other hand, the aforementioned antibody-conjugate can be conjugated by site-specific (homogeneous conjugation chemistries) to effectively control the DAR value, thereby obtaining ADC with better stability and aggregation. In some embodiments, the stretching unit Str covalently linked to Ab in the aforementioned antibody-conjugate is linked to, for example, an anti-folate receptor α (FRA) antibody.

ADCs with homogeneous DAR values of 2 have lower hydrophobicity and aggregation effects than ADCs with average DAR values of 3.4-4 obtained by random binding methods, but the TI (therapeutic index) may be limited (such as PBD and Aur0101).

In some embodiments, k in the antibody conjugates of the present disclosure is selected from 2.0 to 2.5, including 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 or any value between two numbers.

In other embodiments, k in the antibody conjugates of the present disclosure is selected from 2.5 to 3.5, including 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.6, 3.7, 3.8, 3.9, 4.0 or any value between two numbers.

In other embodiments, the stretcher unit Str covalently linked to Ab is linked to, for example, an anti-folate receptor alpha (FRA) antibody by glycosylase catalysis.

On the other hand, the anti-folate antibody conjugates disclosed herein have a good inhibitory effect on the proliferation of FOLR1-positive tumor cells. For example, in some embodiments, the antibody conjugates disclosed herein inhibit KB (Cervix) cell proliferation with an IC ₅₀ of 0.01 to 1000 ng/mL. In some embodiments, the antibody conjugates disclosed herein inhibit KB (Cervix) cell proliferation with an IC ₅₀ of 0.01 to 500 ng/mL. In some embodiments, the antibody conjugates disclosed herein inhibit KB (Cervix) cell proliferation with an IC ₅₀ of <300 ng/mL. In some embodiments, the antibody conjugates disclosed herein inhibit KB (Cervix) cell proliferation with an IC ₅₀ of <200 ng/mL. In some embodiments, the antibody conjugates disclosed herein inhibit KB (Cervix) cell proliferation with an IC ₅₀ of <50 ng/mL.

In other embodiments, the antibody conjugates of the present disclosure inhibit SK-OV-3 (Ovarian) cell proliferation at an IC ₅₀ of 0.01 to 500 ng/mL. In some embodiments, the antibody conjugates of the present disclosure inhibit SK-OV-3 (Ovarian) cell proliferation at an IC ₅₀ of 0.01 to 300 ng/mL. In some embodiments, the antibody conjugates of the present disclosure inhibit SK-OV-3 (Ovarian) cell proliferation at an IC ₅₀ of <200 ng/mL. In some embodiments, the antibody conjugates of the present disclosure inhibit SK-OV-3 (Ovarian) cell proliferation at an IC ₅₀ of <100 ng/mL. In some embodiments, the antibody conjugates of the present disclosure inhibit SK-OV-3 (Ovarian) cell proliferation at an IC ₅₀ of <50 ng/mL.

In other embodiments, the antibody conjugates disclosed herein inhibit T47D cell proliferation at an IC50 of 0.01 to 500 ng/mL. In some embodiments, the antibody conjugates disclosed herein inhibit T47D cell proliferation at an IC50 of 0.01 to 300 ng/mL. In some embodiments, the antibody conjugates disclosed herein inhibit T47D cell proliferation at an IC50 of <200 ng/mL. In some embodiments, the antibody conjugates disclosed herein inhibit T47D cell proliferation at an IC50 of <100 ng/mL. In some embodiments, the antibody conjugates disclosed herein inhibit T47D cell proliferation at an IC50 of <50 ng/mL.

On the other hand, the present disclosure also provides isotope substitutions of the aforementioned antibody-conjugates. In some embodiments, the isotope substitutions are substituted with deuterium atoms.

On the other hand, the present disclosure also provides a pharmaceutical composition, which contains a therapeutically effective amount of the aforementioned antibody-drug conjugate, or an isotope substituted product thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

In certain embodiments, the pharmaceutical composition contains 0.01-99.99% of the aforementioned antibody-drug conjugate, or its isotope substitution, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned antibody-drug conjugate, or its isotope substitution. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned antibody-drug conjugate, or its isotope substitution. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned antibody-drug conjugate, or its isotope substitution. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned antibody-drug conjugate, or its isotope substitution.

In certain embodiments, the pharmaceutical composition contains 0.01%-99.99% of a pharmaceutically acceptable excipient based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 1%-99% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 2%-98% of a pharmaceutically acceptable excipient.

The present disclosure also provides a use of the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition in the preparation of a drug for treating or preventing a tumor. In some embodiments, the tumor is associated with the expression of folate receptor α.

The present disclosure also provides a use of the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition in the preparation of a drug for treating and/or preventing cancer. In some embodiments, the cancer is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

The present disclosure also provides a method for treating or preventing cancer patients associated with folate receptor α expression, by administering to the patient a therapeutically effective amount of the aforementioned antibody-drug conjugate, or an isotope substituted product thereof, or the aforementioned pharmaceutical composition.

The present disclosure also provides the aforementioned antibody-drug conjugate, or an isotope substitution thereof, or the aforementioned pharmaceutical composition for treating or preventing cancer associated with folate receptor α expression.

The active compound may be formulated for administration by any appropriate route, preferably in a unit dosage form, or in a form in which a patient can self-administer a single dose. The unit dosage form of the disclosed compound or composition may be tablets, capsules, cachets, bottled liquids, powders, granules, lozenges, suppositories, reconstituted powders or liquid preparations.

Detailed description of the invention

Unless otherwise defined, all technical and scientific terms used in the present disclosure are consistent with the common understanding of those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described in the present disclosure may be used to implement or test the present disclosure, the present disclosure describes preferred methods and materials. When describing and claiming the present disclosure, the following terms are used in accordance with the following definitions.

When a trade name is used in this disclosure, Applicants intend to include formulations of the trade name product, generic drugs, and the active drug portion of the trade name product.

Unless stated otherwise, the terms used in the specification and claims have the following meanings.

The compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present disclosure. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure form or in racemic form. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral raw materials or chiral reagents.

Optically active (R)- and (S)-isomers as well as D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is usually accomplished by using chromatography, which uses a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamates are generated from amines).

表示未指定构型，即如果化学结构中存在手性异构体，键

可以为

或

或者同时包含

和

两种构型。键

表示未指定构型，包括顺式(E)或反式(Z)构型。或者本公开所描述的

是指双键，以该键键合的结构可为“顺式异构体”或“反式异构体”或者“顺式异构体和反式异构体以任何比例形成的混合物”，例如式E代表E-1、式E-2或两者以任何比例形成的混合物：In the chemical structures of the compounds disclosed herein, the bond

Indicates that the configuration is not specified, that is, if there are chiral isomers in the chemical structure, the bond

Can be

or

or include both

and

Two configurations. Key

indicates an unspecified configuration, including cis (E) or trans (Z) configurations.

Refers to a double bond, and the structure bonded by this bond can be a "cis isomer" or a "trans isomer" or a "mixture of cis isomers and trans isomers in any proportion". For example, Formula E represents E-1, Formula E-2 or a mixture of the two in any proportion:

并未指定构型，即可以为Z构型或E构型，或者同时包含两种构型。In the chemical structures of the compounds disclosed herein, the bond

The configuration is not specified, that is, it can be Z configuration or E configuration, or contain both configurations.

Compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also referred to as prototransfer tautomers) include interconversion via proton migration, such as keto-enol and imine-enamine, lactam-lactim isomerization. Lactam-lactim equilibrium examples are between A and B as shown below.

All compounds in the present disclosure can be drawn as either Form A or Form B. All tautomeric forms are within the scope of the present disclosure. The naming of the compounds does not exclude any tautomers.

The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from that normally found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ^{13 N, 15} N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ¹²³ I, ¹²⁵ I, and ³⁶ Cl, ^respectively .

Unless otherwise specified, when a position is specifically designated as deuterium (D), the position is understood to have deuterium (i.e., at least 10% deuterium incorporation) at least 1000 times greater than the natural abundance of deuterium (which is 0.015%). The natural abundance of the compound in the example may be at least 1000 times greater than deuterium, at least 2000 times greater than deuterium, at least 3000 times greater than deuterium, at least 4000 times greater than deuterium, at least 5000 times greater than deuterium, at least 6000 times greater than deuterium or more. The present disclosure also includes various deuterated forms of the formula (I) compound. Each available hydrogen atom connected to a carbon atom may be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of the formula (I) compound with reference to the relevant literature. In preparing deuterated forms of compounds of formula (I), commercially available deuterated starting materials may be used, or they may be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane, and the like.

"Optionally" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "C _1-6 alkyl optionally substituted with halogen or cyano" means that halogen or cyano may but need not be present, and the description includes instances where the alkyl is substituted with halogen or cyano and instances where the alkyl is not substituted with halogen and cyano.

"Pharmaceutical composition" means a mixture containing one or more compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredients, and thus exert biological activity.

"Pharmaceutically acceptable excipients" include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration for use in humans or domestic animals.

As used herein, an "effective amount" or "therapeutically effective amount" includes an amount sufficient to improve or prevent the symptoms or symptoms of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

The term "drug" refers to a cytotoxic drug or an immunomodulator. Cytotoxic drugs are chemical molecules that have strong ability to destroy the normal growth of tumor cells. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations, but due to the lack of specificity, while killing tumor cells, they can also cause apoptosis of normal cells, leading to serious side effects. The term includes toxins, such as small molecule toxins or enzyme-active toxins from bacteria, fungi, plants or animals, radioactive isotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes. Immunomodulators are inhibitors of immune checkpoint molecules.

The terms "linker", "linking unit", "linker unit", "linker" or "linking fragment" refer to a chemical structure fragment or bond that is connected to a ligand at one end and to a drug at the other end, and can also be connected to other linkers before being connected to the drug.

The joint can include one or more joint components. Exemplary joint components include 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those derived from coupling with joint reagents: N-succinimidyl 4-(2-pyridylthio) valerate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC, also referred to as MCC in this article) and N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB). The joint can include a stretching unit, a spacer unit, an amino acid unit and an extension unit. It can be synthesized by methods known in the art, such as described in US2005-0238649A1. The joint can be a "cleavable joint" that is convenient for releasing drugs in cells. For example, an acid-labile linker (eg, hydrazone), a protease-sensitive (eg, peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020) can be used.

The term "stretcher" refers to a chemical structural fragment that is covalently linked to an antibody via a carbon atom at one end and is linked to an amino acid unit, a disulfide moiety, a sulfonamide moiety, or a non-peptide chemical moiety at the other end.

The term "spacer unit" refers to a bifunctional compound structural fragment that can be used to couple an amino acid unit and a cytotoxic drug to ultimately form an antibody-drug conjugate. This coupling method can selectively link the cytotoxic drug to the amino acid unit.

The term "amino acid" refers to an organic compound that contains an amino group and a carboxyl group in its molecular structure, and both the amino group and the carboxyl group are directly connected to the -CH- structure. The general formula is H ₂ NCHRCOOH, where R is H, substituted or unsubstituted alkyl, etc. Depending on the position of the amino group attached to the carbon atom in the carboxylic acid, it can be divided into α, β, γ, δ, ε...-amino acids. In the biological world, the amino acids constituting natural proteins have their specific structural characteristics, that is, their amino groups are directly connected to the α-carbon atom, i.e., α-amino acids, including glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, proline, etc. Non-natural amino acids such as citrulline. As known to those skilled in the art, non-natural amino acids do not constitute natural proteins and therefore do not participate in the synthesis of antibodies in the present disclosure. The three letter codes and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

The spacer unit in the present disclosure is PAB, the structure of which is a p-aminobenzyloxycarbonyl fragment, the structure of which is shown in formula (VI), connected to D,

Joint components include but are not limited to:

MC = 6-maleimidocaproyl, the structure is as follows:

Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease cleavable linker)

Citrulline = 2-amino-5-ureidopentanoic acid

Me-Val-Cit = N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to protect it from cleavage by cathepsin B)

MC(PEG) ₆ -OH = Maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteine)

SPP = N-succinimidyl 4-(2-pyridylthio)pentanoate

SPDP = N-succinimidyl 3-(2-pyridyldithio) propionate

SMCC = succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate

IT = Iminothiolane

PBS = phosphate buffered saline.

The term "antibody-drug conjugate" refers to a ligand connected to a biologically active drug via a stable linker. In the present disclosure, "antibody-drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment connected to a biologically active toxic drug via a stable linker.

The term "drug loading" can be expressed as the ratio of the amount of drug to the amount of antibody, i.e., the average number of drugs coupled to each antibody in the ADC. The range of drug loading can be 1-20, preferably 1-10 cytotoxic drugs (D) connected to each antibody (Ab). In the embodiments of the present disclosure, drug loading is expressed as k, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the mean of any two values. Preferably 1-10, more preferably 1-8, or 2-8, or 2-7, or 3-8, or 3-7, or 3-6, or 4-7, or 4-6, or 4-5 mean. Conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA test, monoclonal antibody molecular size variant assay (CE-SDS) and HPLC feature identification of the average number of drugs per ADC molecule after the coupling reaction.

The monoclonal antibody molecular size variant determination method (CE-SDS) disclosed in the present invention can adopt the sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method to quantitatively determine the purity of the recombinant monoclonal antibody product according to the capillary electrophoresis method (2015 edition of the "Chinese Pharmacopoeia" 0542) based on the molecular weight under reducing and non-reducing conditions.

The loading capacity of the antibody-drug conjugate can be controlled by the following non-limiting methods, including:

(1) Control the molar ratio of the linking reagent and the monoclonal antibody,

(2) Control the reaction time and temperature,

(3) Select different reaction reagents.

The term "antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain respectively. The same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains or λ chains according to the difference in constant regions. Each of the five classes of Ig can have κ chains or λ chains. The antibodies disclosed herein are preferably specific antibodies against cell surface antigens on target cells. The sequences of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain vary greatly, which is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conservative framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

The antibodies disclosed herein include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, with humanized antibodies and fully human antibodies being preferred.

The term "murine antibody" in this disclosure refers to antibodies prepared in mice according to the knowledge and skills in the art. When preparing, a test subject is injected with a specific antigen, and then a hybridoma expressing an antibody with the desired sequence or functional characteristics is isolated.

The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma that secretes mouse-specific monoclonal antibodies, then clone the variable region gene from the mouse hybridoma cells, and then clone the constant region gene of the human antibody as needed, connect the mouse variable region gene with the human constant region gene into a chimeric gene, and then insert it into an expression vector, and finally express the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.

The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting the mouse CDR sequence into the human antibody variable region framework, that is, different types of human germline antibody framework sequences. The heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components can be overcome. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase), and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity. The humanized antibodies disclosed in the present invention also include humanized antibodies after further affinity maturation of CDR by phage display. References further describing methods for humanizing mouse antibodies include, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and the methods of Winter and colleagues [Jones et al., Nature, 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].

The terms "fully human antibody", "fully human antibody" or "completely human antibody", also known as "fully human monoclonal antibody", are antibodies whose variable and constant regions are both human, eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The present disclosure is a fully human monoclonal antibody. The relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.

The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of an antibody. Examples of binding fragments included in "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') ₂ fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bridge on the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of a single arm of an antibody; (v) single domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) combinations of two or more isolated CDRs, optionally connected by synthetic linkers. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, so that they can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies can be of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibodies.

Fab is an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity among fragments obtained by treating IgG antibody molecules with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which about half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond.

F(ab')2 is an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, obtained by digesting the portion below two disulfide bonds in the hinge region of IgG with the enzyme pepsin, and comprising two Fab regions linked at the hinge position.

Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F(ab')2.

Furthermore, the Fab' fragment of the antibody can be produced by inserting a DNA encoding the Fab' fragment into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic organism or a eukaryotic organism to express the Fab'.

The term "single-chain antibody", "single-chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. 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 variants using 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers useful in the present disclosure 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.

The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDR applies only to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering convention (see Lefranc M.P., Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc. Following the IMGT convention, the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.

The term "antibody framework" refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-continuous amino acids in a unique spatial conformation (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E.Morris, Ed. (1996)).

The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the ^binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of less than about ^10-7 M, for example, less than about ^10-8 M, ^10-9 M or ^10-10 M or less.

The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which other DNA segments can be connected. In another embodiment, the vector is a viral vector, in which other DNA segments can be connected to the viral genome. The vector disclosed herein can be autonomously replicated in the host cell introduced therein (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) or can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome (e.g., non-additional mammalian vectors).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the prior art, such as the Cold Spring Harbor Guide to Antibody Experimental Techniques, Chapters 5-8 and 15. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR region. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulins, 2001 ISBN012441351.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plants or animal cells. Easily transformed bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary Cell Line) and NS0 cells.

The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified by conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product must be immediately frozen, such as -70°C, or lyophilized.

Amino acid sequence "identity" refers to the percentage of amino acid residues in a first sequence that are identical to the amino acid residues in a second sequence, while aligning the amino acid sequences and, if necessary, introducing gaps to achieve maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be achieved in a variety of ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

The term "peptide" refers to a compound fragment between amino acids and proteins. It is composed of two or more amino acid molecules connected to each other by peptide bonds. It is a structural and functional fragment of protein. Hormones, enzymes, etc. are essentially peptides.

The term "sugar" refers to a biological macromolecule composed of three elements: C, H, and O, which can be divided into monosaccharides, disaccharides, and polysaccharides.

The term "fluorescent probe" refers to a class of fluorescent molecules that have characteristic fluorescence in the ultraviolet-visible-near infrared region, and whose fluorescence properties (excitation and emission wavelengths, intensity, lifetime and polarization, etc.) can sensitively change with the properties of the environment, such as polarity, refractive index, viscosity, etc. They non-covalently interact with nucleic acids (DNA or RNA), proteins or other macromolecular structures to change one or several fluorescence properties, and can be used to study the properties and behaviors of macromolecules.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, 1-ethyl-2-methylpropyl, and various branched isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl.

The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.

"Monovalent group" refers to a compound that "formally" eliminates a monovalent atom or group. "Subunit" refers to a compound that "formally" eliminates two monovalent or one divalent atoms or groups. Example "alkyl" refers to the remaining part after removing one hydrogen atom from an alkane molecule, including straight-chain and branched monovalent groups of 1 to 20 carbon atoms. Alkyl groups containing 1 to 6 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl and various branched isomers thereof.

The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of an alkane radical by removing two hydrogen atoms, and is a straight or branched group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, and more preferably an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (—CH ₂ —), 1,1-ethylene (—CH(CH ₃ )—), 1,2-ethylene (—CH ₂ CH ₂ )—, 1,1-propylene (—CH(CH ₂ CH ₃ )—), 1,2-propylene (—CH ₂ CH(CH ₃ )—), 1,3-propylene (—CH ₂ CH ₂ CH ₂ —), 1,4-butylene (—CH ₂ CH ₂ CH ₂ CH ₂ —), and 1,5-butylene (—CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ —). Alkylene groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available attachment point, and the substituents are preferably independently selected from one or more of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, alkoxy, and cycloalkyl. Similarly, “alkenylene” is as defined above.

The term "alkoxy" refers to-O-(alkyl) and-O-(unsubstituted cycloalkyl), wherein the definition of alkyl or cycloalkyl is as described above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyls include spirocyclic, fused and bridged cycloalkyls.

The term "heterocycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are heteroatoms selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocycloalkyls include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocycloalkyls include spirocyclic, fused ring and bridged heterocycloalkyls.

The term "spiro heterocycloalkyl" refers to a polycyclic heterocycloalkyl group that shares one atom (called spiro atom) between 5 to 20 monocyclic rings, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated π electron system. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of spiro atoms shared between rings, spiro heterocycloalkyl is divided into single spiro heterocycloalkyl, double spiro heterocycloalkyl or multi-spiro heterocycloalkyl, preferably single spiro heterocycloalkyl and double spiro heterocycloalkyl. More preferably 4 yuan/4 yuan, 4 yuan/5 yuan, 4 yuan/6 yuan, 5 yuan/5 yuan or 5 yuan/6 yuan single spiro heterocycloalkyl. Non-limiting examples of spiro heterocycloalkyl include:

The term "fused heterocycloalkyl" refers to a polycyclic heterocycloalkyl group of 5 to 20 yuan, each ring in the system shares a pair of adjacent atoms with other rings in the system, one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S (O) _m (wherein m is an integer 0 to 2) heteroatoms, and the remaining ring atoms are carbon. Preferably 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocycloalkyl, preferably bicyclic or tricyclic, more preferably 5 yuan/5 yuan or 5 yuan/6 yuan bicyclic fused heterocycloalkyl. Non-limiting examples of fused heterocycloalkyl include:

The term "bridged heterocycloalkyl" refers to a 5 to 14-membered polycyclic heterocycloalkyl group in which any two rings share two atoms that are not directly connected, which may contain one or more double bonds, but no ring has a completely conjugated π electron system, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocycloalkyl, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocycloalkyl include:

The heterocycloalkyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocycloalkyl, non-limiting examples of which include:

等。

wait.

The heterocycloalkyl radical may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.

The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocycloalkyl or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, non-limiting examples of which include:

The aryl group may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl is preferably 5 to 10 yuan, more preferably 5 or 6 yuan, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to an aryl, heterocycloalkyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include:

The heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

The term "aminoheterocycloalkyl" refers to a heterocycloalkyl group substituted by one or more amino groups, preferably by one amino group, wherein heterocycloalkyl is as defined above, and wherein "amino" refers to -NH ₂ . Representative embodiments of the present disclosure are as follows:

The term "heterocycloalkylamino" refers to an amino group substituted by one or more heterocycloalkyl groups, preferably by one heterocycloalkyl group, wherein the amino group is as defined above, and wherein the heterocycloalkyl group is as defined above. Representative embodiments of the present disclosure are as follows:

The term "cycloalkylamino" refers to an amino group substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein the amino group is as defined above, and wherein the cycloalkyl group is as defined above. Representative embodiments of the present disclosure are as follows:

The term "cycloalkylalkyl" refers to an alkyl group substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein alkyl is as defined above and wherein cycloalkyl is as defined above.

The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

The term "hydroxy" refers to an -OH group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "amino" refers to _-NH2 .

The term "nitro" refers to _-NO2 .

The abbreviation "Me" in the chemical formula is methyl.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocycloalkyl group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and the description includes instances where the heterocycloalkyl group is substituted with an alkyl group and instances where the heterocycloalkyl group is not substituted with an alkyl group.

"Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are replaced independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and the skilled person can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxy groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (e.g. olefinic) bonds.

The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt of the antibody-drug conjugate of the present disclosure, or a salt of a compound described in the present disclosure, which is safe and effective when used in mammals and has the desired biological activity. The antibody-drug conjugate of the present disclosure contains at least one amino group, and thus can form a salt with an acid, and non-limiting examples of pharmaceutically acceptable salts include: hydrochloride.

The term "solvate" refers to a pharmaceutically acceptable solvate formed by the ligand-drug conjugate compound of the present disclosure and one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate.

The term "drug carrier" is used for the drugs disclosed herein and refers to a system that can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug, and deliver the drug to the targeted organ. The drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure, and preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, and can be used as excellent drug carriers.

The term "excipient" refers to the additives in pharmaceutical preparations other than the main drug, which can also be called auxiliary materials. For example, the binders, fillers, disintegrants, and lubricants in tablets; the matrix part in semi-solid preparations such as ointments and creams; the preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. in liquid preparations can all be called excipients.

The term "diluent" is also called filler, and its main purpose is to increase the weight and volume of the tablet. The addition of diluent not only ensures a certain volume, but also reduces the dosage deviation of the main ingredients, improves the compression molding of the drug, etc. When the drug in the tablet contains oily components, an absorbent needs to be added to absorb the oily substance and keep it in a "dry" state to facilitate tableting.

In this article, the preparation method of anti-folate receptor alpha (FRA) antibodies or antigen-binding fragments thereof can be found in WO2017151979 and the relevant content is introduced herein for illustration.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Comparison of the bystander effect of ADC-001 in T47D/A549 cell lines.

DETAILED DESCRIPTION

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

The experimental methods in the examples disclosed herein that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by raw material or product manufacturers. Reagents that do not specify specific sources are conventional reagents purchased from the market.

The structures of the compounds were determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). NMR shifts (δ) are given in units of 10 ^-6 (ppm). NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ), deuterated methanol (CD ₃ OD) as the measuring solvent, and tetramethylsilane (TMS) as the internal standard.

MS was measured using Agilent 1200/1290 DAD-6110/6120 Quadrupole MS liquid spectrometer-mass spectrometer (manufacturer: Agilent, MS model: 6110/6120 Quadrupole MS).

High performance liquid chromatography (HPLC) analysis was performed using Agilent HPLC 1200DAD, Agilent HPLC1200VWD and Waters HPLC e2695-2489.

Chiral HPLC analysis was performed using an Agilent 1260DAD high performance liquid chromatograph.

High performance liquid chromatography was performed using Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 preparative chromatographs.

RP-HPLC analysis was performed using an Agilent 1260 InfinityⅡ liquid chromatograph, chromatographic column: Shimadzu SEC-300 3um×7.8mm×300mm, mobile phase: 0.1% trifluoroacetic acid in water/0.1% trifluoroacetic acid in acetonitrile (ACN) solution.

Chiral preparations were performed using a Shimadzu LC-20AP preparative chromatograph.

The CombiFlash rapid preparation instrument used Combiflash Rf200 (TELEDYNE ISCO).

The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The silica gel plate used in thin layer chromatography (TLC) adopts a specification of 0.15mm-0.2mm, and the specification used for thin layer chromatography separation and purification products is 0.4mm-0.5mm.

Silica gel column chromatography generally uses Yantai Huanghai Silica Gel 200-300 mesh silica gel as the carrier.

The known starting materials disclosed in the present invention can be synthesized by methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, Darui Chemicals and other companies.

Unless otherwise specified in the examples, the reactions can be carried out under an argon atmosphere or a nitrogen atmosphere.

Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a volume of about 1L.

Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.

Unless otherwise specified in the examples, the solution refers to an aqueous solution.

Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

The reaction progress in the embodiment is monitored by thin layer chromatography (TLC), the developing solvent used in the reaction, the system of column chromatography eluent used for purifying the compound and the developing solvent system for thin layer chromatography, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid can be added for adjustment.

Example 1

Step 1: Preparation of compound 1b

Under ice water bath, compound 1a (eribulin, prepared according to ZL201010236637.2) (72.91 mg, 0.1 mmol) was dissolved in 10 mL of tetrahydrofuran, Fmoc-OSu (fluorenylmethoxycarbonyl succinimide, 41 mg, 0.12 mmol) was added, and then stirred at room temperature until the reaction was complete. The crude product was concentrated under reduced pressure and used directly in the next step.

Step 2: Preparation of compound 1c

The crude compound 1b obtained in the previous step was dissolved in 10 mL of anhydrous ether, and silver oxide (34.8 mg, 0.15 mmol) was added, followed by iodomethane (28.4 mg, 0.2 mmol). The mixture was stirred at room temperature until the reaction was complete, filtered, and then concentrated under reduced pressure to obtain a crude product, which was directly subjected to the next step of reaction.

Step 3: Preparation of compound 1

The crude compound 1c obtained in the previous step was dissolved in 10 mL of tetrahydrofuran, and 2 mL of diethylamine was added. The mixture was stirred at room temperature until the reaction was complete, and the crude product was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane/ethyl acetate/petroleum ether) to obtain 3 mg of the target product compound 1.

LC/MS(ESI): m/z 744.2[M+H] ⁺ .

Under ice-water bath, 4a (50 mg, 0.08 mmol, prepared according to the method in WO2017151979) was dissolved in 1.5 mL of N,N-dimethylformamide, and then DIPEA (N,N-diisopropylethylamine, 18 mg, 0.14 mmol) was added, followed by di(p-nitrobenzene) carbonate (49 mg, 0.16 mmol). After stirring at room temperature for 10 min, 20 mL of methyl tert-butyl ether was added, stirred for 20 min, filtered, and dried to obtain 36 mg of a solid, LC/MS (ESI): m/z 784.1 [M+H] ⁺ .

Compound 1 (13.5 mg, 0.018 mmol) was dissolved in 1.5 mL of DMF, DIPEA (7 mg, 0.054 mmol) was added, and then compound 4b (18 mg, 1.3 mmol) was added in batches. The mixture was stirred until the reaction was almost complete, and the crude product was concentrated. 6.5 mg of compound L-1 was obtained by HPLC separation with a purity of 96.95%. LC/MS (ESI): m/z 1388.3 [M+H] ⁺ .

Example 2

At 37°C, add the prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) aqueous solution (10mM, 21.3uL, 21.34mmol) to the PBS buffered aqueous solution of anti-folate receptor α (FRA) antibody (pH=6.5 0.05M PBS buffered aqueous solution; 10.0mg/mL, 1.43mL, 97nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

Compound L-1 (1.07 mg, 771 nmol) was dissolved in 50 uL dimethyl sulfoxide, added to the above reaction solution, placed in a water bath oscillator, and oscillated at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH = 6.5, containing 0.001 M EDTA) to obtain the title product in PBS buffer (1.21 mg/mL, 10.8 mL), which was stored in a refrigerator at 4°C. The average DAR value was calculated by the RP-HPLC detection method: n = 4.

Comparative Example 1

Prepared according to the method of CN201780022381.5, wherein the average DAR value was calculated by the RP-HPLC detection method: n=4.

Test Example 1: In vitro cytotoxic activity screening of compound 1

1.1. Experimental principles and methods

This experiment uses CTG to detect ATP content, reflecting the survival of tumor cells. First, by planting cells at different densities and culturing for 3 and 5 days, the final culture conditions are determined based on IC ₅₀ and maximum inhibition rate. Then, the killing effect of the toxin molecules is tested under these conditions.

1.2. Cell line selection

According to the purpose of the experiment, two disease models of breast cancer and NSCLC were selected, and three cell lines, SKBR3 tumor cells (HER2+, ATCC, catalog number HTB-30), MDA-MB-468 (HER2-, ATCC, catalog number HTB-132) and A549 (human non-small cell lung cancer cells, ATCC, catalog number CCL-185), were selected for screening experiments.

1.3. Determination of cell culture conditions

1) Cell culture: A549, SK-BR-3 and MDA-MB-468 cells were cultured with Ham's F-12K (Kaighn's) medium (Gibco, 21127030) and McCoy's 5A medium (ThermoFisher, Catalog No. 16600108) containing 10% FBS (Gibco, 10099-141) and Leibovitz's L-15 medium (ThermoFisher, Catalog No. 11415-114), respectively.

2) Cell plating: After A549 was digested with trypsin, it was terminated with the above culture medium. After counting, 4.3×10 ⁵ , 7.2×10 ⁵ , and 11.5×10 ⁵ cells were taken and culture medium was added to make the final volume 26 ml. 180ul of cell suspension was added to each well of the 2nd to 11th columns of a 96-well plate (Corning, Cat. No. 3903), so that the cell density was 3K, 5K, and 8K per well, respectively. 200ul of culture medium was added to the 12th column, and the remaining wells were filled with PBS. Repeat the above operation for SKBR3 and MDA-MB-468 cells. Two parallel copies were made.

2) Drug preparation: In a round-bottom 96-well plate (Corning, catalog number 3788), the positive control eribulin and the stock solution of the disclosed compound were prepared with DMSO. 2mM was prepared in the first column of the preparation plate 1 (the stock solution was diluted 10 times with DMSO), and then the gradient was diluted 10 times in DMSO to the 10th column, and the 11th column was DMSO. 95ul of the corresponding culture solution was added to each well of the 2nd to 11th columns of the preparation plate 2, and 5ul of the solution was drawn from the 2nd to 11th columns of the preparation plate 1 to the preparation plate 2. After mixing, 20ul was drawn and added to the plated cells, and the culture was continued for 3 days and 5 days.

3) CTG assay (Cell Titer-GloTM, luminescent cell viability assay, Promega): The cell plates were removed on the 3rd and 5th days, and equilibrated to room temperature. 90ul CTG was added to each well, and the cells were reacted at room temperature in the dark for 10 minutes. The luminescence value was read by a microplate reader and the IC ₅₀ was calculated.

1.4. Data Results

Table 1

Test Example 2: Pharmacokinetic Test of Compound 1

1. Overview

的比格犬为受试动物，应用LC/MS/MS法测定了比格犬静脉注射给予化合物D-1和艾日布林后不同时刻血浆中的药物浓度。研究本公开化合物在犬体内的药代动力学行为，评价其药动学特征。Non-

Beagles were used as test animals, and the drug concentrations in the plasma of beagles at different times after intravenous injection of compound D-1 and eribulin were determined by LC/MS/MS. The pharmacokinetic behavior of the disclosed compound in dogs was studied, and its pharmacokinetic characteristics were evaluated.

2. Experimental plan

2.1 Trial Drugs

Compound 1 and Eribulin

2.2 Experimental animals

Six male beagle dogs were equally divided into two groups and purchased by Medicipia Pharmaceutical Technology (Shanghai) Co., Ltd. for animal drug administration experiments.

2.3 Drug preparation

Compound D-1 was weighed, and 5% volume of DMSO, 20% PG and 20% PEG400 were added to dissolve it, and then 55% physiological saline was added to prepare a 0.25 mg/ml colorless clear solution.

Eribulin was weighed, and 5% volume of DMSO, 20% PG and 20% PEG400 were added to dissolve it, and then 55% physiological saline was added to prepare a 0.25 mg/ml colorless clear solution.

2.4 Administration

A group of dogs were intravenously administered with compound D-1, the dosage was 0.5 mg/kg, and the administration volume was 2 ml/kg.

Another group of dogs were given eribulin intravenously, with the dose of 0.5 mg/kg and the administration volume of 2 ml/kg.

3. Operation

Dogs were injected with compound D-1, and 1 ml of blood was collected before administration and at 5 minutes, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, and 24.0 hours after administration. The collected blood samples were placed in EDTA-K2 anticoagulant blood collection tubes, and the collected whole blood was placed on ice and centrifuged within 1 hour to separate plasma (centrifugal force 2200g, centrifugation 10min, 2-8°C). The plasma samples were stored in a -80°C refrigerator before testing.

Dogs were injected with eribulin compounds, and 1 ml of blood was collected before administration and 5 minutes, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, and 24.0 hours after administration. The collected blood samples were placed in EDTA-K2 anticoagulant blood collection tubes, and the collected whole blood was placed on ice and centrifuged to separate plasma within 1 hour (centrifugal force 2200g, centrifugation 10min, 2-8°C). Plasma samples were stored in a -80°C refrigerator before testing.

Determination of the content of the test compound in dog plasma after drug injection: Take 25 μl of dog plasma at each time after administration, add 50 μl (100 ng/mL) of internal standard solution camptothecin (China National Institute for the Control of Biological Products) and 200 μl of acetonitrile, vortex mix for 5 minutes, centrifuge for 10 minutes (3700 rpm), and take 3-4 μl of the supernatant of the plasma sample for LC/MS/MS (API4000 triple quadrupole tandem mass spectrometer (No. 2), Applied Biosystems, USA; Shimadzu LC-30AD ultra high performance liquid chromatography system, Shimadzu, Japan) analysis.

4. Pharmacokinetic parameter results

The pharmacokinetic parameters of the disclosed compounds are shown in Table 2 below.

Table 2

Test Example 3: Cell proliferation inhibitory activity

1. Test samples

Compound 1 and Eribulin

2. Experimental methods

1) Digest the cells with trypsin, count them, and centrifuge them;

2) Adjust the cell density as needed. For SK-OV-3 and Cal-51 cells, mix the cells with 5% FBS medium and adjust the cell density. For OVCAR3 and T47D cells, mix the cells with 10% FBS medium and adjust the cell density.

3) Inoculate cells into 96-well plates at 75 μL/well. 750 cells per well for SK-OV-3 and Cal-51 cells, and 1000 cells per well for OVCAR3 and T47D cells, overnight, and dilute with 2x drug.

4) Incubate at 37°C for another 72 h, replace with 130 μL of fresh culture medium, add 20 μL of 7.5x concentration drug, and incubate at 37°C for 48 h;

5) Equilibrate the cell plate and CellTiter Glo reagent to room temperature;

6) Add 50 μL of CellTiter Glo reagent to each well and mix at room temperature for 10 min. Place the plate on a multifunctional microplate reader to read the chemiluminescence value.

3. Experimental results

Table 3

Test Example 4: Antibody-dependent cell-mediated cytotoxicity

1. Test samples

Antibody conjugates ADC-001 and ADC-002;

Farletuzumab comes from Shanghai Maijin Biopharmaceutical Technology Co., Ltd.

2. Experimental methods

1) Cal-51 cells were digested into single cells using trypsin and centrifuged;

2) Adjust the cell density to 1×10 ⁵ cells/mL with culture medium, inoculate into 96-well plates at 100 μL/well, incubate overnight, prepare antibodies with RPMI 1640 culture medium containing 20 ng/mL IL-2, add 50 μL to each well, and replicate for each concentration;

3) Remove NK92-CD16A-176V cells and adjust the cell density to 1×10 ⁶ cells/mL using RPMI 1640 medium containing 20 ng/mL IL-2;

4) NK92-CD16A-176V cells were seeded into the corresponding wells at 50 μL/well and the cells were placed in an incubator for 4 h;

5) Take out the plate, take out 100 μL of culture supernatant from each well and equilibrate to room temperature;

6) Add 50 μL CTG to each well, shake at room temperature for 10 min, and then detect chemiluminescence.

Calculate the specific killing ratio, the calculation formula is: specific killing % = (target cells + effector cell control group - target cells + effector cell treatment group) / target cells * 100

3. Experimental results

Table 4

Test Case 5: Bystander Effect

1. Test samples

Antibody conjugates ADC-001 and ADC-002

2. Experimental methods

1) Digest T47D and A549 into single cells with trypsin and centrifuge;

2) The cell density was adjusted to 1×10 ⁶ cells/mL, and T47D and A549 were labeled with CellTrace ^TM Violet (CTV) and CellTrace ^TM CFSE (CFSE), respectively;

3) After labeling, 6.5×10 ⁴ cells T47D and 1.3×10 ⁴ cells A549 were inoculated into each well of a 24-well plate in 500 μL, with 3 replicates for each concentration, overnight;

4) Add 500 μL of 2x drug-containing culture medium (final concentration 20 nM) to each well and treat for 48 h;

5) The cells were digested with trypsin and collected, and the number of CTV or CFSE-positive cells was analyzed by flow cytometry.

4. Experimental results

The experimental results showed that folate receptor-negative cells A549 were insensitive to ADC-001 and ADC-002 when cultured alone. However, when co-cultured with folate receptor-positive cells T47D, A549 cells became more sensitive to ADC-001 and ADC-002 due to bystander killing.

Test Example 6: In vivo anti-tumor inhibitory effect on Cal-51 model

1. Test samples

Antibody conjugates ADC-001 and ADC-002

2. Experimental methods

2.1 Experimental animals

BALB/c-nude mice, 6 weeks old, female, obtained from the Experimental Animal Management Department of Shanghai Institute of Family Planning Sciences.

2.2 Experimental procedures

Each nude mouse was subcutaneously inoculated with 5x10 ⁶ Cal-51 cells (from ATCC). When the tumor volume reached 150-200 mm ³ , the animals were divided into groups according to the tumor volume and the mice were intravenously injected (IV). The specific dosage and dosing schedule are shown in Table 5. The tumor volume was measured twice a week, the mice were weighed, and the data were recorded.

Table 5 Dosage regimen

2.3 Experimental indicators

The experimental index is to examine the effect of the drug on tumor growth, and the specific index is T/C% or tumor inhibition rate TGI (%).

Tumor diameter was measured with a vernier caliper twice a week, and tumor volume (V) was calculated as:

V = 1/2 × a × b ² where a and b represent length and width respectively.

T/C (%) = (T-T0)/(C-C0) × 100, where T and C are the tumor volumes at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.

Tumor inhibition rate (TGI) (%) = 100-T/C (%).

When the tumor regresses, tumor inhibition rate (TGI) (%) = 100-(T-T0)/T0×100

If the tumor is smaller than its initial volume, that is, T<T0 or C<C0, it is defined as partial tumor regression (PR); if the tumor disappears completely, it is defined as complete tumor regression (CR).

At the end of the experiment, when the experimental endpoint was reached, or when the average tumor volume of the solvent group reached 1500 mm ³ , the animals were killed by CO ₂ anesthesia, and then the tumors were dissected and photographed.

2.4 Statistical analysis

Unless otherwise specified, the tumor volumes between the two groups were compared using the two-tailed Student's t test, and P < 0.05 was defined as a statistically significant difference.

3 Experimental results

Table 6

At 1.5 cmpk, the tumor inhibition rates of antibody-drug conjugates ADC-001 and ADC-002 (positive control group) on human breast cancer cells were 47% and 0, respectively. The anti-tumor activity of ADC-001 was better than that of the positive control ADC-002. At 5 mpk, the antibody-drug conjugates ADC-001 and ADC-002 (positive control group) had almost equivalent anti-tumor activity. This indicates that the effective concentration of ADC-001 is lower. At the same time, tumor-bearing mice can tolerate the above drugs well, and there is no significant weight loss.

Claims (24)

1. An antibody-drug conjugate having a structure shown in Formula I: Ab-(LD) _k or a pharmaceutically acceptable salt or solvate thereof (I) Wherein, Ab is an anti-folate receptor α (FRA) antibody or an antigen-binding fragment thereof, L is a linker that covalently links Ab to D, and k is 1 to 20, -D is as follows:

2. The antibody-drug conjugate according to claim 1, wherein k is selected from 1 to 10 and can be an integer or a decimal. 3. The antibody-drug conjugate of claim 1 or 2, wherein the linker comprises a cleavable peptide portion. 4 . The antibody-drug conjugate according to claim 3 , wherein the cleavable peptide portion is cleavable by an enzyme, preferably by a cathepsin, and further the cathepsin is preferably cathepsin B. 5. The antibody-drug conjugate according to claim 3 or 4, wherein the linker comprises an amino acid unit, and the amino acid unit preferably comprises a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, more preferably valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe) or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly). 6. The antibody-drug conjugate of claim 1 or 2, wherein the linker comprises a cleavable sulfonamide moiety. 7. The antibody-drug conjugate according to any one of claims 1 to 3, wherein the linker comprises a cleavable disulfide moiety. 8. The antibody-drug conjugate according to claim 6 or 7, wherein the linker is cleavable under reducing conditions. 9. The antibody-drug conjugate according to any one of claims 1 to 8, wherein the linker comprises a spacer unit connected to D. 10. The antibody-drug conjugate of claim 9, wherein the spacer unit comprises p-aminobenzyloxycarbonyl (pAB). 11. The antibody-drug conjugate according to claim 9, wherein the spacer unit comprises

Wherein, Z ₁ to Z ₄ are selected from carbon atoms or nitrogen atoms; R ⁴ is selected from alkyl, cycloalkyl, aryl and heteroaryl, and the alkyl, cycloalkyl, aryl and heteroaryl are each independently substituted by one or more substituents selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl; R ¹ and R ² are each independently selected from hydrogen, C _1-6 alkyl, haloalkyl or C _3-6 cycloalkyl, preferably hydrogen; or, R ¹ and R ² together with the carbon atom to which they are connected form a C _3-6 cycloalkyl; X is selected from -O- or -NH-; L is selected from an integer between 1 and 4; Q is VE, VE provides a glycosidic bond that can be cleaved by intracellular glycosidases, E is selected from -O-, -S- or -NR ³ -, R ³ is selected from hydrogen or methyl; further, V is selected from

wherein R ⁵ is selected from -COOH or CH ₂ OH. 12. The antibody-drug conjugate according to any one of claims 1 to 11, wherein L-D is a chemical moiety represented by the following formula: -Str-(Pep)-Sp-D, Str is a stretching unit covalently linked to Ab, Sp is a spacer unit, Pep is selected from amino acid units. 13. The antibody-drug conjugate of claim 12, wherein Str is selected from a chemical moiety represented by the following formula:

wherein R ⁶ is selected from -WC(O)-, -C(O)-WC(O)-, -(CH ₂ CH ₂ O) _p1 C(O)-, -(CH ₂ CH ₂ O) _p1 CH ₂ C(O)-, -(CH ₂ CH ₂ O) _p1 CH ₂ CH ₂ C(O)-, wherein W is selected from C _1-8 alkylene, C _1-8 alkylene-cycloalkyl or straight-chain heteroalkylene of 1 to 8 atoms, wherein the heteroalkylene contains 1 to 3 heteroatoms selected from N, O or S, wherein the C _1-8 alkylene, C _1-8 alkylene-cycloalkyl and straight-chain heteroalkylene are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, alkoxy and cycloalkyl; L ¹ is selected from -NR ⁷ (CH ₂ CH ₂ O) _p1 CH ₂ CH ₂ C(O)-, -NR ⁷ (CH ₂ CH ₂ O) _p1 CH ₂ C(O)-, -S(CH ₂ ) _p1 C(O)-, -(CH ₂ ) _p1 C(O)- or a chemical bond, wherein p1 is an integer from 1 to 20, preferably a chemical bond; p1 is an integer from 1 to 20, and R ⁷ is selected from a hydrogen atom, an alkyl group, a halogenated alkyl group and a hydroxyalkyl group. 14. The antibody-drug conjugate of claim 13, wherein ^R6 is selected from _C1-6 alkylene C(O)-, -( _{CH2 - CH2O} ) _2C (O)- _, -(CH2 _{- CH2O} )2CH2C(O)-, _- ( _CH2 - _CH2O ) _2CH2CH2C (O)-, -( _CH2 - _CH2O ) _3C (O)-, _and -( _CH2 - _CH2O ) _4C (O)-. 15. The antibody-drug conjugate according to claim 13 or 14, wherein the linker L comprises: maleimide-(PEG) ₂ -Val-Cit, maleimide-(PEG) ₆ -Val-Cit, maleimide-(PEG) ₈ -Val-Cit, maleimide-(PEG) ₄ -CH ₂ CH ₂ C(O)-Val-lys, maleimide-(CH ₂ ) ₅ -Val-Cit, maleimide-(CH ₂ ) ₅ -Val-lys, maleimide-(CH ₂ ) ₅ -Gly-Gly-Phe-Gly, maleimide-(PEG) ₂ -Ala-Ala-Asn, maleimide-(PEG) ₆ -Val-Cit, -Ala-Ala-Asn, maleimide-(PEG) ₈ -Ala-Ala-Asn, maleimide-(PEG) ₄ -triazole-(PEG) ₃ -sulfonamide, maleimide-(PEG) _2- CH ₂ CH ₂ C(O)-Val-lys, maleimide-(PEG) ₄ -triazole-(PEG) ₃ -sulfonamide or Mal-(PEG) ₄ -triazole-(PEG) ₃ -disulfide. 16. The antibody-drug conjugate of claim 12, wherein Str is selected from a chemical moiety represented by the following formula:

wherein R ⁸ is selected from C _1-10 alkylene, C _2-10 alkenylene, (C _1-10 alkylene)O—, N(R ^d )-(C _2-6 alkylene)-N(R ^d ) and N(R ^d )-(C _2-6 alkylene); and each R ^d is independently H or C ₁ -C ₆ alkyl. 17. The antibody-drug conjugate according to any one of claims 1 to 15, which is represented by the following formula:

k is selected from 1 to 10, and can be an integer or a decimal; P1 is selected from 2, 4, 6 or 8; P2 is selected from 0, 1 or 2;

k is selected from 1 to 10, and can be an integer or a decimal; P1 is selected from 2, 4, 6 or 8; P2 is selected from 0, 1 or 2;

k is selected from 1 to 10, and can be an integer or a decimal; P1 is selected from 2, 4, 6 or 8; P2 is selected from 0, 1 or 2;

k is selected from 1 to 10, and can be an integer or a decimal; P2 is selected from an integer between 1 and 6;

k is selected from 1 to 10, and can be an integer or a decimal; P2 is selected from an integer between 1 and 6;

k is selected from 1 to 10 and can be an integer or a decimal; P2 is selected from an integer between 1 and 6, Ab, D as defined in claim 1. 18. The antibody-drug conjugate according to any one of claims 1 to 17, wherein the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. 19. The antibody-drug conjugate according to any one of claims 1 to 18, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, preferably, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10. 20. The antibody-drug conjugate according to any one of claims 1 to 19, which is represented by the following formula:

Wherein, k is selected from 1 to 10 and can be an integer or a decimal. 21. An isotope-substituted compound according to any one of claims 1 to 20, preferably, the isotope substitution is deuterium atom substitution. 22. A pharmaceutical composition comprising a therapeutically effective amount of the antibody-drug conjugate according to any one of claims 1 to 20, or the isotope-substituted product according to claim 21, and a pharmaceutically acceptable carrier, diluent or excipient. 23. Use of the antibody-drug conjugate according to any one of claims 1 to 20, the isotope-substituted product according to claim 21, or the pharmaceutical composition according to claim 22 in the preparation of a drug for treating or preventing tumors. 24. The use according to claim 23, wherein the tumor is a cancer associated with the expression of folate receptor alpha, and the cancer is preferably breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

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