Antibody drug conjugate targeting folate receptor alpha as well as preparation method and application of antibody drug conjugate

Abstract

The invention provides an antibody drug conjugate targeting folate receptor alpha, and also discloses application of the antibody drug conjugate in preparation of drugs for treating folate receptor alpha-related tumors. The antibody drug conjugate provided by the invention combines the targeting property of the anti-folate receptor alpha antibody to folate receptor alpha expression cells, the strong endocytosis effect of a target spot and the toxic effect of a small molecule chemical drug, so that an excellent tumor killing effect is achieved.

Description

[0001] Cross-reference to related applications

[0002] This patent application claims priority to Chinese Patent Application No. CN202410603763.9, filed on May 15, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to antibody-drug conjugates, and more specifically, to antibody-drug conjugates targeting folate receptor α (FRα), their preparation methods, and uses. Background Technology

[0004] Folic acid receptor α (FRα) is a glycosylphosphatidylinositol protein attached to the cell membrane. Under physiological conditions, it is only present on the apical / luminal surface of a few polarized epithelial cells; however, when it loses its polarization and spreads throughout the cell surface, it is overexpressed in epithelial-derived malignancies. Studies have demonstrated that FRα is overexpressed in various tumor types, including ovarian cancer, endometrial cancer, lung cancer, and triple-negative breast cancer. Notably, FRα is expressed in over 80% of serous ovarian malignancies and is unaffected by chemotherapy. FRα's high expression in tumors and low expression in normal cells have attracted considerable attention from researchers as a potential therapeutic target for cancer.

[0005] Antibody-drug conjugates (ADCs) are a novel type of cancer therapy consisting of an antibody or antibody-like ligand, a small molecule drug, and a linker that conjugates the ligand to the drug. They combine the antitumor activity of small molecule drugs with the high selectivity, stability, and favorable pharmacokinetic characteristics of antibodies or antibody-like ligands, and are considered to be among the most promising antitumor drugs.

[0006] Several companies are currently focusing on developing antibody-drug conjugates (ADCs) targeting folate receptor α (FRα). To date, FRα-targeting ADCs that have entered clinical trials or are marketed include: Eisai's MORAb-202, which consists of the FRα-targeting monoclonal antibody farletuzumab conjugated to eribulin via a cleavable linker, with an average DAR of 4.0; Super's STRO-002, which consists of the FRα-targeting monoclonal antibody conjugated to the microtubule-targeting drug 3-aminophenyl hemiasterlin, with an average DAR of 4.0; ImmunoGen's Mirvetuximab soravtansine (IMGN853, Elahere), which consists of the humanized anti-FRα monoclonal antibody M9346A conjugated to the microtubule-targeting cytotoxin DM4 via a cleavable sulfo-SPDB linker, with an average DAR of 3.5; and so on.

[0007] Existing ADC drugs have shown some therapeutic effects, but they also have certain problems. For example, Eisai's MORAb202 exhibits significant heterogeneity, and the synthesis of eribulin is complex and difficult. Furthermore, according to the latest clinical data released by Super, STRO-002 has a 78% rate of grade 3 or higher adverse reactions, with neutropenia being a particularly significant issue. Similarly, IMGN853 (ELAHERE) failed to meet its primary endpoint in a phase III clinical trial as monotherapy for platinum-resistant ovarian cancer; compared to chemotherapy, IMGN853 did not statistically improve progression-free survival in ovarian cancer patients.

[0008] Currently, the available types of ADCs targeting folate receptor α are still relatively limited, and they suffer from drawbacks such as poor efficacy as monotherapy and strong toxic side effects. Therefore, there is still a need to develop new ADCs to meet clinical needs. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide an anti-folate receptor α antibody that has high specificity for folate receptor α, as well as high biological activity, high endocytic capacity, and better in vivo safety. The antibody is prepared into an antibody-drug conjugate by coupling it with a small molecule chemical drug via a linker with a specific structure. The antibody is used to target folate receptor α-expressing cells and has a strong endocytic effect through the target site. Combined with the action of the small molecule chemical drug, an excellent tumor killing effect is achieved.

[0010] Therefore, one object of the present invention is to provide an antibody against folate receptor α or a fragment thereof. Another object of the present invention is to provide an antibody-drug conjugate targeting folate receptor α prepared using the antibody or a fragment thereof, or a salt thereof.

[0011] The technical solution of the present invention is as follows.

[0012] On one hand, the present invention provides an antibody-drug conjugate or a salt thereof targeting folate receptor α, comprising an antibody or antigen-binding fragment thereof covalently linked to a drug via a linker against folate receptor α.

[0013] Furthermore, the antibody-drug conjugate targeting folate receptor α or its salt provided by the present invention has the molecular structural formula Ab-[L-CPT]m, wherein Ab represents an antibody against folate receptor α or its antigen-binding fragment, L represents a linker, CPT represents a cytotoxic drug, and m represents the average number of drug linkages (average DAR) relative to each Ab molecule.

[0014] About Ab:

[0015] In the antibody-drug conjugate or its salt provided by the present invention, the antibody against folic acid receptor α or its antigen-binding fragment comprises complementarity-determining regions (CDRs) of the heavy chain, namely heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and complementarity-determining regions (CDRs) of the light chain, namely light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3).

[0016] According to a specific embodiment of the present invention, the heavy chain CDRs contained in the antibody against folic acid receptor α or its antigen-binding fragment are derived from the amino acid sequences shown in any one of SEQ ID NOs. 1, 3, 5, 6 and 9; and / or, the light chain CDRs contained in the antibody or its antigen-binding fragment are derived from the amino acid sequences shown in any one of SEQ ID NOs. 2, 4, 7 and 8.

[0017] The amino acid sequences shown in any one of SEQ ID NO.1 to SEQ ID NO.9 provided above are the amino acid sequences of the heavy chain variable region (VH) or light chain variable region (VL) of the exemplary antibody provided in the "Detailed Description" section of this application. Using antibody heavy chain or light chain complementarity-determining region definition tools well-known in the art (e.g., Chothia, Kabat, IMGT, Contact, etc.), those skilled in the art can easily determine the heavy chain CDRs and light chain CDRs contained therein.

[0018] Preferably, in the antibody-drug conjugate or its salt provided by the present invention, the antibody against folic acid receptor α or its antigen-binding fragment comprises heavy chain CDRs and light chain CDRs from the heavy chain variable region and light chain variable region shown in the following amino acid sequence pairings:

[0019] (1) SEQ ID NO.1 + SEQ ID NO.2;

[0020] (2) SEQ ID NO.3 + SEQ ID NO.4;

[0021] (3) SEQ ID NO.5 + SEQ ID NO.2;

[0022] (4) SEQ ID NO.6 + SEQ ID NO.7;

[0023] (5) SEQ ID NO.6 + SEQ ID NO.8;

[0024] (6) SEQ ID NO.9 + SEQ ID NO.7;

[0025] (7) SEQ ID NO.9 + SEQ ID NO.8; and

[0026] (8)SEQ ID NO.9+SEQ ID NO.4.

[0027] As described above, for example, Chothia and / or Kabat can be used to delineate the CDRs in the above amino acid sequence pairings, as shown in the embodiments of the present invention.

[0028] Accordingly, in the antibody-drug conjugate or its salt provided by the present invention, the heavy chain CDRs and light chain CDRs contained in the antibody against folic acid receptor α or its antigen-binding fragment are as follows:

[0029] (1) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21;

[0030] (2) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.16; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21;

[0031] (3) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.22, and SEQ ID NO.21;

[0032] (4) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.16; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.22, and SEQ ID NO.21;

[0033] (5) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.23; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21;

[0034] (6) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.23; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21;

[0035] (7) HCDR1, HCDR2, and HCDR3 sequentially comprising the amino acid sequences shown in SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.23; and LCDR1, LCDR2, and LCDR3 sequentially comprising the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.22, and SEQ ID NO.21; or

[0036] (8) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.23; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.22, and SEQ ID NO.21.

[0037] Preferably, in the antibody-drug conjugate or its salt provided by the present invention, the antibody against folate receptor α or its antigen-binding fragment specifically targets folate receptor α, preferably human folate receptor α.

[0038] Preferably, in the antibody-drug conjugate or its salt provided by the present invention, the antibody against folic acid receptor α or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, both of which include the above-mentioned CDRs and the framework region (FR) therebetween, and the arrangement of each region from the N-terminus to the C-terminus is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0039] Further preferably, in the antibody-drug conjugate or its salt provided by the present invention, the heavy chain variable region of the antibody against folate receptor α or its antigen-binding fragment may contain an amino acid sequence shown in any one of SEQ ID NOs. 1, 3, 5, 6, and 9, or contain an amino acid sequence having at least 75% identity with said amino acid sequence; and / or, the light chain variable region of the antibody against folate receptor α or its antigen-binding fragment may contain an amino acid sequence shown in any one of SEQ ID NOs. 2, 4, 7, and 8, or contain an amino acid sequence having at least 75% identity with said amino acid sequence. In the context of the present invention, the term "at least 75% identity" resulting in a maximum 25% difference in the amino acid sequence may exist in any frame region of the heavy chain variable region or light chain variable region of the antibody or its antigen-binding fragment, or in any domain or sequence other than the heavy chain variable region and light chain variable region of the antibody or its antigen-binding fragment. Such differences may be caused by amino acid deletions, additions, or substitutions at any position, wherein substitutions may be conservative or non-conservative substitutions. The “at least 75% identity” refers to any percentage of identity between at least 75% and 100% identity, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity.

[0040] According to a specific embodiment of the present invention, in the antibody-drug conjugate or its salt provided by the present invention, the antibody against folic acid receptor α or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise a combination selected from the following amino acid sequences:

[0041] (1) The amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.2;

[0042] (2) The amino acid sequence shown in SEQ ID NO.3, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.4, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.4;

[0043] (3) The amino acid sequence shown in SEQ ID NO.5, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.2;

[0044] (4) The amino acid sequence shown in SEQ ID NO. 6, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 7;

[0045] (5) The amino acid sequence shown in SEQ ID NO. 6, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 8, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 8;

[0046] (6) The amino acid sequence shown in SEQ ID NO.9, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.7;

[0047] (7) The amino acid sequence shown in SEQ ID NO. 9, or an amino acid sequence having at least 75% identity with said amino acid sequence; and the amino acid sequence shown in SEQ ID NO. 8, or an amino acid sequence having at least 75% identity with said amino acid sequence; or

[0048] (8) The amino acid sequence shown in SEQ ID NO.9, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.4, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.4.

[0049] Preferably, in the antibody-drug conjugate or its salt provided by the present invention, the antibody against folic acid receptor α can be a mouse antibody, rabbit antibody, or human antibody, or it can be a mouse antibody, a chimeric antibody, or a fully or partially humanized antibody. The antibody can also be a derivatized antibody, such as an antibody obtained by CDR transplantation, affinity maturation, point mutation modification, or chemical modification based on an initial mouse monoclonal antibody. The chemical modification includes glycosylation, acetylation, polyethylene glycol modification, phosphorylation, amidation, protease cleavage, linkage with cellular ligands or effector molecules, protection and / or blocking of active reactive groups, etc. Preferably, the antigen-binding fragment of the antibody can be a single-chain variable fragment (scFv), a bivalent single-chain variable fragment (BsFv), a disulfide-stabilized variable fragment (dsFv), (dsFv)2, an antigen-binding fragment (Fab), Fab' fragment (Fab'), (Fab' fragment)2 (F(ab')2), or a variable fragment (Fv), etc. Regarding the antigen-binding fragment of the antibody described in this invention, it can be any fragment of the antibody capable of specifically binding to folate receptor α; and the folate receptor α is mammalian FRα, preferably primate FRα, more preferably human FRα.

[0050] In addition to the heavy chain and / or light chain variable regions, the antibody-drug conjugate or its salt provided by the present invention may further include a heavy chain constant region (CH) and / or a light chain constant region (CL) in the anti-folate receptor α antibody or its antigen-binding fragment, preferably including a human or mouse heavy chain constant region and / or a light chain constant region. Preferably, the antibody or its fragment includes a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE and / or a κ or λ type light chain constant region.

[0051] Preferably, in the antibody-drug conjugate or its salt provided by the present invention, the antibody against folic acid receptor α is a monoclonal antibody, preferably a murine, chimeric, or humanized monoclonal antibody; more preferably, the heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 subtype, and the light chain constant region is κ type. Alternatively, for example, the antibody is an immunoglobulin, specifically IgA, IgD, IgE, IgG, or IgM, such as human subtypes of IgA, IgD, IgE, IgG, or IgM, more preferably human IgG1, IgG2, IgG3, or IgG4 subtype.

[0052] According to a specific embodiment of the present invention, the antibody against folate receptor α or its antigen-binding fragment comprises a heavy chain constant region, the heavy chain constant region comprising an amino acid sequence as shown in SEQ ID NO. 11 or a variant thereof. Alternatively, the antibody against folate receptor α or its antigen-binding fragment comprises a light chain constant region, the light chain constant region comprising an amino acid sequence as shown in SEQ ID NO. 13 or a variant thereof. "Variation of amino acid sequence" as defined above refers to an amino acid sequence having at least 75% sequence identity with said amino acid sequence.

[0053] Regarding L:

[0054] In the antibody-drug conjugate or its salt provided by this invention, the linker L can be a cleavable or non-cleavable linker. Cleavable linkers can be selected from PEG-Val-Cit-PAB, PEG-Val-Ala, and PEG-Gly-Gly-Phe-Gly, etc.; non-cleavable linkers can be selected from MCC, PEG4Mal, and mc, etc.

[0055] In the context of this invention, "connector" and "joint" are used interchangeably.

[0056] About CPT:

[0057] In the antibody-drug conjugate or its salt provided by the present invention, CPT is a cytotoxic drug.

[0058] Preferably, CPT is selected from one or more of the following: microtubule inhibitors MMAE, DM1, DM4, Tublysin, muscarinic acid, chalcogenide, eribulin and derivatives thereof; topoisomerase inhibitors SN38, eczema and derivatives thereof; DNA binders PBD, doxorubicin and derivatives thereof.

[0059] Regarding m:

[0060] In the antibody-drug conjugate targeting folate receptor α or its salt provided by this invention, m represents the average number of drug linkages (average DAR) relative to each Ab molecule. m can be 1.0-10.0 and can be an integer or a non-integer.

[0061] Preferably, m is an integer or non-integer of 1.0-5.0, more preferably 3.0-4.2, more preferably 3.5-4.5, even more preferably 3.8-4.2, and still more preferably 3.9-4.1. Particularly preferably, m is 4.0.

[0062] Furthermore, the antibody-drug conjugate or its salt provided by the present invention has the structure shown in the following structural formulas Ia and / or Ib:

[0063]

[0064] and / or

[0065]

[0066] In structural formulas Ia and / or Ib, Ab, CPT, and m are as defined above.

[0067] In structural formulas Ia and / or Ib, M is a phenoxy group or a phenoxy group substituted with one or more substituents, or a chemical bond; in the substituted phenoxy group, the substituent is selected from alkyl (e.g., C1-C6 alkyl, preferably C1-C4 alkyl), haloalkyl (e.g., haloC1-C6 alkyl, preferably haloC1-C4 alkyl, such as trifluoromethyl), alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), and halogen.

[0068] In structural formulas Ia and / or Ib, SP1 is selected from C1-C8 alkylene, C1-C8 cycloalkylene, or C1-C21 (preferably C1-C16, more preferably C1-C11) straight-chain heteroalkylene, wherein the C1-C21 straight-chain heteroalkylene comprises 1-11 (preferably 1-6) heteroatoms selected from N, O, or S, wherein each of the C1-C8 alkylene, C1-C8 cycloalkylene, and C1-C21 straight-chain heteroalkylene is independently and optionally substituted by one or more substituents selected from hydroxyl, amino, sulfonic acid, and cyano groups. Preferably, SP1 is selected from C1-C8 alkylene, C1-C8 cycloalkylene, or C1-C16, preferably C1-C11, more preferably C1-C7 straight-chain heteroalkylene, wherein the straight-chain heteroalkylene comprises 1-6, preferably 1-4 heteroatoms selected from N, O, or S; preferably, the heteroatom is O.

[0069] In structural formulas Ia and / or Ib, SP2 is selected from -NH(CH2CH2O)bCH2CH2CO-, -NH(CH2CH2O)bCH2CO-, -S(CH2)bCO- or a chemical bond, wherein b is an integer from 1 to 20, preferably an integer from 1 to 10, and more preferably an integer from 1 to 6.

[0070] In structural formulas Ia and / or Ib, A represents a short peptide structure consisting of 2-4 amino acids or a combination of a short peptide structure consisting of 2-4 amino acids and a self-releasing structural fragment. Wherein, when A represents 2 amino acids, it can be -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Phe-Arg-, or -Gly-Val-, preferably -Phe-Lys-, -Val-Ala-, or -Val-Cit-; when A represents 3 amino acids, it can be -Glu-Val-Ala-, -Glu-Val-Cit-, or -Ala-Ala-Ala-, preferably -Glu-Val-Ala- or -Ala-Ala-Ala-; when A represents 4 amino acids, it can be -Gly-Gly-Phe-Gly- or -Gly-Phe-Gly-Gly-, preferably -Gly-Gly-Phe-Gly-. Preferably, A is -Val-Ala-, -Gly-Gly-Phe-Gly-, -Ala-Ala-Ala-, or Val-Cit-PABC.

[0071] In structural formulas Ia and / or Ib, CPT can be a camptothecin-type compound or anorectatine-type compound.

[0072] Preferably, M can be a phenoxy group substituted with a halogen (fluorine, chlorine, bromine, iodine). More preferably, M is a phenoxy group substituted with a dihalogen (fluorine, chlorine, bromine, iodine).

[0073] Preferably, SP1 can be -(CH2CH2O)a-CH2-, where a is an integer from 1 to 20, more preferably an integer from 1 to 10, more preferably an integer from 1 to 6, and even more preferably an integer from 2 to 4. More preferably, a is 3.

[0074] Preferably, SP2 is a chemical bond.

[0075] According to specific embodiments of the present invention, the antibody-drug conjugate or its salt provided by the present invention has the structure shown in the following structural formulas IIa and / or IIb:

[0076]

[0077] and / or

[0078]

[0079] In structural formulas IIa and / or IIb, CPT can be a camptothecin-type compound or anorectatine-type compound.

[0080] Furthermore, CPT is a camptothecin-type compound, and its structure can be as shown in structural formula I, wherein structural formula I is connected to the carboxyl group in group A of structural formulas Ia and / or Ib or IIa and / or IIb via an amide bond, preferably with the amino group adjacent to group G in structural formula I connected to the carboxyl group in group A via an amide bond:

[0081]

[0082] In structural formula I, R1, R2, R3, and R4 are independently hydrogen, halogen (fluorine, chlorine, bromine, iodine), C1-7 alkyl or substituted C1-7 alkyl, or any two of R1, R2, R3, and R4 together with the carbon atoms they are attached to form a C3-6 cyclic alkyl group. Preferably, R1 and R2 are the same; and / or, R3 and R4 are the same; more preferably, R1 and R2 are halogens (fluorine, chlorine, bromine, iodine), and R3 and R4 are hydrogen.

[0083] In structural formula I, G is hydrogen, halogen, methyl, or methoxy. Preferably, G is hydrogen, fluorine, or chlorine.

[0084] In structural formula I, Y is oxygen, sulfur, methylene, or a substituted methylene group. A substituted methylene group may be formed by replacing one hydrogen atom or both hydrogen atoms of the methylene group. The substituent may be benzyl or alkyl. When the substituent is alkyl, the alkyl group, together with R3 and / or R4 and the carbon atoms attached to them, can form a C3-6 membered fused or spirocyclic structure. When Y is a substituted methylene group, the substituent is preferably an alkyl group, more preferably a straight-chain or branched C1-4 alkyl group. Preferably, Y is methylene.

[0085] In structural formula I, X is oxygen or sulfur.

[0086] In structural formula I, n = 0 or 1.

[0087] Furthermore, the structure of CPT is shown in structural formula IA, wherein structural formula IA is connected to the carboxyl group in group A of structural formulas Ia and / or Ib or structural formulas IIa and / or IIb via an amide bond:

[0088]

[0089] In structural formula IA, groups R1, R2, R3, and R4 are defined in the same way as groups R1, R2, R3, and R4 in structural formula I above.

[0090] Alternatively, further, CPT is an olistatin compound, preferably MMAE or MMAF.

[0091] According to a specific embodiment of the present invention, the antibody-drug conjugate targeting folate receptor α or a salt thereof has the following structure:

[0092]

[0093] and / or

[0094]

[0095] Alternatively, the antibody-drug conjugate targeting folate receptor α or its salt has the following structure:

[0096]

[0097] and / or

[0098]

[0099] On the other hand, the present invention provides a method for preparing the antibody-drug conjugate targeting folate receptor α or a salt thereof, the preparation method comprising the following steps:

[0100] (1) React the antibody against folic acid receptor α or its antigen-binding fragment with a reducing agent in a buffer solution to obtain the reduced antibody or its fragment.

[0101] (2) Crosslink the drug-containing linker with the reduced antibody or its fragment obtained in step (1) in a mixture of buffer and organic solvent to obtain an antibody-drug conjugate targeting folic acid receptor α.

[0102] In the context of this invention, the term "drug-containing linker" refers to a compound obtained by directly or indirectly covalently bonding a linker to a cytotoxic drug, and is used interchangeably with "drug-containing connector".

[0103] In the preparation method provided by this invention, the antibody against folic acid receptor α or its antigen-binding fragment is as defined above; the drug-containing linker has the structure shown in structural formula III:

[0104]

[0105] In structural formula III, M, SP1, SP2, A, and CPT are defined as above.

[0106] In structural formula III, E is selected from the following groups, wherein... Indicates the connection site with M:

[0107] E-1: E-2: E-3:

[0108] E-4: E-5: And E-6:

[0109] When group E is E1, structural formula III further becomes structural formula IIIA:

[0110]

[0111] In structural formula IIIA, R6 and R7 are independently hydrogen, halogen, or Ar'S.

[0112] Wherein, Ar' is a phenyl or a phenyl substituted with one or more substituents, wherein the substituents are selected from alkyl (e.g., C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen, ester, amide, and cyano.

[0113] Preferably, Ar' is a phenyl or 4-methylformyl-substituted phenyl group. Or 4-formylmorpholine substituted phenyl

[0114] Preferably, the compounds represented by structural formulas III and IIIA are further represented by structural formula V.

[0115]

[0116] In structural form V, A and CPT are defined as above; R6 and R7 are independently Ar'S, and Ar' is defined as above.

[0117] In structural formula V, Xh and Yh are independently hydrogen, halogen, haloalkyl (e.g., haloC1-C6 alkyl, preferably haloC1-C4 alkyl, such as trifluoromethyl) or alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy).

[0118] In structural formula V, j is any integer from 1 to 10, preferably 1 to 5, and more preferably 3 to 5.

[0119] More preferably, the compound represented by structural formula V provided by the present invention is further a compound represented by structural formula VI:

[0120]

[0121] In structural VI, A and CPT are defined as above.

[0122] According to a specific embodiment of the present invention, the drug-containing linker is the following compound:

[0123]

[0124] Preferably, the preparation method includes the following steps:

[0125] a. Antibody reduction: Add ≥5.5 molar equivalents of reducing agent to phosphate buffer containing 5-30 mg / mL of antibody and react for 1.5-2 h. The reducing agent is one or more selected from TCEP, DTT, 2-MEA and DTBA.

[0126] b. Antibody conjugation: The reduced antibody from step a is replaced with phosphate buffer at pH 6.5-7.8, and the antibody is diluted to a concentration of 3.5-15 mg / mL. 4.5-6.5 molar equivalents of the drug-containing linker dissolved in an organic co-solvent are added to the antibody dilution. The reaction is carried out at 15-35°C with stirring for ≥0.5 h. The organic co-solvent is one or more selected from DMA, DMSO, DMF, and ACN.

[0127] c. Hydrophobic chromatography: The antibody-conjugate products were purified by hydrophobic chromatography using hydrophobic packing material.

[0128] More preferably, the preparation method further includes the following step after step b or after step c:

[0129] d. Hydrolysis: The antibody-conjugated product was displaced into phosphate buffer at pH 7.4-9.0 and heated at 35±10℃ for 2-24 h to obtain the hydrolysis product.

[0130] Preferably, the displacement in steps a, b, and d is performed using molecular sieves, ultrafiltration centrifuge tubes, or ultrafiltration membrane packs. Preferably, the hydrophobic packing material used in step c is Butyl Sepharose HP, Capto Phenyl Impres, or Butyl Sepharose FF.

[0131] The antibody-drug conjugate targeting folate receptor α or its salt provided by this invention can be included in compositions, and more particularly in pharmaceutical formulations, for use in various purposes as needed. Therefore, in another aspect, this invention also provides a composition comprising the antibody-drug conjugate targeting folate receptor α or its salt provided by this invention. Preferably, the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier, excipient, or excipient.

[0132] In another aspect, the present invention also provides the use of the antibody-drug conjugate targeting folate receptor α or its salts or compositions thereof in the preparation of a medicament for treating tumors. Alternatively, the present invention also provides a method for treating tumors, the method comprising administering the antibody-drug conjugate targeting folate receptor α or its salts or compositions thereof provided by the present invention to a subject in need of treatment. The subject is a mammal, preferably a primate, and more preferably a human.

[0133] Preferably, the tumor is a folate receptor α-associated tumor, such as a solid tumor. Preferably, the tumor is selected from any of the following: ovarian cancer, endometrial cancer, lung cancer (e.g., non-small cell lung cancer), breast cancer, cervical cancer, choriocarcinoma, colon cancer, mesothelioma, and oral cancer (e.g., oral epithelial carcinoma).

[0134] In another aspect, the present invention provides a kit comprising an antibody-drug conjugate targeting folate receptor α, or a salt thereof, or a combination thereof, provided by the present invention. The kit can be used for the aforementioned therapeutic purposes. Depending on the intended application, the kit may also contain other reagents.

[0135] This invention provides antibody-drug conjugates (ADCs) targeting folate receptor α, obtained by conjugating an antibody against folate receptor α with a cytotoxic drug. Experiments have demonstrated that the anti-folate receptor α antibody provided by this invention can be conjugated with linker-payloads of different structures (cytotoxic drugs are exemplarily camptothecin and oliquistatin compounds) to prepare ADCs. The prepared ADCs have an average DAR of approximately 4.0 and high purity. Furthermore, against folate receptor α-positive tumor cells, the antibody-drug conjugates provided by this invention exhibit comparable or stronger activity than reference ADC drugs in terms of cell binding, induction of apoptosis, inhibition of tumor growth, induction of cellular DNA damage, and inhibition of microtubule aggregation.

[0136] Therefore, the antibody-drug conjugates provided by this invention have significant application potential in targeted therapy or cell killing of folate receptor α-positive tumors, as well as in combined immunotherapy. Attached Figure Description

[0137] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0138] Figure 1 The dose-response curve of the antibody-drug conjugate according to the present invention binding to FRα-positive tumor cells is shown.

[0139] Figure 2 The antibody-drug conjugate according to the present invention is shown to induce apoptosis in FRα-positive tumor cells.

[0140] Figure 3The bystander effect of the antibody-drug conjugate according to the present invention is illustrated.

[0141] Figure 4 The activity of the antibody-drug conjugate according to the present invention in inducing DNA damage in FRα-positive tumor cells is demonstrated.

[0142] Figure 5 The antibody-drug conjugate according to the present invention exhibits activity in inhibiting microtubule aggregation in FRα-positive tumor cells. Detailed Implementation

[0143] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0144] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available products.

[0145] The antibodies against folic acid receptor α used in the examples are described in Chinese patent application CN202311533599.0, the entire contents of which are incorporated herein by reference.

[0146] Example Group 1: Preparation of Anti-human FRα Antibody

[0147] Example 1 Preparation of anti-human FRα mouse antibody

[0148] Mice were immunized with commercially available recombinant human FRα-his protein (Nearshore Pharmaceutical Group, catalog number C784) using standard methods. Multiple cell clones secreting antibodies that specifically bind to human FRα were obtained using hybridoma cloning or phage display technology. The resulting cloned cells were cultured in a large-scale manner, total RNA was extracted, reverse transcribed into cDNA, and the antibody light variable region (VL) and heavy chain variable region (VH) sequences were amplified by PCR. The PCR amplification products were purified and ligated into a T-vector, and DNA sequencing was performed to obtain the variable region sequence of the mouse monoclonal antibody.

[0149] Two mouse antibodies derived from clones 45B1 and Pha3 were selected for the construction of chimeric and humanized antibodies.

[0150] Example 2 Preparation of anti-human FRα chimeric antibodies and their humanized antibodies

[0151] The heavy chain variable region (VH) coding nucleic acid sequence of the anti-human FRα mouse antibody was prepared by DNA synthesis. This sequence was then spliced ​​together with the published heavy chain constant region coding nucleic acid sequence of the human monoclonal antibody IgG1 subclass (SEQ ID NO.10) and constructed into the mammalian cell expression vector pcDNA. TM In 3.1(+)(Invitrogen#V790-20); the light chain variable region (VL) encoding nucleic acid sequence of anti-human FRα mouse antibody was prepared, and it was spliced ​​together with the published light chain constant region encoding nucleic acid sequence of human monoclonal antibody κ subclass (SEQ ID NO.12) to construct the mammalian cell expression vector pcDNA. TM In 3.1(+)(Invitrogen#V790-20), the constructed chimeric antibody heavy chain vector and light chain vector were paired and mixed, and HEK293 cells were transfected with polyethyleneimine (PEI). After about 7 days, the cell supernatant was collected and purified using Protein A to obtain the anti-human FRα chimeric antibody protein.

[0152] High-affinity and high-blocking-capacity mouse antibody molecules were selected. Combining the antibody coding schemes of Kabat and Chothia, the amino acid sequence regions of the six antigen complementarity determinants (CDRs) of the heavy and light chains of the mouse antibody, as well as the framework region supporting the conserved three-dimensional conformation of the antibody, were determined. By analyzing known human antibody sequences, the variable region sequence of the heavy chain of the human antibody most similar to that of the mouse antibody was selected. Using its antibody framework region sequence as a template, the mouse antibody heavy chain CDRs were combined with the human antibody framework region to generate the humanized antibody heavy chain variable region sequence. The same process was used to generate the humanized antibody light chain variable region sequence. Optionally, individual amino acids in the framework region were reverse-mutated, i.e., changed from human to mouse. Using the same procedures as in the preparation of chimeric antibodies, the constructed humanized antibody heavy chain variable region sequence and light chain variable region were spliced ​​together with the heavy chain constant region of the human monoclonal antibody IgG1 subclass and the light chain constant region of the human monoclonal antibody κ subclass, respectively. After expression and purification, the anti-human FRα humanized antibody protein was obtained.

[0153] The mouse anti, chimeric, and humanized antibody sequences used in this invention are shown in Table 1 and Table 2, respectively.

[0154] Table 1. Variable region and CDR sequences of anti-human FRα antibody derived from clone 45B1

[0155]

[0156]

[0157] Table 2. Variable region and CDR sequences of anti-human FRα antibodies derived from clone Pha

[0158]

[0159]

[0160]

[0161]

[0162] The nucleic acid sequence encoding the light chain constant region of the human monoclonal antibody κ subclass (SEQ ID NO.12): Cgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatccccgcgaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagcagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaaccgcggagagtgt

[0163] The amino acid sequence of the light chain constant region of the human monoclonal antibody κ subclass (SEQ ID NO.13): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0164] Example Group 2 Preparation of antibody-drug conjugates

[0165] Example 1 Synthesis of drug-containing linker MWF-L6

[0166] The drug-containing linker MWF-L6 was synthesized according to the method described in patent application publication WO2023109965A1, and MWF-L6 was obtained as an orange-red solid with LC-MS (ESI): M+1=1450.

[0167]

[0168] Example 2 Synthesis of drug-containing linker BL20E

[0169] The drug-containing linker BL20E was synthesized according to the method described in patent application publication WO2018095422A1. BL20E was obtained as a yellow solid with LC-MS (ESI): M+1 = 1963.

[0170]

[0171] Example 3 Control drug-containing linker sulfo-SPDB-DM4

[0172] The control drug-containing linker sulfo-SPDB-DM4 was purchased from MCE.

[0173] Example 4 Preparation and physicochemical characterization of antibody-drug conjugates

[0174] The schematic diagram of the site-specific conjugation preparation process for antibody-drug conjugates is as follows:

[0175]

[0176] The antibody is reduced to open the disulfide bond, then coupled with a drug-containing linker to form a bridging antibody-drug conjugate. The maleimide ring is then opened by hydrolysis, and finally purified to obtain an antibody-drug conjugate with an average DAR of 4.

[0177] 4.1 General preparation method of antibody-drug conjugates

[0178] a. General preparation method for site-directed coupling

[0179] The ADC coupled to MWF-L6 is prepared according to the method described in patent application publication WO2023109965A1. A specific exemplary method is as follows:

[0180] Antibody reduction: 120 mg of antibody sample was used with a Sephadex G25-supported NAP-25 column, purged to pH 7.0 in a buffer solution containing 50 mM sodium chloride and 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, and the antibody concentration was diluted to 10 mg / ml. 10 ml of a total of 100 mg of antibody sample was taken, and 2.1 ml of 10 mg / ml TCEP (Sigma-Aldrich) aqueous solution was added at an antibody-TCEP molar ratio of 1:10. After incubation for 2 hours, the sample was purged using Sephadex G25 column reaction solution to a buffer solution at pH 6.5 containing 50 mM sodium chloride and 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate.

[0181] Antibody-drug-linker coupling and hydrolysis: The reduced antibody was diluted to 5 mg / mL, and 0.38 mL of N,N-dimethylacetamide (DMA) (2% of the total reaction volume) was added as a pre-solvent. A DMA-drug-linker mixture containing 10 mg / mL of drug-linker was added at an antibody-small molecule drug molar ratio of 1:5.5 as the reaction solution. The mixture was stirred at room temperature for 30 minutes. The reaction solution was then replaced with a pH 8.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer using a Sephadex G25 support NAP-25 column to remove excess drug-linker. The mixture was then heated in a 37°C water bath for 3 hours.

[0182] Purification of the antibody-drug conjugate: The above sample was concentrated using an AMICOM ultrafiltration centrifuge tube to approximately 15 mg / mL. A 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate + 3 M ammonium phosphate buffer solution was added until the conductivity reached 100 mS / cm. The sample was loaded onto a TOYOPEAL Butyl-650M hydrophobic column (purchased from TOSOH). Phase A consisted of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate + 0.6 M ammonium sulfate, and Phase B consisted of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution. Elution was performed using an 8-fold column volume gradient from 0-100% in Phase B, and the main peak was collected.

[0183] The final sample was replaced with a 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 using an AMICOM ultrafiltration centrifuge tube and filtered using a 0.22 μm filter membrane (Sartorius stedim Ministart).

[0184] The ADC coupled to BL20E is prepared according to the method described in patent application publication WO2022228563A1. A specific exemplary method is as follows:

[0185] Sample Reduction and Coupling: The antibody sample was replaced with a Sephadex G-25 carrier-based NAP-25 desalting column to a pH 7.4 buffer solution containing 50 mM sodium chloride and 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, and the antibody concentration was diluted to 10 mg / ml. 10 ml of a total of 100 mg of antibody sample was taken, and 10 mg / ml of TCEP (Sigma-Aldrich) aqueous solution was added at an antibody-reducing agent molar ratio of 1:10. After incubation for 2 hours, the reaction solution was replaced with a Sephadex G25 desalting column to a pH 7.0 buffer solution containing 50 mM sodium chloride and 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate.

[0186] The reduced antibody was diluted to 5 mg / mL, and 1.33 mL of N,N-dimethylacetamide (DMA) (7.4% of the total reaction volume) was added as a presolvent. A DMA-drug linker mixture containing 10 mg / mL of drug linker was added at an antibody-small molecule drug molar ratio of 1:5.5. The mixture was stirred at room temperature for 60 minutes. The reaction solution was then replaced with a pH 8.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer using a Sephadex G-25 carrier NAP-25 desalting column to remove excess drug linker. The mixture was then heated in a 37°C water bath for 3 hours. Mass spectrometry analysis of the conjugate structures was performed before and after water bath heating. The analysis results of different conjugates all showed that the conjugate before water bath heating was of formula Ia, and after water bath heating, a completely ring-opening formula Ib structure was obtained, indicating 100% hydrolysis.

[0187] Sample purification: The above sample was concentrated using AMICOM ultrafiltration centrifuge tubes to approximately 15 mg / mL. A 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate + 3 M ammonium phosphate buffer solution was added until the conductivity reached 74 mS / cm. The sample was loaded onto a Butyl-Sepharose 4FF hydrophobic column (purchased from GE Healthcare), using phase A (50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate + 0.45 M ammonium sulfate) and phase B (50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution). Elution was performed using a 0-80% linear gradient of 12 column volumes followed by a 100% isocratic gradient in phase B, and the main peak was collected.

[0188] The final sample was replaced with 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 using an AMICOM ultrafiltration centrifuge tube and filtered using a 0.22 μm filter membrane (Sartorius stedim Ministart).

[0189] b. Preparation method of ELAHERE (as opposed to ADC)

[0190] The ADC ELAHERE was prepared according to the method described in patent application publication WO2011106528A1.

[0191] 4.2 General Analytical Methods for Antibody-Drug Conjugates

[0192] a. Determination of drug antibody-drug conjugate ratio and concentration by ultraviolet spectrophotometry (UV-DAR method)

[0193] The concentration of antibody-drug conjugates can be obtained by measuring the UV absorbance at 280 nm and the characteristic absorption wavelength of small molecules, and then performing the following calculations.

[0194] a1. Determination of the drug-antibody conjugate ratio (DAR) of antibody-drug conjugates

[0195] According to the literature [Clin Cancer Res. 2004 Oct 15; 10(20):7063-70], DAR in, A is the molar absorbance coefficient of the antibody at 280 nm. 280 A represents the UV absorbance of the antibody-drug conjugate at 280 nm. z The value represents the UV absorbance of the antibody-drug conjugate at the characteristic absorption wavelength Z nm of the drug-containing linker. The molar absorptivity of the antibody at the characteristic absorption wavelength Z nm of the drug-containing linker is given. The molar absorption coefficient of the drug-containing linker at its characteristic absorption wavelength Z nm is given. The molar absorption coefficient of the drug-containing linker at 280 nm is given.

[0196] in:

[0197]

[0198] a2. Drug concentration determination

[0199] Since the total absorbance at a given wavelength is equal to the sum of the absorbances of all absorbing chemical substances present in the system (additive nature of absorbance), assuming that the molar absorptivity of the antibody and the drug-containing linker remains unchanged before and after conjugation, the antibody-drug conjugate concentration has the following relationship:

[0200]

[0201] Therefore, the molar concentration (mol / L) of antibody-drug conjugates This yields the antibody-drug conjugate concentration (g / L). Substituting the DAR value into the formula yields the protein concentration.

[0202] Among them, MW ADC The molecular weight (MW) of the antibody-drug conjugate is given. Ab The molecular weight (MW) of the antibody. D The molecular weight is the drug-containing linker.

[0203] b. Hydrophobic chromatography

[0204] b1. Determination of DAR value of antibody-drug conjugates by hydrophobic chromatography-HIC-HPLC

[0205] Sample preparation: The sample was diluted to 2.0 mg / ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was used for HPLC analysis.

[0206] Chromatographic column: Sepax Proteomix HIC Butyl-NP5, 5μm, 4.6mm*35mm;

[0207] Mobile phase A: 1.5M (NH4)2SO4 + 25mM PB, pH 7.0

[0208] Mobile phase B: 25 mM PB + 20% IPA, pH 7.0

[0209] Flow rate: 0.6 mL / min;

[0210] Detection wavelength: 280nm;

[0211] Column temperature: 30℃;

[0212] Sample loading volume: 10 μL;

[0213] HIC chromatographic gradient

[0214] time ％A ％B 0 100 0 10 80 20 20 40 60 25 0 100 25.1 100 0 35 100 0

[0215] DAR calculation formula:

[0216] DAR = ∑(weighted peak area) / 100, that is, DAR = (D0 peak area ratio * 0 + D1 peak area ratio * 1 + D2 peak area ratio * 2 + D3 peak area ratio * 3 + D4 peak area ratio * 4 + D5 peak area ratio * 5 + D6 peak area ratio * 6 + D7 peak area ratio * 7 + D8 peak area ratio * 8) / 100.

[0217] b2. Determination of DAR value of antibody-drug conjugates by hydrophobic chromatography-HIC-HPLC

[0218] Sample preparation: The sample was diluted to 2.0 mg / ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was used for HPLC analysis.

[0219] Chromatographic column: Sepax Proteomix HIC Butyl-NP5, 5μm, 4.6mm*35mm;

[0220] Mobile phase A: 1.2M (NH4)2SO4 + 25mM PB, pH 7.0

[0221] Mobile phase B: 25 mM PB + 20% IPA, pH 7.0

[0222] Flow rate: 0.6 mL / min;

[0223] Detection wavelength: 280nm;

[0224] Column temperature: 30℃;

[0225] Sample loading volume: 10 μL;

[0226] HIC chromatographic gradient

[0227] time ％A ％B 0 100 0 1 100 0 20 0 100 20.1 100 0 30 100 0

[0228] DAR calculation formula: same as b1.

[0229] c. Determination of DAR value by mass spectrometry LC-MS

[0230] Sample preparation: Take an appropriate amount of sample into an ultrafiltration tube, replace it with 50mM NH4HCO3 replacement buffer (pH 7.1), add buffer, and ultrafilter and centrifuge (13000g*5min).

[0231] The sample was diluted to 1 mg / mL, and the injection volume was 5 μg. MS-DAR was calculated based on the peak abundance of the deconvolution mass. The DAR of the ADC sample was analyzed using liquid chromatography-time-of-flight mass spectrometry (LC-MS). The LC-MS detection parameters are as follows:

[0232]

[0233]

[0234] d. Determination of molecular size heterogeneity by size exclusion chromatography-HPLC

[0235] Sample preparation: The sample was diluted with the mobile phase to about 1.0 mg / ml, centrifuged at 12000 rpm for 10 min, and the supernatant was injected for analysis.

[0236] Chromatographic column: TOSOH, TSKgel G3000SWXL, 5μm, 7.8mm*300mm;

[0237] Mobile phase: 100 mM PB + 200 mM arginine hydrochloride, 5% isopropanol (pH 6.8);

[0238] Flow rate: 0.6 mL / min;

[0239] Detection wavelength: 280nm;

[0240] Column temperature: 30℃;

[0241] Sample loading volume: 20 μL;

[0242] Washing time: 20 min;

[0243] Elution gradient: isocratic elution.

[0244] 4.3 Preparation and Characterization of Antibody-Drug Conjugates Targeting FRα

[0245] As described in “a. General preparation method of site-directed conjugation” above, anti-FRα antibodies HZB1, HZB1-H1L0, HZB1-H1L1, phage3, phage3-H0L0, phage3-H0L1, phage3-H1L0, and phage3-H1L1 were conjugated with drug-containing linkers BL20E and MWF-L6, respectively, to prepare site-directed conjugated ADCs.

[0246] As described in "b. Preparation method of control ADC ELAHERE" above, anti-FRα antibody Mirvetuximab was conjugated with sulfo-SPDB-DM4 to prepare control ADC ELAHERE.

[0247] Using methods a, b, and d from “4.2 General Analytical Methods for Antibody-Drug Conjugates” above, the DAR value, concentration, and purity of site-conjugated ADCs were determined, respectively.

[0248] Using methods a, c, and d from “4.2 General Analytical Methods for Antibody-Drug Conjugates” above, the DAR value, concentration, and purity of the control ADC ELAHERE were determined.

[0249] The results are shown in Table 3.

[0250] Table 3. Characterization results of antibody-drug conjugates

[0251]

[0252] Example Group 3 Activity evaluation of antibody-drug conjugates

[0253] Example 1 In vitro antitumor activity of antibody-drug conjugates targeting FRα

[0254] When using adherent cells, the effect of antibody-drug conjugates on the proliferation of tumor cells in vitro was detected using the sulfonylrhodamine B staining method (SRB method).

[0255] A certain number of logarithmically growing cells were seeded into 96-well culture plates and allowed to adhere overnight. Different concentrations of antibody-drug conjugates, antibodies, or cytotoxic drugs were then added. After incubation for 144 hours, the cells were fixed with trichloroacetic acid. After staining with SRB solution (prepared with 1% glacial acetic acid), 10 mM Tris solution was added to each well to dissolve the cells. OD values ​​were read at 510 nm using a microplate reader.

[0256] Inhibition rate (%) = (OD value control well - OD value drug administration well) / OD value control well × 100%

[0257] Based on the inhibition rate at each concentration, the half-maximal inhibitory concentration (IC50) was calculated using Graphpad Prism software. 50 The experiment was repeated once, and the data are expressed as mean ± SD, and a concentration (logarithm)-inhibition rate plot was generated. The results are shown in Table 4.

[0258] The results showed that HZB1-MMAE had a significant and selective inhibitory effect on the proliferation of FRα-positive cells, and its selectivity was correlated with the FRα expression level of cells and the cytotoxicity of the small molecule toxin MMAE. HZB1-MF6 did not fully exert its efficacy due to its slow release characteristics, but it has the potential to achieve sustained efficacy.

[0259] Table 4. Inhibitory effects of HZB1-MF6 and HZB1-MMAE on the proliferation of cultured tumor cells in vitro. 50 (n=2)

[0260]

[0261] Example 2 The binding activity of antibody-drug conjugates targeting FRα with FRα-positive or negative tumor cells

[0262] Different concentrations (3, 10, 30, 100, 300, 1000, 3000, 10000, 30000, 10000, 100000 ng / mL) of fluorescently labeled HZB1-MF6, HZB1-MMAE, Elahere, HZB1, and isotype control antibody hIgG1 were incubated with FRα-positive tumor cells KB, MC38 / FRα, or FRα-negative tumor cells MC38 on ice in the dark for 1 hour (the isotype control antibody hIgG1 was set at 100000 ng / mL). After centrifugation, the supernatant was removed, and the cells were washed with PBS and resuspended. The fluorescence intensity of the cells was detected by flow cytometry (BD ACCURI C6 PLUS). Each experiment was repeated once. Data are expressed as mean ± SD. Results are shown in Table 5. Figure 1 .

[0263] The results showed that HZB1-MF6, HZB1-MMAE, Elahere, and HZB1 all bound to FRα-positive tumor cells (KB) in a concentration-dependent manner. HZB1 had a stronger binding affinity to KB cells than the reference drug Elahere. HZB1-MF6 and HZB1-MMAE had a weaker maximum binding affinity to KB cells than HZB1.

[0264] Table 5. Binding of HZB1-MF6, HZB1-MMAE, HZB1, and Elahere to KB cells EC 50 (n=2)

[0265]

[0266] Example 3 Antibody-drug conjugates targeting FRα induce apoptosis in FRα-positive tumor cells

[0267] KB cells (purchased from ATCC) were seeded in 6-well plates and incubated for 144 hours with antibody-drug conjugate HZB1-MF6 (0.660, 6.597, 65.967, 659.664, 1978.991 nM), antibody HZB1 (678.32 nM), and cytotoxic drug MF-6 (0.1, 1, 10 nM).

[0268] KB cells were seeded in 6-well plates and incubated for 48 hours with antibody-drug conjugate HZB1-MMAE (0.651, 6.511, 65.115, 651.151 nM), Elahere (0.671, 6.709, 67.089, 670.885 nM), antibody HZB1 (678.32 nM), and cytotoxic drug MMAE (0.1, 1, 10 nM).

[0269] Supernatant and cells were collected separately, Annexin-V-FITC and PI were added, mixed well, and stained at room temperature in the dark for 15 minutes. Finally, 1× Bindig buffer was added for resuspending. Apoptosis was detected by flow cytometry (BD Accuri™ C6 Plus flow cytometer). The gating density for each sample group was 1×10⁻⁶. 4 Cells. Experimental data were obtained using BD CSampler. TM Analysis using Plus C6 Plus software. Results are shown below. Figure 2 .

[0270] The results showed that HZB1-MF6 and HZB1-MMAE induced apoptosis in KB cells in a concentration-dependent manner. The apoptosis induced by HZB1-MMAE was comparable to that elahere, the positive reference drug. MF-6 and MMAE also induced apoptosis in a concentration-dependent manner, while HZB1 did not have a significant apoptosis-inducing effect.

[0271] Example 4 Bystander effect of antibody-drug conjugates targeting FRα

[0272] FRα-positive KB cells and FRα-negative NCI-H929 / luc cells (NCI-H929 cells transfected with and expressing luciferase) were seeded into 96-well plates, or FRα-negative NCI-H929 / luc cells were seeded alone into 96-well plates. After incubating with antibody-drug conjugates HZB1-MF6 (30, 100, 300, 1000, 3000, 10000, 30000, 100000, 300000 ng / mL), HZB1-MMAE (1, 3, 10, 30, 100, 300, 1000, 3000, 10000 ng / mL), or Elahere (10, 30, 100, 300, 1000, 3000, 10000, 30000, 100000 ng / mL) for 144 hours, the cells were centrifuged, the supernatant was discarded, Lysis buffer was added to lyse the cells, centrifuged again, the supernatant was transferred to a microplate reader, the substrate luciferin was added, and the fluorescence intensity was detected using a microplate reader.

[0273] Inhibition rate (%) = (OD value control well - OD value drug administration well) / OD value control well × 100%

[0274] The experiment was repeated once, and the data are expressed as mean ± SD. Results are shown below. Figure 3 .

[0275] The results showed that HZB1-MF6, HZB1-MMAE, and Elahere all exhibited strong bystander effects, significantly killing FRα-negative NCI-H929 / luc cells while simultaneously killing FRα-positive KB cells. HZB1-MF6, HZB1-MMAE, and Elahere had no significant effect on the proliferation of FRα-negative NCI-H929 / luc cells cultured alone.

[0276] Example 5 Antibody-drug conjugates targeting FRα have the activity of inducing DNA damage in FRα-positive tumor cells.

[0277] KB cells were seeded in 6-well plates and incubated for 48 hours with antibody-drug conjugate HZB1-MF6 (0.660, 6.597, 65.967, 659.664, 1978.991 nM), antibody HZB1 (678.32 nM), and cytotoxic drug MF-6 (1, 10 nM).

[0278] KB cells were seeded in 6-well plates and incubated for 48 hours with antibody-drug conjugate HZB1-MMAE (0.651, 6.511, 65.115, 651.151 nM), Elahere (0.671, 6.709, 67.089, 670.885 nM), antibody HZB1 (678.32 nM), and cytotoxic drug MMAE (0.1, 1 nM).

[0279] Cells were collected and lysed using lysis buffer (0.1M MES, pH 6.6, 1mM MgCl2, 1mM EGTA, 0.5% Triton X-100, 10% glycerol). After centrifugation at 12000 rpm for 5 minutes, the supernatant was discarded, and 1×SDS sample buffer (50mM Tris, pH 6.8, 100mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol) was added. The cells were then denatured in a boiling water bath for 10 minutes, and the γ-H2AX level was detected by Western blot. Results are shown below. Figure 4 .

[0280] The level of γ-H2AX reflects the extent of intracellular DNA damage. The results showed that HZB1-MF6, like MF-6, HZB1-MMAE, Elahere, and MMAE, could induce DNA damage in KB cells, while HZB1 did not show a significant DNA damage-inducing effect.

[0281] Example 6 Antibody-drug conjugates targeting FRα inhibit the activity of microtubule aggregation in FRα-positive tumor cells.

[0282] KB cells were seeded in 6-well plates and incubated for 48 hours with antibody-drug conjugate HZB1-MMAE (0.651, 6.511, 65.115, 651.151 nM), Elahere (0.671, 6.709, 67.089, 670.885 nM), antibody HZB1 (678.32 nM), and cytotoxic drug MMAE (3, 10 nM).

[0283] Collect the supernatant and cells, centrifuge, add lysis buffer (0.1M MES, pH 6.6, 1mM MgCl2, 1mM EGTA, 0.5% Triton X-100, 10% glycerol), centrifuge at 12000 rpm for 5 minutes, discard the supernatant, add 1×SDS sample buffer (50mM Tris, pH 6.8, 100mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol), denature in a boiling water bath for 10 minutes, and then detect the level of tubulin aggregates in the cells using Western blot. Results are shown below. Figure 5.

[0284] The level of tubulin aggregates reflects the intracellular tubulin aggregation status. The results showed that HZB1-MMAE, Elahere, and the small molecule toxin MMAE, all inhibited intracellular tubulin aggregation in KB cells, while HZB1 did not show a significant inhibitory effect on tubulin aggregation.

[0285] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

Claims

1. An antibody-drug conjugate targeting folate receptor α or a salt thereof, comprising an antibody or antigen-binding fragment of the anti-folate receptor α covalently linked to a drug via a linker, characterized in that, The antibody-drug conjugate or its salt has the molecular formula Ab-[L-CPT]m, where Ab represents an antibody against folate receptor α or its antigen-binding fragment, L represents a linker, CPT represents a cytotoxic drug, and m represents the average number of drug linkages (average DAR) relative to each Ab molecule. Furthermore, the antibody against folic acid receptor α or its antigen-binding fragment comprises complementarity-determining regions (CDRs) of the heavy chain, namely heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and complementarity-determining regions (CDRs) of the light chain, namely light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), as shown below: (1) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21; (2) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.16; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21; (3) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.22, and SEQ ID NO.21; (4) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.16; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.22, and SEQ ID NO.21; (5) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.23; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21; (6) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.23; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21; (7) HCDR1, HCDR2, and HCDR3 sequentially comprising the amino acid sequences shown in SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.23; and LCDR1, LCDR2, and LCDR3 sequentially comprising the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.22, and SEQ ID NO.21; or (8) HCDR1, HCDR2, and HCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.23; and LCDR1, LCDR2, and LCDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.22, and SEQ ID NO.

21.

2. The antibody-drug conjugate or its salt according to claim 1, characterized in that, The antibody against folic acid receptor α or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising a combination of amino acid sequences selected from: (1) The amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.2; (2) The amino acid sequence shown in SEQ ID NO.3, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.4, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.4; (3) The amino acid sequence shown in SEQ ID NO.5, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.2; (4) The amino acid sequence shown in SEQ ID NO. 6, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 7; (5) The amino acid sequence shown in SEQ ID NO. 6, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 8, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 8; (6) The amino acid sequence shown in SEQ ID NO.9, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.7; (7) The amino acid sequence shown in SEQ ID NO. 9, or an amino acid sequence having at least 75% identity with said amino acid sequence; and the amino acid sequence shown in SEQ ID NO. 8, or an amino acid sequence having at least 75% identity with said amino acid sequence; or (8) The amino acid sequence shown in SEQ ID NO.9, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.4, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.4; Preferably, the antibody against folic acid receptor α or its antigen-binding fragment further comprises a heavy chain constant region (CH) and / or a light chain constant region (CL), preferably comprising a human or mouse heavy chain constant region and / or a light chain constant region.

3. The antibody-drug conjugate or its salt according to claim 1 or 2, characterized in that, In the molecular structural formula Ab-[L-CPT]m: a. The connector L can be a cuttable or non-cuttable connector; b. The CPT is selected from one or more of the following: microtubule inhibitors MMAE, DM1, DM4, Tublysin, muscarine, chalcogenide, eribulin, and derivatives thereof; topoisomerase inhibitors SN38, eczema, and derivatives thereof; DNA binders PBD, doxorubicin, and derivatives thereof; and / or cm is an integer or non-integer in the range of 1.0-10.0; preferably, m is an integer or non-integer in the range of 1.0-5.0, more preferably 3.0-4.2, more preferably 3.5-4.5, further preferably 3.8-4.2, and even more preferably 3.9-4.1; particularly preferably, m is 4.

0.

4. The antibody-drug conjugate or its salt according to any one of claims 1 to 3, characterized in that, The antibody-drug conjugate or its salt has the structure shown in the following structural formulas Ia and / or Ib: and / or In structural formulas Ia and / or Ib M is a phenoxy group or a phenoxy group substituted with one or more substituents, or a chemical bond; in the substituted phenoxy group, the substituent is selected from alkyl (e.g., C1-C6 alkyl, preferably C1-C4 alkyl), haloalkyl (e.g., haloC1-C6 alkyl, preferably haloC1-C4 alkyl, e.g., trifluoromethyl), alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen; SP1 is selected from C1-C8 alkylene, C1-C8 cycloalkylene, or C1-C21 (preferably C1-C16, more preferably C1-C11) straight-chain heteroalkylene, wherein the C1-C21 straight-chain heteroalkylene comprises 1-11 (preferably 1-6) heteroatoms selected from N, O, or S, wherein each of the C1-C8 alkylene, C1-C8 cycloalkylene, and C1-C21 straight-chain heteroalkylene is independently and optionally substituted by one or more substituents selected from hydroxyl, amino, sulfonic acid, and cyano groups; SP2 is selected from -NH(CH2CH2O)bCH2CH2CO-, -NH(CH2CH2O)bCH2CO-, -S(CH2)bCO- or chemical bonds, wherein b is an integer from 1 to 20, preferably an integer from 1 to 10, and more preferably an integer from 1 to 6; A represents a short peptide structure consisting of 2-4 amino acids or a combination of a short peptide structure consisting of 2-4 amino acids and a self-releasing structural fragment.

5. The antibody-drug conjugate or its salt according to claim 4, characterized in that, In structural formulas Ia and / or Ib: SP1 is selected from C1-C8 alkylene, C1-C8 cycloalkylene, or C1-C16, preferably C1-C11, more preferably C1-C7 straight-chain heteroalkylene, wherein the straight-chain heteroalkylene comprises 1-6, preferably 1-4, heteroatoms selected from N, O, or S; preferably, the heteroatom is O; and / or, A can be -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Phe-Arg-, or -Gly-Val-; -Glu-Val-Ala-, -Glu-Val-Cit-, or -Ala-Ala-Ala-; -Gly-Gly-Phe-Gly- or -Gly-Phe-Gly-Gly-; or Val-Cit-PABC.

6. The antibody-drug conjugate or its salt according to claim 4 or 5, characterized in that, In structural formulas Ia and / or Ib: aM is a halogen-substituted phenoxy group; preferably, M is a dihalogen-substituted (fluorine, chlorine, bromine, iodine) substituted phenoxy group; b. SP1 is -(CH2CH2O)a-CH2-, where a is an integer from 1 to 20, preferably an integer from 1 to 10, more preferably an integer from 1 to 6, and even more preferably an integer from 2 to 4; further preferably, a is 3; and / or c.SP2 is a chemical bond.

7. The antibody-drug conjugate or its salt according to any one of claims 1 to 6, characterized in that, The antibody-drug conjugate or its salt has the structure shown in the following structural formulas IIa and / or IIb: and / or Optionally, CPT is a camptothecin-type compound; Preferably, the structure of CPT is as shown in structural formula I, wherein structural formula I is connected to the carboxyl group in group A of structural formula Ia and / or Ib or structural formula IIa and / or IIb via an amide bond, preferably with the amino group adjacent to group G in structural formula I connected to the carboxyl group in group A via an amide bond. In structural form I, R1, R2, R3, and R4 are independently hydrogen, halogen (fluorine, chlorine, bromine, iodine), C1-7 alkyl or substituted C1-7 alkyl, or any two of R1, R2, R3, and R4 together with the carbon atoms they are attached to form a C3-6 cyclic alkyl group; preferably, R1 and R2 are the same; and / or, R3 and R4 are the same; more preferably, R1 and R2 are halogen (fluorine, chlorine, bromine, iodine), and R3 and R4 are hydrogen; G is hydrogen, halogen, methyl or methoxy, preferably hydrogen, fluorine or chlorine; Y is oxygen, sulfur, methylene, or a substituted methylene group; when Y is a substituted methylene group, the substituent is benzyl or alkyl. X represents oxygen or sulfur; n = 0 or 1.

8. The antibody-drug conjugate or its salt according to claim 7, characterized in that, The structure of CPT is shown in structural formula IA, wherein structural formula IA is connected to the carboxyl group in group A in structural formulas Ia and / or Ib or structural formulas IIa and / or IIb via an amide bond: Optionally, CPT is an olistatin compound; Preferably, CPT is MMAE or MMAF.

9. The antibody-drug conjugate or its salt according to any one of claims 1 to 8, characterized in that, The antibody-drug conjugate or its salt has the following structure: and / or Alternatively, the antibody-drug conjugate or its salt may have the following structure: and / or 10. A method for preparing the antibody-drug conjugate targeting folate receptor α or a salt thereof according to any one of claims 1 to 9, the preparation method comprising the following steps: (1) React the antibody against folic acid receptor α or its antigen-binding fragment with a reducing agent in a buffer solution to obtain the reduced antibody or its fragment. (2) Crosslink the drug-containing linker with the reduced antibody or its fragment obtained in step (1) in a mixture of buffer and organic solvent to obtain an antibody-drug conjugate targeting folic acid receptor α. The drug-containing linker has the structure shown in Formula III: In structural form III, M, SP1, SP2, A and CPT are as defined in any one of claims 1 to 11; E is selected from the following groups, wherein... Indicates the connection site with M: E-1: E-2: E-3: E-4: E-5: Japanese E-6:

11. A composition comprising an antibody-drug conjugate targeting folate receptor α or a salt thereof, as described in any one of claims 1 to 9.

12. Use of the antibody-drug conjugate targeting folate receptor α or a salt thereof, or the composition of claim 11, in the preparation of a medicament for treating tumors, according to any one of claims 1 to 9.

13. A kit comprising an antibody-drug conjugate targeting folate receptor α according to any one of claims 1 to 9, or a salt thereof, or a composition according to claim 11.

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